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PMC11699711 | In recent years, regenerative endodontic therapy (RET) has been suggested for the treatment of immature necrotic teeth . The primary aim of RET is apical healing; secondary aims include continued root development, demonstrated by both root elongation and an increase in the canal wall thickness, as well as a positive response to sensibility tests . In a follow-up of teeth one year after RET, apical healing and complete apical closure were observed in 100% and 55% of the teeth, respectively, which were accompanied by an increase in the canal’s width and lengthening of roots . Similar survival rates were reported in a recently published meta-analysis . Clinically, during the final steps of RET, a layer of bio-ceramic material barrier is applied on the scaffold within the root canal space . There is limited evidence as to the effect of the scaffold, either platelet-rich plasma (PRP), platelet-rich fibrin (PRF), platelet pellet, or blood clot, on the secondary outcomes, although all treatment modalities resulted in healing of the apical rarefaction . A retrospective study reported lower success and survival rates after RET in traumatized immature necrotic teeth. The type of traumatic injury had an effect on the treatment outcome, with teeth after avulsion having the lowest survival and success rates (66.7% and 33.3%, respectively) . The etiology of pulp necrosis, which led to treatment, either trauma, dens evaginatus, or caries, did not affect the success of the treatment, which ranged between 93.1% to 96% . Histological examinations of extracted human teeth that have undergone regenerative endodontic therapy (RET) have shown that the newly formed tissues primarily consist of fibrous connective tissue and cementum-like connective tissue . Failures after RET may be observed during follow-up examination. In a systematic review of failed RET cases, 39% of the 67 failures were detected more than two years after initiation of the RET . According to a study that analyzed 16 failed cases, 37.5% of the failures were identified within six months after the procedure. The main etiologic factors that can lead to failure are dental trauma, dens evaginatus, and dental caries . Interventions after failures include extraction, nonsurgical root canal treatment, repeated RET, and Ca(OH)₂ apexification , as well as a surgical approach . There is scarce clinical evidence regarding the outcome of treatment of failed RET cases . In a report of three failed root canal treatment (RET) cases, the teeth were retreated after repeated debridement of the root canals. All the cases were treated in a single visit using negative-pressure irrigation. Orthograde root canal treatment was performed in one case, and in a second case, orthograde retreatment was supplemented by an apical mineral trioxide aggregate (MTA) prior to obturation. In the third case, repeated RET was performed. In all three cases, resolution of the periapical lesion was observed . Successful repeated RET was observed in another case report describing the failure of a previously healed immature incisor tooth. The tooth was retreated in one visit and showed complete apical repair after two years . The main reason for failure is a persistent bacterial infection . A more significant decrease in primary microflora was found in successful RET cases than in failed cases treated with either Ca(OH)₂ or chlorhexidine. After dressing with Ca(OH)₂ or chlorhexidine, a greater reduction in microflora was observed in successful RET cases compared to failed treatments . It is assumed that lack of mechanical debridement during the regenerative procedure allows for relapse of the apical pathology after a temporary resolution of the signs and symptoms . This study aims to describe the outcome of retreatment in failed cases of regenerative treatment, describe the reasons for the failure, discuss the optional treatment options, and present a decision-making flowchart. The study was conducted in the Hebrew University Hadassah School of Dental Medicine in Jerusalem. Ethical approval was obtained from the Institutional Helsinki Ethics Committee . Data collection was based on the digital medical records of patients treated by endodontic post-grad students between 2015 and 2020. From patients aged six to 17, 414 endodontically treated immature teeth (Nolla stages 7-9) were screened to allocate patients with a failed RET. Data including patients' ages, sex, medical, and dental history were collected. Their dental records, specifically regarding the treated tooth, were compiled. Cases with incomplete records or insufficient follow-up were excluded. Around 48 teeth underwent RET and were followed for one year or more. Eight failed cases were observed among the teeth that underwent RET. Two teeth were referred for extraction due to a root fracture. Six patients (aged seven to 16 years) who were referred for the management of apical periodontitis that developed after a previous RET failed were retreated and followed for more than one year. The teeth included five maxillary incisor teeth and one maxillary second premolar (Table 1 ). Reviewing the medical records of the young patients treated in the endodontic post-grad clinic revealed 48 well-documented cases of RET, eight of which failed. Initially, 46 of the cases were successful, with one immediate failure recorded and one tooth extracted because of infection after one year. This resulted in an initial survival rate of 97.9% (47 out of 48 teeth) and an initial success rate of 95.8% (46 out of 48 teeth). Over time, one additional tooth was extracted due to root fracture, leading to a long-term survival rate of 95.8% (46 out of 48 teeth) and a long-term success rate of 83.3% (40 out of 48 teeth). A seven-year-old female patient who was diagnosed with pulp necrosis and asymptomatic apical periodontitis of the left maxillary central incisor following a complicated crown fracture and traumatic extrusion was treated by RET according to the American Association of Endodontists (AAE) guidelines. The immature tooth was classified as Nolla stage 7, and the plug placed in the canal during the second visit was MTA ProRoot (Dentsply Sirona, Johnson City, TN). The girl was scheduled for follow-up appointments, and after 1.5 years, continued root development, and the periapical lesion has healed. Six years later, a periapical lesion reemerged, and her dentist referred her after unsuccessfully attempting to perform a root canal treatment. The root was fully developed and was now classified as Nolla 10, and pulp canal obliteration (PCO) was observed. During the orthograde retreatment, the root canal was negotiated using ReadySteel C+ files (Dentsply Maillefer, Switzerland) under the operative microscope. After ultrasonic irrigation, the canal was obturated with gutta-percha and AH Plus root canal sealer (Dentsply, DeTrey, Konstanz, Germany), and the access cavity was sealed with a composite restoration. After a one-year follow-up, the periapical lesion healed . An eight-year-old male patient who was diagnosed with asymptomatic apical periodontitis for the right maxillary central incisor following lateral luxation was treated by RET according to the AAE guidelines. The immature tooth was classified as Nolla state 8, and the plug placed in the canal during the second visit was MTA ProRoot (Dentsply Sirona Johnson City, TN). The boy was scheduled for follow-up appointments, and after three years, the periapical lesion had healed, and a calcified barrier was observed apical to the MTA plug. The root canal remained without any additional change. Seven years after the RET, he was referred for treatment by his orthodontist due to a reemerging periapical lesion. The MTA plug appeared to be disintegrated. After the composite restoration of the access cavity was removed, the remnants of the MTA plus were removed. Following chemomechanical preparation, and since the apical foramen diameter was very wide, an apical plug was placed using EndoSequence® RRM (Brasseler, USA). After a one-year follow-up, the periapical lesion healed . A 16-year-old male patient was diagnosed with pulp necrosis and symptomatic apical periodontitis for the right maxillary central incisor following a traumatic dental injury that occurred two and a half years earlier. The exact nature of the dental injury was unknown. According to his dental records, the boy was treated by RET following the AAE guidelines. The immature tooth was classified as Nolla state 8, and the plug placed in the canal during the second visit was MTA ProRoot (Dentsply Sirona, Johnson City, TN). The boy returned 2.5 years after completion of the RET due to the re-emergence of the periapical lesion. Considering the large MTA plug, the calcified barrier apical to the plug, and the thin dentinal walls, apical surgery was considered. The patient was scheduled for apical surgery. During the procedure, the canal was only minimally resected, and the canal walls were irrigated and scrubbed with a micro-brush with chlorhexidine 2%. The empty root canal space was filled with EndoSequence® RRM. At the two-year follow-up, incomplete healing was observed . A 15-year-old male patient who was diagnosed with asymptomatic apical periodontitis of the right maxillary second premolar was referred by his orthodontist before the beginning of an orthodontic treatment. RET was performed two years earlier, following the AAE guidelines using MTA, because of pulp necrosis that developed because of the irregular type II dens invaginatus anatomy. Upon arrival, it was evident that the root continued to develop, although a periapical lesion was now evident. A CBCT scan revealed that the dens had been only partially sealed, and the canal apical to the intra-coronal plug was obliterated. Following the replacement of the coronal seal, apical surgery was performed. The short root canal was only minimally resected. Debridement of the apical root canal was done using ultrasonic tips, and retrograde obturation was done using EndoSequence® RRM. After one year, complete healing of the apical radiolucency was observed, and orthodontic treatment was initiated. Three years after the completion of the RET, the periapical tissue remained normal . A 10-year-old male patient who suffered a crown fracture and pulp necrosis with asymptomatic apical periodontitis in the right maxillary central incisor was treated by RET using MTA following the AAE guidelines. Apical repair was observed during the follow-up examination, and the tooth remained asymptomatic. Two years later, his orthodontist referred the patient due to an acutization of an apical radiolucency. A periapical radiograph and a CBCT scan demonstrated an increase in the thickness of the root canal walls, with the root end becoming blunt and almost closed. Repeated RET resulted in complete repair of the periapical lesion and a calcified apical tissue, observed two years later. After apical healing, the orthodontic treatment was continued . A nine-year-old boy presented with a horizontal root fracture in the right central maxillary incisor following the tooth's extrusion. A periapical radiograph revealed a type II dens invaginatus. The tooth was diagnosed with pulp necrosis, and inflammatory root resorption was observed. RET was performed according to the AAE guidelines using calcium hydroxide dressing between visits. After bleeding was allowed into the canal space, a collagen plug was placed as a barrier covered by EndoSequence® RRM. Eight weeks after its initiation, treatment was completed, and healing of the radiolucent lesion was evident. During follow-up, the tooth exhibited recurrent pathology at the fracture line. The patient was scheduled for retreatment of the tooth. Repeated RET included additional ultrasonic irrigation and calcium hydroxide dressing. One year after retreatment, the apical rarefaction resolved with continued maturogenesis of the apical fragment and approximation of the coronal and apical fragments attached by a calcified tissue and surrounded by the continuous periodontal ligament . Regenerative endodontics has become a promising alternative for treating immature teeth with apical pathology . A recent meta-analysis reported a 100% survival rate for immature teeth after RET for at least one year . Similar success rates that ranged between 93.1% and 96% were found for teeth that underwent RET due to trauma, dens evaginatus, or caries . These results are similar to the one-year survival and success rates reported in the current study (97.9% and 95.8%, respectively). Despite high survival and success rates , failures can still occur, presenting clinicians with challenging treatment decisions, particularly in immature teeth . Despite initial signs of healing and continued root development observed during short-term follow-up examinations, six cases ultimately failed. These cases were successfully managed through secondary interventions. The timeframe during which RET failures are detected varies significantly, ranging from less than six months following treatment to as long as four, eight, and even 11 years . In the literature, the average follow-up time reported was shorter: 19.9 months for the mineral trioxide aggregate plug and 16.7 months for the RET group . In the current study, one tooth was extracted immediately after the RET due to persistent infection, and one failure was observed three months after the initial follow-up visit, while the other five cases showed failures between two and seven years after the RET was completed, and another tooth was extracted due to a root fracture, despite initial signs of success. This study emphasizes that while short-term success is encouraging, it does not guarantee long-term stability in RET cases. The success rate dropped over time from 95.8% to 83.3%. The study strongly advocates for extended, diligent monitoring to ensure the best possible outcomes for patients undergoing regenerative endodontic procedures. The initial success of the regenerative treatment was observed during the follow-up in the cases described above. It was manifested in the healing of the periapical lesion and the continued development of the roots. However, over time, a new periradicular rarefaction reemerged. RET failures have been attributed to persistent infection or inadequate root canal disinfection due to a lack of mechanical debridement, which is associated with an effort to avoid further weakening of the thin and fragile dentinal walls . Indeed, histological examination after RET’s failure demonstrated bacteria and biofilm on the canal walls . It was reported that most of these failures were following a secondary dental trauma (56%), as observed in case number 6; abnormal anatomy of the dens invaginatus (25%), as observed in case number 3; and a result of persistent infection, as observed in case 5 . Failures that occur after the initiation of orthodontic treatment , as reported in case number 2, raise concerns as to the immune competency of the revitalized tissue after regenerative endodontic procedure (REP) . Teeth treated by RET may not withstand biting forces and fracture, especially if they are poorly developed and the canal walls are short and thin . Thus, they necessitate extraction, as they may not be restorable . Repeated RET should be considered in an attempt to improve the low prognosis resulting from the thin root walls and the unfavorable crown-to-root ratio. RET is likely to fail without adequate root canal disinfection . Efforts are being made to improve the efficacy of the antibiotic paste placed in the canals during RET . Current disinfection protocols applied during RET cannot eliminate bacterial infection from the root canals . Supplementary agitation of sodium hypochlorite by means such as ultrasonic activation or the use of XP finisher (FKG Dentaire SA, La Chaux-de-Fonds, Switzerland) can effectively reduce the biofilm and enhance disinfection of the root canal , especially in wide root canals . In the event of a failed regenerative endodontic procedure (REP), the procedure can be repeated, offering another opportunity for success in cases where the initial REP did not yield the desired outcomes . Modifying the protocol during repeated procedures and treatments may be necessary, allowing for variations in the procedure. The current protocol includes the use of several antimicrobial agents . Alternatives like calcium hydroxide or modified TAP can be considered, along with a different intra-coronal barrier. Another bioceramic material, such as Biodentine, which has different characteristics and setting times, may perform better . RET was successfully repeated in two cases. The successful treatment outcome in case number 6 after repeated RET can be attributed both to the patient's young age and the wide diameter of the canal at the fracture line . This may imply that when a failure occurs in teeth with a large apical diameter, especially when the patient is young, the preferred treatment modality will be a repetition of the RET. However, a surgical approach may be required when a calcified barrier has formed apical to the bioceramic plug in the canal . Surgical endodontics can be a viable option to address regenerative endodontic failures. The main advantage of this procedure in teeth with short roots is maintaining the crown-to-root ratio. It is usually recommended to resect 3 mm of the root end during apical surgery to remove the majority of ramifications and lateral canals . Since the apical canal is quite wide, it may be advisable to minimally resect the root by one mm or to smooth out any uneven edges of the root. Favorable outcomes were reported after minimal root resection and extended retrograde root end preparation . This approach can also be suggested when the orthograde access to the infected tissue in the canal is blocked. Removal of the bioceramic plug from the canal may risk the tooth in fracture. When anatomical constraints prevent apical surgery, intentional replantation (IR) can be an alternative treatment option. A case report highlights the successful intentional replantation of an immature central incisor that had undergone RET and later developed a chronic apical abscess. Despite signs of external root resorption, the replanted tooth remained symptom-free throughout a four-year follow-up period . In most cases, failure occurred after the healing of the periapical lesion and continued root development. Despite the late failure, it can be assumed that teeth with a better crown-to-root ratio and the increase in the root wall thickness are advantageous compared to more immature teeth, which are more susceptible to fracture . Additionally, the time between regenerative endodontic therapy (RET) and the decision to perform a full root canal treatment (RCT) due to reemerging pathology may allow for greater patient cooperation, particularly among young individuals. Teeth that have undergone successful RET and demonstrated treatment failure at the follow-up appointments can undergo additional treatment that will allow the preservation of the teeth and healing of the periapical lesion. The treatment options include ReRET, orthograde root canal treatment with or without an apical plug, or surgical root canal treatment. Despite the initial treatment failure, a successful outcome was observed after a secondary intervention. | Study | biomedical | en | 0.999997 |
PMC11699719 | Iron administration in piglets in their first days of life is a basic zootechnical measure relevant for welfare and health. Dependent on body weight, pigs are marginally equipped with iron at birth ranging from 124 to 180 mg iron/kg dry matter in piglets from 0.8 kg to 1.6 kg body weight . For piglet growth with daily weight gains of 250 g, approximately 10 mg iron/day is needed , resulting in iron depletion within the first three weeks of the suckling period without iron supplementation. Iron concentrations of 1.7–5.4 µg/mL in sow colostrum and 1.3–4.6 µg/mL in milk are not adequate to fill this supply gap . Although study outcomes about oral iron supplementation vary with respect to efficacy oral iron uptake from external sources is assessed to be insufficient due to low intestinal iron absorption . The immaturity of the duodenal mechanisms of iron absorption was found to be related to low expression of iron transporters (duodenal divalent metal transporter (DMT) 1 protein, ferroportin) and a high expression of hepcidin as the major iron-regulatory hormone during the first days of life . A physiological tight control of iron metabolisms and regulation of iron release into extracellular body compartments is essential to prevent toxic effects of iron . It can be assumed, that breeding success with regard to high piglet growth rates and high number of piglets born alive overstrain genetically determined and evolutionary meaningful iron homeostasis control mechanisms. The naturally low foetal iron reserves are still low in litters of hyperprolific sows, while in parallel growth rate and feed efficacy of piglets have increased, and also the demand for essential nutrients as iron . While in adults iron homeostasis is dependent on absorption in the duodenum and its recycling by macrophages, iron deficiency anaemia (IDA) in piglets with the consequence of growth retardation, depression and a disturbance of cognitive development can only be prevented by iron supplementation . Immediately after birth a haemodilution caused by colostrum uptake and subsequent shifts in body fluid to the circulatory system leads to low haemoglobin (Hb) concentrations in piglets. The heaviest piglets are considered to be at higher risk to become anaemic due to their larger blood volume . If not treated, a latent iron deficiency in the first living days will finally result in a microcytic hypochromic anaemia characterized by Hb concentrations below 90 g/L and small erythrocytes . Cut-off Hb concentrations considered critical for piglet health differ among experts and are still under discussion. This might be due to a lack of currently valid values for Hb corrected for breed, production phase and age . Some authors suggest a Hb of 110 g/L to exclude latent iron deficiency , which corresponds to the global cut-off for children younger than 5 years defined in 1968 in a WHO technical report . In swine, either Hb concentrations below 80 g/L are considered critical for piglet health or Hb concentrations below 90 g/L . Some authors recommended for diagnostic of IDA the determination of iron-related parameters (e.g. ferritin, transferrin and transferrin saturation, iron, total iron binding capacity) next to established haematological and reticulocyte parameters [ 7 , 15 – 17 ]. Iron-related parameters are more difficult to interpret with respect to health status, because iron homeostasis is tightly regulated. Due to the biological duality of iron, balancing of risks and benefits of iron administration in suckling piglets is under research in different production systems with varying available iron sources from feed, water and environment. A comparison of piglets raised in intensive indoor and outdoor production systems and supplemented with 100 mg iron dextran on day three of age resulted in higher Hb concentrations on day 28 of life in outdoor pigs . Some authors conclude that no prophylactic iron treatment is necessary in outdoor pig production . A comparison of organic piglets either raised indoor with iron supplementation or outdoor without iron supplementation resulted in higher Hb concentrations in outdoor pigs, because soil contained sufficient bioavailable iron . In outdoor pigs with physiologically low Hb concentrations on their third day of life and receiving no iron treatment the majority of piglets was not anaemic at weaning . Treated pigs showed lower Hb levels at weaning, indicating reduced iron absorption from the duodenum due to downregulation of ferroportin . Other authors conclude that iron administration in general is necessary in outdoor pig production . In organic farms with prolonged suckling periods one iron injection was found to be not sufficient in spite of iron uptake from external sources as soil and piglet starter diet . Contradictory results might be explainable by differences in bioavailable iron in soil or by other factors as genotype of the sow or season . In intensive pig production daily iron requirements of suckling piglets are usually complied by subcutaneous or intramuscular (IM) administration of iron dextran or gleptoferron in the first days of life . Currently, combined iron products containing an active substance against coccidia are also used on farms. The advantage of these products combining two routine measures in one is that they save time and effort. The parenteral application of a coccidiostat is more comfortable than the formerly established oral application with same efficacy . However, when using a combination product targeting two diseases, an adjustment of the necessary amount of iron to prevent anaemia in the frame of a farm-specific treatment protocol is not possible. The aim of this study was to test for non-inferiority of iron dextran (Uniferon ® , Pharmacosmos A/S, Holbaek, Denmark), which can be used in flexible volumes, versus to the combinatory product Forceris ® (Forceris ® , Ceva Santé Animale, Libourne, France) containing gleptoferron and toltrazuril. In addition, different administration schemes for iron dextran with respect to time point and dosage were compared on a conventional farm. The animal experiment was performed in a farrow-to-finish farm with 230 sows (Danbred genetics) and 2550 fattening places in a swine dense region in North-Western Germany. Farrowing intervals were five weeks with a four-week suckling period and farrowing groups of approximately 57 sows. Sows of one farrowing group farrowed in three farrowing units with 20 places each. No nurse sows were used, and split suckling was not performed. The farm was known positive but stable for porcine reproductive and respiratory syndrome virus (PRRSV) according to category II-vx and for Actinobacillus ( A. ) pleuropneumoniae and Mycoplasma (M.) hyopneumoniae . No disease outbreaks were observed in the year before start of the study. All sows were vaccinated every four months with a live attenuated PRRSV-1 vaccine (Suvaxy ® PRRS MLV, Zoetis Deutschland GmbH, Berlin, Germany) and against influenza virus with a triple vaccine containing H1N1, H1N2 and H1N3 (Respiporc FLU3, Ceva, Düsseldorf, Germany) as well as a vaccine against H1panN1 (Respiporc FLUpanH1N1, Ceva). Sows were further vaccinated every six months against parvovirus and erysipelas (Parvoruvac ® , Ceva). Piglets are vaccinated routinely against edema disease on day five of life (Ecoporc SHIGA, Ceva) and on day 23 of life against PRRSV-1 (Suvaxy ® PRRS MLV), porcine circovirus 2 (PCV2) and M . hyopneumoniae in combination (Porcilis ® PCV M Hyo, MSD Tiergesundheit, Unterschleißheim, Germany). In addition, piglets were vaccinated twice on day 23 and three weeks later with an autogenous vaccine containing killed Streptococcus ( S .) suis and A. pleuropneumoniae strains. On average 16.7 live and 1.8 dead born piglets with 15% suckling piglet losses were recorded on the farm in the study year. The piglet´s average weights at weaning were 7 kg and the average daily weight gain in the fattening period 0.9 kg. Study piglets were selected randomly and stratified by weight and sex out of 17 litters of sows in different farrowing units, which had farrowed on the same day. A total number of 136 piglets out of the 17 litters were included in the study on their second day of life. Sows farrowed in conventional crates on a fully slatted floor. Farrowing was not induced medically, supervised on the main farrowing days from 6am to 10pm and assisted if necessary. The ambient temperature in the farrowing room during farrowing was 26 °C due to warm weather conditions in summer. Piglets had access to a water-heated resting area with a surface temperature of 33 °C. No cross-fostering was performed during the experiment in the 17 litters. In all farrowing pens an automatic replacement milk system was implemented to provide milk supplement from day 5 to 11 of life. Creep feeding started at day 12 of life ad libitum with a supplementary feed as training mash (wet creep feed) provided via the automatic replacement milk system until weaning. Since day 16 of life additional dry creep feed was provided in an extra trough ad libitum until three days after weaning. Since day 3 after weaning creep feeding was paralleled by feeding nursery starter diet ad libitum (Table 1 ). At weaning on day 26 of life piglets were brought litter-wise to the nursery pens with three litters stalled in one pen with place for 33 piglets on fully slatted plastic floor. Pens were equipped with automatic dry feeders with eight feeding places and four nipple drinkers. In addition temporary drinking trays were provided as well as three chains with wooden beams as enrichment objects. Lightweight piglets were collected from different litters and commingled in separate nursery pens. Ambient temperature in the nursery unit was 30 °C for two days at the beginning of the nursery period and decreased to 28 °C until end of the first week. In the following six weeks temperature was decreased by 1 °C to 22 °C until end of nursery. After a seven-week nursery period, pigs were brought to the fattening unit in a neighbouring building with place for ten pigs per pen on fully slatted floors. Water was used from a well and feed for pigs of all production stages was bought from a commercial feed company. Analysis of drinking water showed low nitrate (127 mg/L), low sulphate (107 mg/L) and low iron (< 0.1 mg/L) concentrations, and low number of colony forming units (CFU) of 33–49 CFU/mL after incubation at 22 °C and 17–39 CFU/mL after incubation at 36 °C without any detection of coliforms. Feed iron contents are recorded in Table 1 . From the current farrowing group only litters from those sows were included in the study, which had farrowed on the same main farrowing day. Out of this subgroup of 34 sows 17 sows with at least ten and less than 18 live born piglets were selected. In the study group 88% of the sows were in parity 4–7, one sow had the first and one the ninth litter. Within each of the 17 litters eight healthy piglets (four males and four females if available) weighing at least 1 kg were allocated to four treatment groups by target randomization based on weight and sex strata resulting in two piglets per group in each litter, which were sampled and treated according to Table 2 . For intramuscular (IM) treatment disposable syringes with a maximum volume of 2 mL (HSW HENKE-JECT ® , Henke Sass Wolf, Tuttlingen, Germany) connected to disposable needles (20-gauge, 0.90 × 40 mm (Sterikan ® , B.Braun, Vet Care GmbH, Tuttlingen, Germany) were used. Needles were only used once. Piglets were examined, sampled and weighed at start of the study on day 2 of life, on day 11 of life, at weaning, end of nursery and end of fattening. Ear tagging was performed on day 2 of life. On day 3 of life piglets were tail-docked and on day 6 male piglets were castrated after pain treatment with 0.4 mg meloxicam (Metacam ® , Boehringer Ingelheim Vetmedica GmbH, Ingelheim, Germany)/kg body weight (BW) and under general anaesthesia by IM injection of 25 mg ketamine (CP-Pharma Handelsgesellschaft mbH, Burgdorf, Germany)/kg BW and 2 mg azaperon (Stresnil ® , Elanco, Kiel Germany)/kg BW. No further zootechnical measures or treatments were performed routinely and no treatment was necessary in piglets of the study group. Study design was elaborated to prove (i) non-inferiority of IM administration of 200 mg iron dextran (1 mL Uniferon ® ) compared to IM administration of 200 mg iron (III)-gleptoferron and 45 mg toltrazuril (1.5 mL Forceris ® , Ceva Santé Animale, Libourne, France) with respect to the major target variable Hb measured prior to weaning on day 24 of life and (ii) to test if additional amount of iron given on day 2 of life or a repeated iron administration on day 11 of life were beneficial for growth and Hb concentrations. An effect of toltrazuril on gut microflora and therefore indirect on intestinal iron absorption cannot be excluded, so that toltrazuril was given in all experimental groups . Toltrazuril as one component in the combinatory product Forceris ® was orally administered as a singular product (Cevazuril ® , Ceva Santé Animale, Libourne, France) in groups 1, 3 and 4. Similar approaches have been performed previously in other comparative studies . For oral administration a disposable syringe with a maximum volume of 2 mL (HSW HENKE-JECT ® ) were used. The syringe was placed laterally through the piglet`s mouth cleft behind the tongue ground before 0.9 ml Cevazuril ® was applied. The piglet’s mouth was closed with gentle pressure until the product was swallowed. Table 2 Experimental groups of piglets provided with different iron treatment strategies Group Treatment Sample size ( n ) 1 Single products: 200 mg iron dextran IM, oral application of 45 mg toltrazuril on day 2 34 2 Combinatory product: 200 mg gleptoferron and 45 mg toltrazuril IM on day 2 34 3 Single products: 300 mg iron dextran IM, oral application of 45 mg toltrazuril on day 2 34 4 Single products: 200 mg iron dextran IM, oral application of 45 mg toltrazuril on day 2 and 200 mg iron dextran IM on day 11 34 Within each of 17 litters eight healthy piglets were allocated to the four treatment groups by target randomization based on weight and sex strata resulting in two piglets per group in each litter Blood samples were collected from the Vena cava cranialis in volumes of 2 mL in collection tubes containing ethylenediaminetetraacetic acid (EDTA) (Kabevette ® , Kabe Labortechnik, Mümbrecht-Elsenroth, Germany) using 21-gauge, 0.80 × 40 mm needles (Sterikan ® , B.Braun). Blood samples were placed in an isolated box with cool pads until end of sampling, transported to the laboratory at the Clinic for Swine and Small Ruminants, University of Veterinary Medicine, Hannover, stored at 6 °C overnight and analysed within 24 h. Haematological variables (Hb, packed cell volume (PCV), erythrocytes, mean corpuscular haemoglobin concentration (MCHC), leucocyte count, thrombocyte count) were automatically analysed in a haematology analyser . Table 3 Clinical variables recorded on days 2, 11, 24, 74 and 160 of life Variables Clinical scores 0 1 2 3 4 5 Claws No claw lesions Pododermatitis haemorrhagica Panaritium - - - Skin lesions head No skin lesions Slight and superficial scratches Dominant red scratches Wet wounds - - Skin dorsal on carpal joints No skin lesions Slight and superficial scratches and crusts Extended crusts Wet wounds - - Navel 1 Physiological diameter and consistency Diameter 0.8–1.5 cm Diameter > 1.5 cm Wet navel Purulent secretion or abscess Hernia umbilicalis Diarrhoea No Pulpy feces Liquid feces - - - Ear Intact skin Dry scratches Bloody wound - - - Tail lesion Intact skin Dry scratches Bloody wound - - - Tail losses Length after tail docking ≥ half length of tail ≥ two third of tail Total tail loss - - Clinical scores were used in suckling piglets for skin lesions dorsal on carpal joints and head, claw lesions and navel. In nursery and fattening pigs tail and ear lesions were scored. All pigs were examined for any other deviations from physiological findings 1 only suckling piglets A sample size calculation for a non-inferiority t-test between experimental groups 1 (200 mg iron dextran) and 2 (200 mg gleptoferron) was performed using NCSS PASS with target power (1-β) 0.8 and target significance level α of 0.05. The calculation was based on a non-inferiority assumption according to expected Hb concentrations as the major target variable deduced from results in three published comparison trials [ 32 – 34 ]. According to these studies a difference of 5 g Hb/L between treatment groups was considered equivalent and defined as a margin for non-inferiority testing between different schemes of iron administration in this study (Table 2 ). Spearman`s rank correlation coefficients were calculated for all variables with a quantitative outcome. Frequencies of clinical findings were compared by chi-square testing between the groups. For the main primary hypothesis under study non-inferiority testing was performed to prove that treatment of piglets in group 1 (200 mg iron dextran) is non-inferior to treatment of piglets in group 2 (gleptoferron) with respect to Hb at weaning. A boundary threshold of 5 g/L Hb was chosen for this test (see above). Procedure of non-inferiority testing was based on Schuirmann’s method of two one-sided tests (TOST). Within explorative analyses for secondary endpoints group differences with respect to red blood cell measurements and weight gain were tested in an analysis of variance model followed by least square means tests for group effects. Due to the explorative nature of these analyses no multiple adjustments were conducted. In addition, it was explored if treatment effects were influenced by birth weights. To include the potential effects of birth weight in the model, piglets were allocated to three birth weight classes. Three weight groups were defined, i.e. “low weight piglets” from the minimum to the 25% quantile, “medium weight piglets” from the 25% to the 75% quantile, “high weight piglets” from the 75% quantile to the maximum. Allocation of animals by birth weight class and treatment to the groups are shown in Table 4 . General linear models with fixed effects group and birth weight class and the combined effects of both were analysed with respect to red blood cell variables at weaning. Table 4 Number of piglets in experimental groups and range of body weights Experimental groups Group 1 Group 2 Group 3 Group 4 Weight classes Body weight (kg) n Body weight (kg) n Body weight (kg) n Body weight (kg) n Weight class 1 (low) 1.14–1.48 9 1.12–1.48 7 1.26–1.46 9 1.10–1.48 10 Weight class 2 (medium) 1.52–1.94 16 1.50–1.94 20 1.50–1.94 14 1.56–1.94 14 Weight class 3 (high) 1.98–2.20 8 2.02–2.30 7 1.96–2.46 8 1.96–2.36 9 Number of piglets allocated in experimental groups and weight classes: sample size and range of weights at study start on day 2 of life, n: number of piglets within weight class The 17 sows included in the study had in average 15.6 live born, 8.9 dead born and 14.4 weaned piglets. The suckling piglet mortality was 7.5% in the litters of the study group. During the suckling period four animals out of the study groups were euthanized and sent for necropsy. Due to chronic disease (starvation, arthritis, ascites) these pigs were considered as biological outliers and were excluded from the final statistical examination. One additional pig with severe arthritis was excluded from the study. All injection sites were inconspicuous in all animals during the whole course of the study. In the majority of suckling piglets, a slight pododermatitis haemorrhagica (reddening of the heels) was found, while only one animal showed a panaritium (swelling, reddening and pain sensitivity of the coronary skin) on day 24 of life. Head skin lesions were mainly superficial scratches on day 2 of life. About 30% of the animals showed more severe head skin lesions (score 2 and 3) on day 11 of life. Skin lesions dorsal on carpal joints were as well most severe on day 11 of life with 7.6% of pigs showing open wounds. Most dorsal carpal skin wounds were superficial. Navel inflammations decreased during the suckling period (Table 5 ). Two pigs were diagnosed with umbilical hernia. A severe purulent inflammation of the navel was found on day 11 in three animals. The incidence of diarrhoea was very low during the whole suckling period and was not treated. During nursery a severe outbreak of disease caused by S . suis led to animal losses. During the second half of the nursery period a severe tail biting outbreak occurred. In total, due to animal losses and loss of ear tags, 42 animals were excluded from further evaluation. Among the remaining 89 pigs at the end of nursery tail injuries were found in more than half of the pigs. Due to ear tag losses only 76 pigs were identified and examined at the end of fattening. Acute tail wounds were diagnosed still in 5.3% of pigs at the end of fattening and 6.6% of the pigs had a total loss of tail. A summary of clinical findings is shown in Table 5 . There were no differences in frequencies of various clinical findings between different treatment groups. Table 5 Selected clinical findings in all examined pigs (%) in the respective stage of production Proportion of pigs with clinical findings (%) Day of life 2 ( n = 131) 11 ( n = 131) 24 ( n = 131) 74 ( n = 89) 160 ( n = 76) Pododermatitis haemorrhagica 64.9 66.4 3.1 - - Head skin lesions (score 2 and 3) 0.8 30.5 5.3 - - Dorsal carpal joint skin lesions (score 2 and 3) 0.0 36.6 58.0 - - Navel inflammation 27.5 24.4 14.5 - - Diarrhoea 3.8 1.5 0.8 - - Tail injuries - - - 55.1 5.3 Tail losses - - - - 14.5 Bloody ear injuries - - - 18.0 0.0 Selected clinical findings in % from all examined pigs (n) in the respective stage of production until end of fattening irrespective of treatment group Table 6 Body weight and average daily weight gain (ADWG) in different treatment groups (mean ± standard deviation) Group 1 Group 2 Group 3 Group 4 Day of life n Body weight (kg) n Body weight (kg) n Body weight (kg) n Body weight (kg) 2 33 1.71 ± 0.32 34 1.72 ± 0.3 31 1.71 ± 1.86 33 1.69 ± 0.34 24 33 6.85 ± 1.45 34 6.62 ± 1.57 31 6.61 ± 1.86 33 6.61 ± 1.81 74 23 31.17 ± 6.36 23 30.15 ± 5.57 20 29.78 ± 5.92 23 29.02 ± 7.52 160 17 116.82 ± 12.65 20 110.13 ± 20.07 19 109.29 ± 14.99 20 108.23 ± 22.65 Group 1 Group 2 Group 3 Group 4 n ADWG (kg) n ADWG (kg) n ADWG (kg) n ADWG (kg) Suckling period (day 2–24) 33 0.23 ± 0.06 34 0.22 ± 0.07 31 0.22 ± 0.07 33 0.22 ± 0.08 Nursery period (day 24-day 74) 23 0.50 ± 0.12 23 0.48 ± 0.10 20 0.47 ± 0.10 23 0.46 ± 0.13 Fattening period (day 74–160) 17 0.97 + 0.19 20 0.91 + 0.19 19 0.92 + 0.13 20 0.91 + 0.2 Suckling, nursery and fattening period (day 2-160) 17 0.73 ± 0.08 20 0.68 ± 0.19 19 0.68 ± 0.09 20 0.67 ± 0.14 On day 2 of life 33 pigs (25%) and at weaning only 2 pigs showed Hb values below 90 g/L. Both anaemic piglets at weaning suffered either from purulent navel inflammation or an umbilical hernia. Blood cell parameters are visualized in Figs. 1 , 2 , 3 , 4 and 5 and summarized in Table 7 . Manifold correlations between red blood cell parameters were found (data not shown). Hb at weaning showed a significant correlation with body weight at weaning ( P < 0.01) and the ADWG in the suckling period ( P = 0.002). The PCV at weaning was significantly correlated with weight at weaning, at end of nursery, at end of fattening and ADWG ( P < 0.01). MCHC at weaning was significantly negatively correlated with weight at weaning, at end of nursery, at end of fattening and ADWG ( P < 0.01). Table 7 Mean (±standard deviation) haematological measures in different groups on day 2 and 24 of life Day of life Group 1 ( n = 33) Group 2 ( n = 34) Group 3 ( n = 31) Group 4 ( n = 33) Haemoglobin (g/L) 2 95.4 (±11.1) 95.2 (±11.9) 98.4 (±8.5) 97.7 (±11.5) 24 120.6 (±8.5) 121.8 (±11.6) 123.4 (±13.3) 126.3 (±11.1) Packed cell volume (L/L) 2 0.28 (±0.03) 0.30 (±0.11) 0.29 (±0.03) 0.29 (±0.04) 24 0.38 (±0.03) 0.38 (±0.04) 0.38 (±0.04) 0.39 (±0.04) Mean corpuscular haemoglobin concentration (g/L) 2 335 (±11) 331 (±42) 339 (±15) 338 (±23) 24 321 (±11) 323 (±12) 326 (±11) 325 (±10) Red blood cell count (10 12 /L) 2 4.53 (±0.65) 4.42 (±0.52) 4.61 (±0.47) 4.55 (±0.661) 24 5.92 (±0.45) 5.78 (±0.51) 5.88 (±0.52) 5.93 (±0.44) Thrombocyte count (10 9 /L) 2 199 (±68) 221 (±61) 214 (±65) 206 (±50) 24 466 (±85) 442 (±72) 464 (±87) 434 (±107) Leucocyte count (10 9 /L) 2 8.05 (±1.77) 8.23 (±2.17) 8.09 (±1.83) 8.02 (±1.75) 24 13.93 (±4.47) 14.11 (±4.67) 13.88 (±3.55) 14.33 (±5.17) To underline the equivalence of treatments a non-inferiority test was conducted (TOST) as described. Based on statistical results shown in Table 8 the non-inferiority testing resulted in a P-value of 0.008, so that the first hypothesis, that iron dextran is non inferior to gleptoferron with respect to Hb at weaning was confirmed. Table 8 Descriptive outcome of non-inferiority testing (TOST) with regard to haemoglobin concentrations (g/L) at weaning Group N Mean (±standard deviation) Mean 95% confidence limits 1 33 120.6 (±8.5) 117.6 123.7 2 34 121.8 (±11.6) 117.8 125.8 Difference group 1–2 (Satterthwaite) -1.16 -6.12 3.79 In a second explorative analysis the four treatment groups were compared with respect to red blood cell variables and weight gain. The analysis of variance models with fixed effect group did not result in any statistical significant group effect on variables listed in Tables 6 and 7 . A group wise least square means comparison resulted in a significantly higher Hb concentration at weaning ( P = 0.04) in piglets of group 4 (twice administration of 200 mg iron dextran on day 2 and day 11) compared to piglets of group 1 (one administration of iron dextran on day 2) as shown in Fig. 1 . No significant group effect was found for PCV , erythrocytes , leucocytes or thrombocytes . Finally, data exploration targeted to reveal different treatment effects in different birth weight classes of piglets. The two-way analysis of variance for the dependent variable Hb at weaning with fixed effects treatment group and weight class revealed no statistically significant effects. Visualization in an interaction plot hints towards countervailing effects as of Hb was affected in different ways by different treatments within the different weight classes . While lowest and highest birth weight classes represent 25% of data each including extreme values, the median birth weight group represents the interquartile range and therefore 50% of the data without extreme values. In this subgroup a treatment response becomes visible with higher Hb concentrations in pigs treated on day 2 and day 11 of life with a full dose of iron dextran . In these medium weight piglets (interquartile weights at study start) significant differences in treatment effects on Hb at weaning were found between group 1 and 4 ( P = 0.01) as well as between group 2 and 4 ( P = 0.03). A significant difference in PCV at weaning was found between group 1 and 4 ( P = 0.02). These findings reflect an effect of the repeated iron treatment on day 11 . Fig. 7 (a) Haemoglobin concentration (g/L) and (b) Packed cell volume (L/L) at weaning in medium weight piglets Medium weight piglets (1.48–1.94 kg body weight at study start) of group 1 and 4 differed in Hb concentration and PCV. Different letters right to the boxes indicate significant differences between respective groups Iron (III)-hydroxide-dextran complex (Uniferon ® , Pharmacosmos A/S, Holbaek, Denmark) is an injectable 20% iron dextran for prevention and treatment of anaemia in swine. In spite of the standard dose of 200 mg iron administered routinely to piglets within the first days of life, IDA can develop due to the high growth rates of modern crossbred pigs and especially in heavy pigs . Next to anaemia, iron deficiency can negatively impact organ functions as especially the intestinal absorption and digestion capacities, due to e.g. destruction of tight junctions and decrease in villus height . The benefit of a second iron dose was therefore examined in several studies, mostly resulting in increased Hb concentrations at weaning, while contradictory results were found for an effect on ADWG . This might be due to known detrimental effects of routine iron bolus administrations, which can be of significance for piglet performance under certain factor constellations. Some authors therefore see no advantage in an additional iron dosage later in the suckling period . Several studies have focused on toxic effects of iron and the beneficial effects of split iron dosages to maintain the tightly balanced physiological iron metabolism . Hepcidin increased significantly after intramuscular administration of 150 mg Fe/kg BW on day three of life, which was not shown with lower doses of 35.5 mg Fe/kg BW given twice on day three and 14 of life . High hepcidin concentrations can block ferroportin with the consequence of lower iron utilization from external sources as creep feed or soil . In one study, the administration of less iron in the first days of life (40 mg) was adequate to improve iron status of piglets without severely increasing the hepcidin expression. A split iron supplementation in piglets was therefore recommended to minimize DNA damage and oxidative stress . These manifold different study results indicate that different protocols in iron administration might be suitable. Next to genetically defined growth intensity real iron requirements for piglets are dependent on several other farm-specific factors as e.g. blood loss during zootechnical measures, iron sources in the environment and health status of piglets. Therefor suitable protocols in iron administration might differ between farms, groups and individuals . The exact need for iron supplementation is considered to be farm specific due to various factors influencing iron availability and requirements, so that diagnosis of IDA should be based on laboratory diagnostical findings . Any parenteral iron supplementation should always target at a balance between the benefit of anaemia prevention and risk related to oxidative damage. These two sides of iron supplementation in piglets are still controversially discussed . In this study, flexible iron dosages and two iron sources (iron dextran and gleptoferron) were compared. Iron dextran was non-inferior to gleptoferron in a combinatory product with respect to Hb concentrations at weaning. Following the non-inferiority concept, if there are benefits in dosage flexibility and costs - while the efficacy is not worse (non-inferior) than gleptoferron in a combinatory product - iron dextran is a reasonable and effective treatment. In the past, comparative studies between both iron sources came to different results. In a study with piglets out of 26 litters no differences in red blood parameters or ADWG were found between pigs treated with iron dextran or gleptoferron . In two recent comparative studies with piglets from only four litters each, differences in various parameters were recorded, which were difficult to interpret due to a lack of sample size justification. In the first study, a comparison between the administration of 200 mg gleptoferron and 200 mg iron dextran in 25 piglets resulted in significant differences in mean Hb concentrations at weaning of 113 g/L in piglets treated with gleptoferron and 101 g/L in piglets treated with iron dextran . Comparable to our study on day 2 of life 28% of the piglets had Hb concentrations < 90 g/L. On day 18 Hb values were all above 90 g/L and higher than at weaning on day 31, which was 7 days later than in our study. In the second study with 32 piglets mean Hb concentrations of 114 g/L at weaning did not differ between piglets treated with 200 mg gleptoferron or iron dextran, while mean concentration-time profiles of iron in serum were significantly increased in the gleptoferron group . From serum iron levels no beneficial effect of iron administrations for the piglets can be deduced, because any bolus iron injection is reflected by increased tissue hepcidin expression as the systemic iron-regulatory hormone . In case that more iron is supplemented than can be bound by transferrin in serum, free toxic iron can catalyze reactive oxygen species . The intracellular iron storage protein ferritin in serum was also compared between the treatment groups but can also be interpreted as an inflammation marker due to its function as an acute phase protein . For this reason, in our study Hb was defined as the target variable. Study design and number of pigs examined in our study differed from those in the cited comparative studies. No differences in ADWG were observed in the cited comparative studies, which is in accordance with our study. A higher dosage of iron on day 2 of life or a second full dosage of iron on day 11 of life had unexpectedly minor effects in our study. A second iron dose led to higher Hb concentrations but not ADWG at weaning. This outcome was similar to other studies, as e.g. in a comparison of gleptoferron injections at different points of time and oral iron supplementation, which led to different Hb concentrations but not ADWG between pigs with one or two iron injections . A second iron dosage of 100 mg on day 11 of life did neither improve red blood cell parameters nor ADWG at weaning on day 21 in a study testing different iron dosages . In contrast to that, in a recent study in organic pig production piglets profited clearly from a second dose of iron on day 14 of life due to the longer suckling period and subsequently later iron intake from feed . If ADWG can be increased by additional administration of iron is still a matter of debate and might depend on several factors, as availability of iron from the sows, blood loss during zootechnical measures, start and iron content of creep feed. Different studies come to different results as reviewed previously . A dose-dependent effect of iron dosage on ADWG in the suckling period was found up to a dosage of 100 mg iron on day 3 after birth and for up to 200 mg in the nursery period . In most studies, no effect of an additional iron administration next to a primary dose of 200 mg on ADWG in the suckling period was found [ 43 – 45 ]. In some studies, the effect of a second iron dosage in the suckling period was only significant on ADWG in the post-weaning period . This is not in accordance with findings in our study. In the post-weaning period several pigs suffered from S.-suis -related disease and in a later time period pig health was impacted by a tail-biting outbreak. Both diseases lead to inflammatory responses with influence on growth rates . Piglets with different birth weights and treated with the same dose of iron responded differently to iron provided in the nursery diet and the effect of a second iron injection was also influenced by red blood cell parameters at the time point of second iron injection . It can be hypothesized, that the significant effect of the second iron administration in medium weight piglets in our study might be due to a different Hb concentration at the respective time point in this group. Although pigs of different weight classes did not differ in their red blood cell parameters on their second day of life, differences can be expected on day 11 of life. Iron supply by only one injection in low weight piglets with lower ADWG might had been adequate, while high weight piglets might have profited from a second dose of iron resulting in higher Hb concentrations in the post-weaning period not examined in this study. For the medium weight piglets the beneficial effect of a second iron dose on Hb concentration was already detectable at weaning. In general, a positive association between ADWG in the post-weaning period and the Hb concentration at weaning exists . Also in our study several red blood cell parameters were positively correlated with the ADWG. In a recent study, a second iron injection resulted in a 4% increase in ADWG from weaning to slaughter . In that study, piglets had no access to creep feed during the suckling period and diets contained less iron (100 mg/kg) than diets in our study (Table 1 ). Mean Hb concentrations at weaning in pigs with only one iron injection (107 g/L) were lower than in our study (group 1 and 2: 121 ± 10 g/L), where piglets had access to external iron sources in milk supplement and creep feed. In our study, iron content in creep feed was below 240 mg/kg as a published recommendation , but was within the range of iron concentrations in published creep feed studies . Creep feed containing 175 mg iron/kg and fed from day 7 after birth was found to be adequate for good growth rates irrespective of parenteral iron administration . Voluntary iron intake by creep feed with appropriate iron content is considered an important iron source during the suckling period allowing iron absorption precisely regulated according to the needs of the piglets . On the other hand, the regulation of intestinal iron absorption is not fully functional before 5–6 weeks of age . Physiologically restricted iron absorption in the gut in young piglets is a protective mechanism to avoid injury of the intestinal barrier and negative influence on the gut microbiota favouring growth of pathogenic bacteria . Any disturbance of gut development and gut flora, which can be due to a lack of colostrum especially in large litters, can have severe consequences for piglets’ health, especially in presence of pathogenic microorganisms. As pathogens require iron for their metabolism, any iron supplementation can interfere with the iron regulation of the host based on internalization of iron in intracellular compartments during inflammation . In our study group the average numbers of live born and weaned piglets were in the upper range of piglet producing farms with high herd productivity in Europe . Large litters need intensive care in the suckling period, usually including cross-fostering, which was not performed in this study . The high proportion of pigs with head and carpal joint lesions in our study indicated increased teat fighting although milk supplement was provided. This finally did not negatively impact the suckling piglet mortality in the litters of the study group, so that management of these large litters can be assessed as successful in this farm. Nevertheless, the ADWG of 226 ± 69 g in the study pigs during the suckling period was relatively low . Next to birth weight as the most important factor various factors related to sow, pen, litter and piglet have impact on ADWG in suckling piglets . It was reported that foreleg lesions in piglets can affect weight gain in piglets. In our study carpal joint lesions were 37% in the second week of life, which is due to intense teat stimulation in the large litters with high competition. It can be assumed, that more cross fostering resulting in no more than one piglet per functional teat in a litter would have led to less carpal joint lesions and a higher ADWG. In three herds with ADWG in piglets ranging from 204 to 210 g sow-related factors had a significant impact on ADWG, as e.g. poorly milking sows three weeks after farrowing . These sow-related factors were not analysed in our study in detail, but all sows were always in a good health status. S. suis -related disease was responsible for 7.5% piglet mortality in the nursery period, so that an inadequate colostrum supply due to the large litter sizes might have been a potential cofactor for the detrimental course of disease. Skin lesions at the head and carpal joints in weaned piglets were additional risk factors for entry of S. suis and subsequent bacteriaemia. A post-weaning ADWG of 475 ± 132 g in surviving pigs was within a physiological range . To find a compromise between benefits and drawbacks of additional iron supplementation, new and flexible iron supply strategies are necessary taking different pig breeds, husbandry and management conditions into account. The advantage of combinatory products addressing anaemia and coccidiosis by one single shot can be counteracted by the lack of adjustment of the necessary amount of iron to prevent anaemia as long as possible. Additional scientific data are necessary to reassess iron supplementation strategies in pigs and other livestock animals with respect to the double-edged character of this element. In large litters of hyperprolific sows, a high heterogeneity of piglet birth weights is accompanied by a high growth potential resulting in varying demands for iron between individuals. While some individuals might benefit from 200 mg Fe, heavier piglets might be undersupplied, and smaller piglets might be burdened by this amount of iron due to its toxic effects. In our study iron dextran as a traditional substance was non-inferior to gleptoferron in a combinatory product. Iron dextran can be used in flexible dosage and treatment schemes according to varying requirements on different farms as an alternative product in case that no treatment against coccidiosis is necessary. In the study farm, Hb was increased at weaning by a second full dose of iron on day 11 of life, but without improvement of ADWG. Differences in published study outcomes about the effects of additional iron dosages might in part depend on piglet`s access to creep feed containing iron already in the suckling period superimposing the effect of additional parenteral iron administration. The consequences of metabolic changes by iron administration balancing its benefits should be addressed in further clinical studies comparing different protocols of iron administration. | Study | biomedical | en | 0.999999 |
PMC11699721 | Prescribing beta-blocker treatments during pregnancy raises concerns due to their potential effects on both the mother and the fetus. Indeed, beta-blockers can interfere with the physiological adjustments necessary for pregnancy: increased cardiac output, heart rate, and blood volume, and decreased peripheral vascular resistance. 5 Previous studies have suggested several potential adverse effects associated with the use of beta-blockers during pregnancy. These adverse effects include increased risks of intrauterine growth restriction, 6 neonatal hypoglycemia, 7 neonatal bradycardia, 7 hypotension, 8 and prolonged neonatal hospitalization. 9 Due to their inhibitory action on beta-adrenergic receptors, beta-blocker treatment can lead to a decrease in maternal heart rate. This hemodynamic change in the mother raises questions about the treatment's impact on fetal heart rate. 10 An in vivo study in mice showed that administration of propranolol resulted in a decrease in fetal heart rate associated with an increase in heart rate variability, whereas saline had no effect on either mothers or fetuses. 11 These results, which seem contradictory at first glance, testify to the lack of knowledge about beta-blocker pharmacology, particularly during pregnancy. Any potential alteration in fetal heart rate in humans could have significant implications for obstetric monitoring and particularly for the interpretation of fetal heart rate monitoring. Indeed, a significant variation in fetal heart rate can be an indicator of fetal distress, requiring rapid medical intervention to prevent serious complications such as fetal asphyxia and hypoxia. 12 Due to the underlying maternal conditions for which beta-blockers are prescribed, there is an increased risk of fetal distress. In this context, accurate interpretation of fetal heart rate monitoring becomes even more crucial. This study aims to determine whether the use of beta-blockers during pregnancy alters the mean fetal heart rate among patients treated with beta-blockers, compared to fetuses whose mothers receive no treatment. For this study, we conducted a retrospective case-control study involving 90 patients, divided into two groups of 45 patients each. The first group consisted of pregnant patients treated with beta-blockers, while the second group was comprised of pregnant women receiving no treatment. Patients on Labetalol only and those treated with Propranolol for Graves’ disease were excluded from the analysis, as the mechanism of action of Labetalol (a noncardio-selective alpha/beta-blocker) and the impact of Graves’ disease on maternal and fetal heart rates 13 could introduce biases into our results. The demographic characteristics (age, parity, and body mass index) of the patients are presented in Table 1 . Information regarding the type of beta-blocker treatment, its indication, and dosage were extracted from medical records and presented in Table 2 . In case of changes in dosage during pregnancy, the highest dosage was retained. Table 1 Main characteristics of untreated patients and patients with beta-blockers Table 1 Untreated patients n =45 (%) Patients with beta-blockers n =45 (%) P Age, median (IQR) 31 (27–34) 31 (27–34) 1 Parity I parous 25 (56) 25 (56) 1 II parous 18 (40) 18 (40) III parous 2 (4.4) 2 (4.4) BMI (kg/m 2 ) <18.5 2 (4.4) 9 (20) 0.11 18.5–24.9 23 (51) 24 (53) 25–29.9 11 (24) 5 (11) 30–34.9 5 (11) 2 (4.4) 35–39.9 0 (0) 1 (2.2) >40 2 (4.4) 1 (2.2) Hautier. Effect of maternal beta-blocker treatment. AJOG Glob Rep 2024. Table 2 Details of treatment received by patients Table 2 Treatment (INN) n (%) Daily dose mg Indication Atenolol 14 (31) 50–100 mg Marfan syndrome 14 Bisoprolol 21 (46) 2.5–10 Marfan syndrome 18 Others 3 Labetalol 1 (2) 200 Marfan syndrome Nebivolol 1 (2) 10 Marfan syndrome Propranolol 8 (17) 40–120 mg Marfan syndrome 2 Others 6 Hautier. Effect of maternal beta-blocker treatment. AJOG Glob Rep 2024. The evaluation of mean fetal heart rate was performed by reading the fetal heart rate monitoring and manually assessing the baseline heart rate on the day of delivery, during labor, or before scheduled C-section, depending on the obstetric context. Situations of suspected intrauterine infection or maternal fever were excluded due to their potential influence on fetal heart rate. It was classified into three categories: less than 110 bpm, between 110 and 150 bpm, and greater than 150 bpm, following the usual definition of normal fetal heart rate. 14 Maternal heart rate on the day of delivery was also recorded from the fetal monitoring recordings or surveillance sheets in the delivery room or operating room. This rate was categorized into three categories for statistical comparison: less than 60 bpm, between 60 and 100 bpm, and greater than 100 bpm. A descriptive analysis was conducted to describe the characteristics of the study population. Quantitative variables are expressed as mean with confidence interval or median with interquartile range, while qualitative variables are expressed as number and percentage. Comparison of qualitative variables was performed using a Chi-squared test or Fisher's exact test when assumptions were not met, and means were compared using Student's t test. An alpha risk of 5% and a significance level of 0.05 were considered in all analyses. Data were processed using R software via the P value interface. 16 For the treatment received by patients on beta-blockers, as depicted in Table 2 , a large majority were treated for Marfan syndrome, accounting for 80% of the cohort ( n =36/45). Other indications for treatment included sinus tachycardia ( n =2), migraine pathology ( n =2), and cardiomyopathy ( n =2). One patient was treated due to dilated cardiomegaly, another for rheumatic mitral insufficiency, and the last for Bouveret-Hoffmann syndrome. Bisoprolol was the most frequently prescribed treatment ( n =21, 46%), at doses ranging from 2.5 to 10 mg per day, followed by atenolol, mainly for Marfan syndrome. Notably, the majority of patients, 37/45 (82%), were already on beta-blocker treatment before pregnancy. Two patients (4.4%) started beta-blockers during the first trimester of pregnancy, two (4.4%) during the second trimester, and 4 (8.8%) during the third trimester. One patient received bisoprolol followed by labetalol due to poor tolerance. The main analysis of the study, presented in Table 3 , revealed no significant difference in the distribution of mean fetal heart rate between the two groups across the three categories mentioned earlier. Most fetuses, regardless of maternal beta-blocker use, had heart rates between 110 and 150 beats per minute, with similar percentages in both groups (87% vs 93%, respectively), P =.71. These findings suggest that beta-blocker use does not significantly impact the average fetal heart rate. Table 3 Maternal and mean fetal heart rate the day of delivery among untreated patients and patients with beta-blockers Table 3 Colonne1 Untreated patients n =45 (%) Patients with beta-blockers n =45 (%) P Maternal heart rate <60/min 1 (2.2) 7 (18) <.01 60–100/min 37 (82) 30 (79) >100/min 7 (16) 1 (2.6) Mean fetal heart rate <110/min 2 (4.4) 0 (0) .71 110–150/min 39 (87) 27 (93) >150/min 4 (8.9) 2 (6.9) Hautier. Effect of maternal beta-blocker treatment. AJOG Glob Rep 2024. However, a significant difference was observed in maternal heart rates between the two groups. There was a notably higher prevalence of maternal heart rates above 100 beats per minute in untreated patients compared to those on beta-blockers (16% vs 2.6%, respectively), P <.01. This result aligns with the known bradycardic effect of beta-blockers. When the analysis focused on the subgroup of patients treated with bisoprolol, fetal heart rates were not significantly different between the groups. Specifically, 100% of fetuses from patients on bisoprolol had heart rates between 110 and 150 bpm, compared to 93% of fetuses from untreated patients ( P =1). The secondary analysis on obstetric and neonatal outcomes revealed several significant differences between the groups, as shown in Table 4 . Significant differences were noted in the onset of labor and mode of delivery. Specifically, cesarean sections were more frequent in patients on beta-blockers (53%) compared to untreated patients (8.9%), P <.01. This difference predominantly concerns cesarean deliveries before labor, linked to the underlying chronic pathology of these patients, leading to specific labor management and a risk of cesarean section due to cardiac decompensation. Table 4 Secondary Analysis. Obstetric and neonatal outcome among untreated patients and patients with beta-blockers Table 4 Colonne1 Untreated patients n =45 (%) Patients with beta-blockers n =45 (%) P Gestational age SA, median (IQR) 38 (36.6–38.4) 38 (36.6–38.4) 1 Onset of labor Spontaneous 27 (60) 7 (16) <.01 Induction 14 (31) 14 (31) C-section 4 (8.9) 24 (53) Mode of delivery Spontaneous vaginal 28 (62) 13 (29) <.01 Instrumental vaginal 6 (13) 5 (11) C-section 11 (24) 27 (60) Birth weight g, median (IQR) 2890 2625 <.01 Birth percentile <10th 6 (13) 16 (36) .014 Hautier. Effect of maternal beta-blocker treatment. AJOG Glob Rep 2024. Regarding birth weight, the results suggest that newborns from mothers on beta-blockers tend to have a lower birth weight than those from untreated mothers, P <.01. These findings suggest a potential association between beta-blocker use during pregnancy and an increased risk of neonatal hypotrophy. Our study revealed a differential effect of beta-blockers on maternal and fetal heart rates in patients treated with beta-blockers during pregnancy compared to untreated pregnant women. Our findings complement existing research on labetalol (alpha-beta-blocker treatment), which, due to its use in hypertensive disorders of pregnancy, has a richer literature. For instance, a systematic review published in 2004 gathered studies on the impact of major antihypertensive drugs on fetal heart rate during pregnancy. 18 The review included studies on labetalol, methyldopa, nifedipine, and hydralazine primarily. The authors did not find adverse effects of these treatments on fetal heart rate. In the neonatal period, the effects of Labetalol have been described, by Thewissen et al 19 who found no differences in newborns of mothers treated with Labetalol compared to the control group concerning heart rate, blood pressure, and the need for vasopressor support. Regarding the molecules studied in our research, Montan et al's 20 1984 trial compared the average fetal heart rate in 40 full-term pregnant women before and after initiating atenolol treatment for hypertension. They found a statistically significant decrease in the average fetal heart rate: 143 vs 133 ( P <.001) after atenolol initiation. However, the clinical impact of such a decrease appears minimal, as the fetal heart rate remains within normal limits, not raising concerns during monitoring. Montan et al also observed a decrease in variability of 5 to 10 bpm for at least 20 minutes in 13.1% of fetuses after atenolol introduction compared to 2.3% before treatment ( P <.01). This parameter could falsely alert during monitoring, especially among fetuses suspected of being hypotrophic in patients treated with beta-blockers. Our study did not investigate this parameter, limiting the applicability of our findings. A more comprehensive or automated approach to interpreting fetal heart rate in patients on beta-blockers could offer more precise information for better monitoring interpretation in these high-risk obstetric patients. Among patients with Marfan syndrome, two were treated with nebivolol. There is no data on the specific effect of this molecule on fetal hemodynamic parameters. A study in rats published in 2016 by Altoama et al 21 compared the effects of bisoprolol and nebivolol on fetuses and found a decrease in average fetal weight gain when the mother was treated with nebivolol ( P <.01). The effects on heart rate were not studied. More broadly, patients on beta-blocker treatment during pregnancy require close monitoring and appropriate clinical management to minimize risks to both the mother and the fetus. Further studies are needed to validate our findings and understand the underlying pathophysiological mechanisms. From a pathophysiological perspective, it is established that so-called “cardio-selective” beta-blockers inhibiting beta-1 receptors like bisoprolol and atenolol cross the placental barrier. 22 These molecules could, therefore, have a potential negative chronotropic or inotropic effect on the fetus. One hypothesis we can propose to explain the absence of effects of these molecules is the immaturity of the fetus's beta-adrenergic receptors. Further research is needed to validate this hypothesis. Contradictory results exist in animals, notably in the 2022 article by Khandoker et al. 11 In this work, conducted in mice, subcutaneous injection of propranolol in pregnant females led to a decrease in fetal heart rate compared to saline injection, with no effect on maternal heart rate. Given that the transplacental passage of beta-blockers in mice has not been demonstrated, 23 the extrapolation of animal models to human pregnancy remains limited. It is important to note that this study has certain inherent limitations due to its design. It is a retrospective, bicentric case-control study, which potentially limits the external validity of our results. Furthermore, the retrospective nature of the study could introduce selection and data collection biases. However, thanks to the computerized collection of our data, the comprehensive nature of the data is ensured. Despite these limitations, this study has several strengths worth noting. The specificity of recruitment of maternal pathologies from the two maternity involved in this study provides genuine originality to our results. The matching between groups on important variables such as age, parity, and gestational age strengthens the robustness of the obtained results. The observed difference in heart rate in mothers on beta-blockers compared to the control group supports good treatment adherence by patients, reinforcing our findings. Similarly, the significant increase in neonatal hypotrophy in treated patients, a known effect of beta-blockers, 6 aligns with fetal exposure to these treatments. Finally, excluding patients with pathologies that could interfere with fetal heart rate constitutes a rigorous methodological approach, allowing for better isolation of the beta-blockers’ effect on fetal heart rate. Our study did not find a significant effect of beta-blockers on fetal heart rate in mothers treated during pregnancy compared to the control group. These women require specialized care management, and the interpretation of fetal monitoring should not be biased by maternal treatment. Further research is needed to understand the underlying physiological mechanisms. | Review | biomedical | en | 0.999997 |
PMC11699727 | Aging is an independent risk factor for cardiovascular diseases, especially for heart failure (HF). Cardiac diastolic dysfunction (CDD) underlies HF with preserved ejection fraction (HFpEF) and is generally considered an aging-related condition. CDD is characterized by impaired relaxation with normal systolic function, however, it impacts approximately 3 million people in the United States and up to 32 million people worldwide. 1 , 2 As reported, CDD has been one of the leading causes of cardiovascular mortality in the elderly. 3 , 4 Patients with CDD have a very poor prognosis with more than 50% deaths 5 years after diagnosis. 5 Nevertheless, effective treatments to improve clinical outcomes and long-term survival in elderly patients with CDD are scarce. The underlying mechanisms are still being explored. 6 , 7 , 8 Cardiac troponin I (cTnI) plays a key role in regulating diastolic function. Changes including deficiency and mutations in cTnI are associated with CDD. 9 , 10 In elderly human hearts, the cytoplasmic concentration of cTnI is decreased in the left ventricular (LV) myocardium. 11 Aging-related CDD is also associated with decreased cTnI expression caused by abnormal histone acetylation modification. 10 Epigallocatechin gallate (EGCG), the main green tea catechin extracted from Camellia sinensis , has emerged as a mediator of cardiovascular health. 12 Cardioprotective effects of EGCG have been documented in several clinical studies. 13 , 14 In our recent clinical trial, cardiac functions and HF symptoms were significantly improved in cardiomyopathy patients who received EGCG capsules daily for 12 months. 15 Green tea intake was also related to enhanced blood vessel function and lower heart disease risk. 12 Several studies have shown that EGCG, as a histone acetylation modification regulator, could alter gene expression via inhibiting histone deacetylase 1 (HDAC1) and regulate organ functions. 16 , 17 So far, little attention has been paid to whether specific long-term intervention with EGCG could prevent the onset and progression of CDD and cTnI expression decline. In this study, we investigated the effects of three doses of EGCG on CDD and the underlying mechanisms. Female C57BL/6 mice were purchased from the Animal Center of Chongqing Medical University. All studies were carried out in strict accordance with the recommendations approved by Chongqing Medical University (Chongqing, China). All mice were housed in five per cage in the controlled environment with a 12 h strict light/dark cycle at 23 ± 1 °C and fed a standard diet from 12 to 18 months of age for 6-month administrations. Mice had free access to drinking water and food. As summarized in Figure 1 A, forty 12-month-old mice were randomized into four groups: aged, 50 mg EGCG (50 mg/kg/day), 100 mg EGCG (100 mg/kg/day), and 200 mg EGCG groups (200 mg/kg/day), containing 10 animals each. EGCG-treated mice were orally administered EGCG (purity: 99%, Selleck, USA) mixed with drinking water. Moreover, the aged mice were orally administered ordinary drinking water without an intervention agent. Mice aged 3 months were assigned as young control group ( n = 10). Drinking water containing EGCG was freshly prepared at about 5:00 p.m. every day at room temperature for EGCG-treated groups, and water left in the bottle was measured to determine the amounts consumed and then replaced. All mice were subjected to transthoracic echocardiography for assessment of cardiac functions at the age of 12 and 18 months, as well as a treadmill test. Heart tissues were harvested for histological analysis and determination of cardiac proteins, histone acetylation modification, and myocardial apoptosis and fibrosis. Figure 1 Long-term administration with EGCG prevents diastolic dysfunction later in life in the aged mice. (A) Diagram of the study design: mice were subjected to transthoracic echocardiography for assessment of cardiac functions, to treadmill test for evaluation of exercise ability, and to mechanistic studies. (B, C) Changes in transcription and protein expression of cardiac troponin I (cTnI) at different ages. (D) Representative M-mode echocardiographic images acquired from LV short-axis views. (E) Representative Doppler echocardiographic tracings. (F, G) Quantitative measurements of cardiac functions including ejection fraction (EF), fractional shortening (FS), the ratio of early ventricular filling to late ventricular filling caused by atrial contraction (E/A), and isovolumetric relaxation time (IVRT) ( n = 8–10). All results are represented as mean ± standard deviation. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. Figure 1 Mice were subjected to conventional transthoracic echocardiography and Doppler analyses using GE Vivid E9 color Doppler ultrasound system (USA) equipped with a high-frequency probe (11 MHz) before (12 months old) and after (18 months old) interventions. Experimental animals were anesthetized with 2% isoflurane for 3–5 min and then maintained with 1%–1.5% during data collection. Ventricular dimension, septal and ventricular wall thickness at end-diastole and end-systole were measured to calculate systolic function using standard M-mode images under a short axis view. The mitral blood inflow and diastolic function were evaluated using the pulse-wave Doppler. Additionally, the following parameters were measured as well: early ventricular filling (E) to late (A) ventricular filling caused by atrial contraction (E/A), isovolumetric relaxation time (IVRT), and isovolumetric contraction time (IVCT). All data were analyzed using in vivo echocardiography report software. Both the echocardiography operator and data analyzer were blinded to the groups. A treadmill test with a six-lane was used to assess exercise capacity, as previously described. 18 After one-week training, the exhaustion test was performed, which comprised 3 exercise tests on the treadmill for consecutive days. All mice received a 1.2-mA shock if they stopped running and slid onto an electric grid placed at the rear end of the treadmill. An exercise test was started at a speed of 10 m/min on a 5° incline. The speed was increased by 1 m/min every 3 min until the mice fatigued, that is, the animals could no longer keep pace with the belt, and at this point, the running distance was recorded. Mice at 3, 10, 12, 14, 16, and 18 months of age were anesthetized with carbon dioxide and sacrificed. Heart tissues were washed with phosphate-buffered saline and stored at −80 °C. After administrations, heart tissues from young (3 months old), aged (18 months old), and EGCG groups were harvested. LV samples were prepared for the transmission electron microscope (TEM), cut into small pieces about 1 mm 3 , fixed immediately with 3% glutaraldehyde, and sent to the TEM center of Chongqing Medical University. All samples were sliced by ultramicrotome (Leica, German) and observed using a Transmission Electron Microscope (Hitachi, Japan). Formalin-fixed and paraffin-embedded tissues were cut into sections and stained with hematoxylin-eosin (HE). Cardiac fibrosis was observed using Masson's trichrome and collagen volume fraction (CVF) was calculated by NIH ImageJ Software as the ratio of fibrosis to total tissue area. TUNEL assay was carried out according to the manufacturer's instructions (Roche, Germany) for observing cardiomyocyte apoptosis. The nuclei were marked in blue, while TUNEL-positive cells were stained in green. The fluorescence intensity of apoptotic cardiomyocytes was documented by a fluorescence microscope (Nikon, Japan). The apoptotic index was estimated as the ratio of the number of apoptotic cells to the total number counted. Primary cardiomyocyte culture from neonatal C57BL/6 mice was performed as previously described. 19 The collected cells were divided into Blank, DMSO, NC, HDAC1 overexpression, and HDAC1 overexpression + EGCG groups. Cardiomyocytes were transfected with empty vector or overexpression HDAC1 adenovirus. In HDAC1 overexpression + EGCG group, cardiomyocytes were treated with EGCG (40 μM) right after transfection with HDAC1 overexpression adenovirus. Cells were collected 24 h after administrations for subsequent analysis. Total RNA was isolated from heart tissues using an RNA Extraction kit (Bioteck, China). Single-strand cDNA synthesis was performed using oligo dT-Adaptor primers and an AMV reverse transcriptase kit (TaKaRa, Japan). cDNA amplification was performed according to a quantitative real-time PCR assay employing a SYBR Green RealMasterMix kit (Tiangen, China). β-actin was used as a housekeeping gene to normalize transcription levels of the genes of interest. Genes of interest, including cTnI, A/B-type natriuretic peptide (ANP and BNP), HDAC1/2/3, sarcoplasmic reticulum Ca 2+ -ATPase (SERCA2a), sarcalumenin (SAR), and control were detected with the corresponding gene-specific pairs of primers, as detailed in Table S1 . Western blotting was performed in accordance with previous protocol. 20 The homogenized samples were obtained using ice-cold RIPA buffer (Beyotime, China) containing protease and phosphatase inhibitors, and the protein concentration was determined using the Bradford Assay (Thermo Fisher, USA). Proteins of interest and control transferred to the PDVF membrane were analyzed using primary antibodies against cTnI (Abcam, USA), HDAC1 (CST, USA), or GAPDH (Arigo, China). The membranes with proteins were incubated with corresponding HRP-labeled secondary antibodies (Beyotime, China). Immunoblots were visualized by enhanced chemiluminescence luminal reagent (Mishushengwu, China) through Imaging System (Bio-Rad, USA) and quantitative analyses were acquired using Quantity One software (Bio-Rad, USA). Chromatin was collected using the Chromatin Extraction Kit (Abcam, England) and immunoprecipitated in accordance with the instructions of the chromatin immunoprecipitation (ChIP) one-step kit (Abcam, England). The chromatin was fragmented into 200–1000 bp DNA fragments by ultrasonication. Protein-DNA complexes were incubated using antibodies against acetylated histone 3 (AcH3, Abcam, England), acetylated lysine 9 on histone H3 (AcH3K9, Abcam, England), acetylated lysine 27 on histone H3 (AcH3K27, Abcam, England), HDAC1 (Cell Signaling Technology, USA), histone deacetylase 2 (HDAC2, Cell Signaling Technology, USA), histone deacetylase 3 (HDAC3, Cell Signaling Technology, USA), GATA4 (Abcam, England) or MEF2c (Abcam, England), alongside non-immune IgG (as a negative control) and RNA polymerase II (as a positive control). The primer sequences targeting the −1000 bp upstream regions of the cTnI promoter were listed in Table S2 . HDAC1 activity was detected using an HDAC1 Activity Assay Kit (Biovision, USA). Proteins were extracted as per the manufacturer's instructions and the protein concentrations were tested using the Bradford Assay (Thermo Fisher, USA). A total of 50 μg protein for each immunoprecipitation was added with 6 μL HDAC1 antibody in sample groups or 6 μL IgG in control groups, followed by incubation at 4 °C overnight. After incubation, the protein-A/G bead slurry of 25 μL was added and incubated at 4 °C for 1 h. The beads were collected by centrifugation and wash. For each reaction, sample and control groups were mixed and incubated with Reaction Mix containing HDAC Assay Buffer and HDAC Substrate at 37 °C for 2 h. The Positive Control, Developer, and Standard Curve were prepared according to the manufacturer's instructions. Reaction supernatant of 100 μL was transferred to individual wells and the fluorescence value was read at Ex/Em = 380/500 nm. Then the AFC standard curve was gained. Sample HDAC Activity = (2 × B)/(T × S) = pmol/min/mg = mU. B was AFC amount from the Standard Curve (pmol). Sample dilution factor was 2. T represented reaction time (120 min) and S represented sample amount (50 × 10 −3 mg). All data were presented and analyzed using SPSS 23.0 software (USA) and GraphPad Prism 8.0 (USA). The results were shown as mean ± standard deviation (SD) and analyzed by one-way ANOVA followed by Tukey's or Bonferoni's or Sidak's multiple comparison post hoc. At least three independent triplicated experiments were performed for each experimental set-up. P values less than 0.05 were considered statistically significant, indicated as ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. To identify the key temporal point of cTnI expression decline during cardiac aging, the transcript and protein levels were measured in mice at 3, 10, 12, 14, 16, and 18 months of age. As shown in Figure 1 B and C, cTnI mRNA expression declined markedly at 14 months of age (14 m, 16 m, or 18 m vs . 12 m, P < 0.01, respectively), and its protein expression was decreased significantly at 16 months of age (16 m or 18 m vs . 12 m, P < 0.05, respectively). This study was aimed to investigate the preventive effects of EGCG on cardiac aging. Therefore, adult mice at 12 months of age were given EGCG for 6 months. As previously described, cTnI expression had not yet decreased at 12 months. Echocardiographic data showed that diastolic function was preserved at the age of 12 months ( Table S3 ), along with equivalent IVRT and E/A ratio to the young mice, which also suggested EGCG interventions should be initiated at this age. We then assessed heart functions in the young and aged mice treated with or without EGCG ( Table S3 ; Table 1 ). In the present study, the LV systolic function, as shown by pEF and fractional shortening, remained unchanged in the aged mice (18 months old), the same as in the young mice . However, a deteriorative and noticeable reduction of LV diastolic function was observed in the aged mice, as reflected by a significant increase of the E/A ratio ( P < 0.001) and a prolongation of IVRT ( P < 0.05) compared to the young mice . Fortunately, long-term administration of 100 and 200 mg EGCG effectively prevented CDD: the E/A ratio was 0.74-fold and 0.48-fold lower, and IVRT was 0.79-fold and 0.65-fold lower in 100 and 200 mg EGCG-treated mice, respectively, as compared with the aged mice, but no significant improvement of both was observed after 50 mg EGCG administration . Table 1 Cardiac functions after 6-month EGCG administration (18 months). Table 1 Parameters Aged Young Aged+50 mg EGCG Aged+100 mg EGCG Aged+200 mg EGCG IVSd (cm) 0.089 ± 0.020 0.086 ± 0.017 0.091 ± 0.010 0.089 ± 0.014 0.085 ± 0.008 LVIDd (cm) 0.305 ± 0.062 0.223 ± 0.046 0.271 ± 0.021 0.270 ± 0.030 0.269 ± 0.029 LVPWd (cm) 0.072 ± 0.025 0.075 ± 0.010 0.081 ± 0.010 0.080 ± 0.018 0.068 ± 0.005 LVIDs (cm) 0.153 ± 0.066 0.100 ± 0.025 0.119 ± 0.014 0.104 ± 0.023 0.116 ± 0.034 EDV (ml) 0.082 ± 0.050 0.030 ± 0.020∗∗ 0.054 ± 0.013 0.055 ± 0.017 0.051 ± 0.014∗ ESV (ml) 0.012 ± 0.016 0.002 ± 0.004 0.004 ± 0.005 0.004 ± 0.005 0.003 ± 0.005 EF (%) 86.52 ± 8.97 90.09 ± 3.90 90.01 ± 4.69 92.06 ± 2.21 90.11 ± 7.62 FS (%) 51.76 ± 11.39 55.03 ± 5.75 55.59 ± 7.94 58.21 ± 4.28 57.09 ± 11.19 SV (ml) 0.066 ± 0.031 0.030 ± 0.020∗∗ 0.049 ± 0.012 0.046 ± 0.015 0.047 ± 0.015 E/A 2.17 ± 0.24 1.19 ± 0.53∗∗∗ 1.89 ± 0.24 1.61 ± 0.42∗ 1.05 ± 0.39∗∗∗ IVRT (ms) 16.38 ± 3.68 11.07 ± 3.79∗ 13.02 ± 5.36 10.24 ± 2.72∗∗ 10.64 ± 3.25∗∗ A, late ventricular filling caused by atrial contraction; EDV, end-diastolic volume; EF, ejection fraction; ESV, end-systolic volume; FS, fractional shortening; IVSd, intraventricular septum at end-diastole; LVIDd, left ventricular internal dimensions at end-diastole; LVIDs, left ventricular internal dimensions at end-systole; LVPWd, left ventricular posterior wall thickness at end-diastole; All values are presented as mean ± SD. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001; another four groups compared to 3 m, respectively. Furthermore, ANP and BNP in LV myocardium, classical molecular markers of HF, were significantly enhanced in the aged mice, along with a marked reduction of heart aging markers SERCA2a and SAR compared to the young subjects . Long-term EGCG intakes at all doses effectively improved cardiac aging phenotype as shown by a conspicuous reduction of HF markers and relieved the aging-related decreases of SERCA2a and SAR in the aged heart, compared with the aged mice . Figure 2 Long-term EGCG supplementation alleviates age-associated heart failure. (A, B) Gene expression of A-type natriuretic protein (ANP) and B-type natriuretic peptide (BNP). (C, D) Gene expression of sarcoplasmic reticulum Ca 2+ -ATPase (SERCA2a) and sarcalumenin (SAR). (E) Treadmill test for assessment of exercise capacity ( n = 8–10). All results are represented as mean ± standard deviation. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. Figure 2 A running exhaustion test was conducted to evaluate exercise capacity by treadmill test . Our findings showed that the average running fatigue distance of the aged mice was 54.52% lower than that of the young mice ( P < 0.001). Concurrently, the aged mice supplemented with 50, 100, and 200 mg EGCG, respectively, exhibited increases in running fatigue distance by 37.01%, 31.36%, and 58.45% (each of EGCG groups vs . the aged group, P < 0.001), indicating EGCG intakes augmented exercise performance in the aged mice, with the most pronounced therapeutic effect observed in those treated with 200 mg EGCG. As the heart aging, cardiac structural and morphological changes became evident . Heart tissue sections were stained with HE, Masson, and TUNEL to detect the degrees of myocardial fibrosis and cardiomyocyte apoptosis, and evaluate the effects of EGCG on the aged mice. And ultrastructure of the myocardium was also observed under TEM. Our data disclosed that heart aging was accompanied by cardiac fibrosis with a markedly increased interstitial fibrotic area , however, no significant inhibitory effect of all EGCG doses on myocardial fibrosis was observed in the aged mice . There was a slight decrease in CVF in the aged mice treated with 100 and 200 mg EGCG, but the difference was not statistically significant. Intriguingly, a remarkable increase of cardiomyocyte apoptosis rate in the aged mice was revealed by TUNEL staining, as opposed to the young mice , but it was normally reduced to the level of youth in the aged mice treated at all EGCG doses , suggesting EGCG could prevent myocardial apoptosis in the aged heart. Meanwhile, the TEM showed that the sarcomeres were arranged in complex patterns and partially dissolved in the aged mice, accompanied by disrupted Z lines and an increased mitochondrial flameng score compared to the young mice . Nevertheless, destruction of myofilaments and swollen mitochondria were alleviated in the aged mice administered with EGCG supplementations at all doses . Figure 3 Long-term EGCG intake alleviates aging-related cardiomyocyte apoptosis and mitochondrial damage. (A, B) Representative histology of heart tissue sections stained with hematoxylin-eosin stain and Masson's trichrome stain (yellow arrows collagen fibrosis; scale bar: 100 μm). (C) TUNEL stain for evaluation of cardiomyocyte apoptosis (red arrows apoptotic cells; 400 × ). (D) Transmission electron microscope (TEM) for the display of myocardial ultrastructure . (E–G) Quantitative analysis of myocardial fibrosis, cardiomyocyte apoptosis, and mitochondrial destruction ( n = 3–4). All results are represented as mean ± standard deviation. ∗∗∗ P < 0.001. Figure 3 Abnormal cTnI expression, as reflected by its deficiency, low levels, and mutations has been associated with CDD. 9 , 10 Emerging evidence revealed gene expression is consistent with histone acetylation modification. 10 , 16 In this study, we detected the levels of cTnI expression and histone acetylation modification near its promoter both in young and aged mice with or without EGCG, to identify the impacts of EGCG on cTnI regulation. The cTnI mRNA and protein levels were significantly lower in the aged mice compared to the young mice . Supplementation with 100 and 200 mg EGCG increased these reduced levels to those observed in the youth, however, 50 mg EGCG supplementation did not alter the protein decline . Furthermore, a significantly declined AcH3 near cTnI's promoter ( P < 0.001) was found in the aged group, along with decreased AcH3K9 ( P < 0.001) and unchanged AcH3K27 ( P > 0.05) compared with the young mice . Supplementations with 100 and 200 mg EGCG notably increased the downregulated AcH3 and AcH3K9 near cTnI's promoter (100 or 200 mg EGCG vs . the aged group: P < 0.01 for both; 200 mg EGCG vs . the young group: P > 0.05) and did not affect AcH3K27 (each of EGCG groups vs . the aged group: P > 0.05). However, no significant improvement of AcH3 and AcH3K9 was found after 50 mg EGCG intake . In addition, the lower transcription factor binding levels of MEF2c and GATA4 near cTnI's promoter were confirmed in the aged mice and improved to the levels as seen in the young mice by 100 and 200 mg EGCG administrations . The results indicated that EGCG could increase cTnI expression via enhancing AcH3 and AcH3K9 near cTnI's promoter and increasing the recruitment of MEF2c and GATA4 in the aged mice. Figure 4 Long-term administration of EGCG promoted cTnI expression via inhibiting HDAC1 and enhancing AcH3K9. (A, B) Quantitative analysis of cTnI transcription and protein expression by quantitative PCR and Western blot, respectively. (C) The enrichment levels of AcH3K4, AcH3K9, and AcH3K27 near cTnI's promoter by chromatin immunoprecipitation (ChIP) assays. (D) The enrichment levels of MEF2c and GATA4 near cTnI's promoter by ChIP assays. (E) Quantitative analysis of class I HDACs by quantitative PCR. (F) The binding levels of class I HDACs near cTnI's promoter by ChIP assays ( n = 8–10). All results are represented as mean ± standard deviation. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. Figure 4 To better understand the target of EGCG, we detected Class I HDACs, the upstream modifier of AcH3K9 in the heart tissues. The data showed the transcript level of HDAC1 was significantly higher in the aged mice than in the young mice ( P < 0.001), and there was no difference in HDAC2 and HDAC3 between the two groups . The elevated protein level of HDAC1 was consistent with its transcription level . But the boosted transcript and protein levels of HDAC1 were markedly inhibited by all doses of EGCG . Furthermore, the HDAC1 binding level near cTnI's promoter was significantly higher in the aged mice than in the young mice ( P < 0.001), but 100 and 200 mg EGCG supplementations significantly inhibited the elevated HDAC1 binding level and had no effects on HDAC2 and HDAC3 . These results suggested that EGCG might enhance AcH3 and AcH3K9 near cTnI's promoter by inhibiting HDAC1. To directly confirm the regulatory effects of HDAC1 on cTnI expression, primary cardiomyocytes transfected with HDAC1 overexpression adenovirus were cultured in vitro . As shown in Figure 5 A and B, cTnI mRNA and protein expression levels were significantly reduced in the HDAC1 overexpression group compared with the Blank group ( P < 0.001 for transcript and P < 0.01 for protein). The transcription and protein levels for HDAC1, the enrichment of HDAC1 near cTnI's promoter, and its activity in cardiomyocytes were elevated in the HDAC1 overexpression group compared with the Blank group, along with decreased AcH3K9 . These data indicated that HDAC1 induced-histone deacetylation modification played a key role in low cTnI expression regulation. Figure 5 EGCG elevates cTnI expression via suppressing HDAC1 and increasing AcH3K9. (A) Quantitative analysis of cTnI and HDAC1 transcription expression by quantitative PCR. (B) Quantitative analysis of cTnI and HDAC1 protein expression by Western blot. (C) The enrichment levels of AcH3K9 and HDAC1 near cTnI's promoter by chromatin immunoprecipitation assays. (D) HDAC1 activity. All results are represented as mean ± standard deviation. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. Figure 5 To better understand the effects of EGCG, primary cardiomyocytes were treated with 40 μM EGCG in the HDAC1 overexpression group . The results showed the transcription and protein levels for cTnI were increased by EGCG treatment in cardiomyocytes with HDAC1 overexpression ( P < 0.05 for transcript and P < 0.01 for protein). We then detected the histone acetylation modification levels. The results revealed the protein for HDAC1 and its enrichment near cTnI's promoter were significantly reduced ( P < 0.001 for both), and the subsequent improvement of AcH3K9 was detected in HDAC1 overexpression group with EGCG administration ( P < 0.001), accompanied by depressed HDAC1 activity ( P < 0.01). Nevertheless, EGCG had no inhibitory effects on the mRNA expression of HDAC1 in primary cardiomyocytes with overexpressed HDAC1 ( P > 0.05). These data confirmed that EGCG could enhance cTnI expression via inhibiting the HDAC1 protein expression, its enrichment near cTnI's promoter and the activity of HDAC1, and then elevating AcH3 and AcH3K9, as shown in Figure 6 . Figure 6 The schematic illustrating that long-term EGCG administration prevents CDD and cTnI decline in aging hearts. The scheme depicts the role of physiological aging in the pathophysiology of CDD. The hallmarks of HFpEF such as CDD, reduced exercise performance, increased heart failure markers, myocardial fibrosis, cardiomyocyte apoptosis, and mitochondrial destruction, which might be associated with cTnI decline induced by HDAC1. According to our results, long-term EGCG administration counteracts aging-associated cardiomyocyte apoptosis and mitochondrial destruction, through which it improves the physiological cardiac function and structure. In the aging mice, EGCG regulates cTnI gene expression via inhibiting the expression of HDAC1, reducing its binding level near cTnI's promoter region and lowering the activity, as well as enhancing AcH3 and AcH3K9. The black blue arrow indicates lower and the red arrow indicates higher. See text for details. AcH3, acetylated histone 3; AcH3K9, acetylated lysine 9 on histone H3; CDD, cardiac diastolic dysfunction; cTnI, cardiac troponin I; EGCG, epigallocatechin gallate; HDAC1, histone deacetylase 1; HFpEF, heart failure with preserved ejection fraction. Figure 6 In recent years, EGCG, the major polyphenol component of green tea extract, has been widely reported to have extensive biological activities in various organs. 21 , 22 , 23 In our study, we found that cTnI expression and diastolic function were decreased gradually during cardiac aging, therefore, EGCG was administered in drinking water before their declines occurred. Then the effects of different concentrations of EGCG were observed. The results showed that 100 and 200 mg EGCG supplementations in drinking water for 6 months prevented the development of aging-related CDD and cTnI expression decline. This effect was due to long-term intervention with 100 and 200 mg EGCG up-regulating cTnI expression via inhibiting HDAC1 and increasing AcH3K9 near cTnI's promoter. However, there was no significant difference in the performance between the 50 mg EGCG group and the aged group. We speculated that the concentrations of EGCG or its derivatives in the blood circulation of mice administered with 50 mg EGCG were too low to generate the cardioprotective effects. In our previous study, intraperitoneal injection of 50 mg EGCG could improve CDD in restrictive cardiomyopathy (RCM) mice, indicating that a higher dose of oral EGCG may be necessary to avoid first-pass elimination. CDD, a clinical syndrome associated with aging, is becoming increasingly prevalent. Recent studies have indicated that abnormal expression of cTnI contributes to the pathophysiology and development of CDD. 9 , 10 , 11 , 24 Homozygous knock-in mice with cTnI R21C mutation developed hypertrophy after 12 months of age and exhibited abnormal diastolic function that is characterized by longer filling times and impaired relaxation. 24 Mice carrying cTnI R193H mutation manifested bi-enlarged atria and severe CDD. 25 A clinical study found that the myocardial cytoplasmic cTnI concentration was decreased in the elderly human hearts, which was consistent with the occurrence of CDD in the elderly population. 11 In hemodialysis patients with pEF, serum cTnI levels were significantly associated with CDD and risk of mortality independent of echocardiographic variables and other biomarkers. 26 Our data showed that cTnI mRNA and protein expression declined markedly at 14 and 16 months of age, respectively, with the lowest expression occurring at 18 months. In fact, histone acetylation is a dynamic and reversible process that regulates gene expression and is mediated by histone acetylase and HDACs. 27 , 28 Our data demonstrated that the expression of HDAC1 and its enrichment near cTnI's promoter were increased in the aged mice, and the level of AcH3K9, a marker for gene activation, was reduced, indicating aging-related low cTnI expression was associated with HDAC1 induced deacetylation of H3K9 near cTnI's promoter. We wonder whether blocking cTnI decline could prevent the occurrence and development of CDD. In Asia, especially in China, there is a tradition of drinking tea for thousands of years. EGCG has been reported to have multiple effects on cardiovascular diseases and could improve CDD in RCM mice and patients with abnormal cTnI. 15 , 25 EGCG dissolved in drinking water was used in the present study. This feeding regimen is noninvasive and well tolerated by animals and mimics the daily consumption of green tea by an average adult human. 29 Based on the daily water consumption and weight of mice, we calculated the mean dose to be about 50, 100, and 200 mg/kg/day. Our data showed that long-term EGCG administration blunted aging-related CDD and exercise performance decline, accompanied by significant reductions of HF markers and increases of SERCA2a and SAR in the EGCG-treated aged mice, suggesting EGCG could prevent cardiac aging and improve HF. SAR, a luminal Ca 2+ buffer protein, is located in the longitudinal sarcoplasmic reticulum of muscle and heart and regulates Ca 2+ reuptake by interacting with SERCA and maintaining its stabilization. 30 , 31 Senescent SAR knock-out mice exhibited typical systolic dysfunction due to the decreases in SERCA2a expression and activity. 30 AAV/SERCA2a gene therapy has been performed in patients with advanced heart failure due to systolic dysfunction. 32 However, in our study, no difference was found in myocardial contractility between the young and aged mice. Systolic dysfunction may not be a major issue in older mice and the population. 2 , 3 , 6 With increasing age, hearts present undesirable structural and morphologic phenotypes, characterized by elevated collagen fiber deposition, myocardial apoptosis, and mitochondrial destruction. Here, we found that EGCG may attenuate myocardial apoptosis and mitochondrial destruction in the aged mice, but had no effects on cardiac fibrosis. EGCG could maintain the baseline mitochondrial function and integrity, which is essential for preventing myocardial apoptosis during aging. 33 Taken together, long-term EGCG supplementation is associated with ameliorative diastolic function and a repressible aging process. Epigenetically, EGCG has been classified as an HDAC inhibitor, which can repress Class I HDACs to enhance histone acetylation and increase the transcription of target genes. 34 , 35 In this study, long-term 100 and 200 mg EGCG treatment could alleviate the decreases in cTnI transcript and protein levels. Enrichment of HDAC1 near cTnI's promoter was reduced, and AcH3 and AcH3K9 were elevated after EGCG treatment in the aged mice, along with elevated recruitments of transcription factors. Although 50 mg EGCG inhibited HDAC1 expression, it did not increase cTnI mRNA expression nor improve histone acetylation and enrichment of transcription factors near cTnI's promoter. To better understand the effect of EGCG on HDAC1, primary cardiomyocytes with HDAC1 overexpression were treated with EGCG. We found both cTnI mRNA and protein were decreased in cardiomyocytes with HDAC1 overexpression, however, EGCG could reverse cTnI expression. The level of AcH3K9 was increased, and protein expression of HDAC1 and its enrichment near cTnI's promoter were significantly reduced by EGCG treatment. Additionally, the activity of HDAC1 was also markedly suppressed. These data strongly suggested that EGCG could increase cTnI expression via inhibiting HDAC1. Unfortunately, there were some limitations in the present study. EGCG blood concentrations were not measured due to a lack of corresponding devices. Water containing EGCG was freshly prepared daily, its blood concentration might be maintained at a certain level to ensure the effectiveness of EGCG. Here, we did not detect the methylation levels near cTnI's promoter. Our previous study revealed no significant changes in methylation levels in mice of different ages, and histone acetylation might be more important as an epigenetic regulator than gene methylation in regulating cTnI gene expression. 10 The primary finding of this study is that EGCG may be an effective agent for preventing aging-related CDD and cTnI expression decline by inhibiting HDAC1 and increasing AcH3 and AcH3K9 near cTnI's promoter in aged mice . We provide a novel insight into the fact that long-term administration of EGCG is an effective strategy in preventing the onset and development of aging-related CDD and cTnI expression decline. Drinking green tea daily may improve diastolic function in elderly individuals and enhance their quality of life. | Review | biomedical | en | 0.999996 |
PMC11699737 | Chronic respiratory diseases are a leading cause of disability and mortality globally . They include a range of infectious and non-infectious health conditions such as tuberculosis, chronic obstructive pulmonary disease (COPD), asthma, occupational lung diseases and pulmonary hypertension. As with many other chronic conditions, their prevalence is increasing . Despite being a heterogeneous group, chronic respiratory diseases share similar risk factors, such as smoking, air pollution, dust and occupational chemicals . Early diagnosis, close monitoring, appropriate therapy, and easy access to health care can improve outcomes . Rurality may be a determinant of risk and outcome of chronic respiratory disease. For example, COPD incidence is higher in populations living in rural and remote areas, who are, on average, also more exposed to risk factors such as indoor pollution and the use of biomass fuel . More than half of all rural residents lack access to critical healthcare and many need to travel long distances to reach facilities. Patients may be reluctant to seek care due to travel costs, time expenditure and loss of potential income, resulting in healthcare-seeking delays and limited access to specialist services and pulmonary rehabilitation . Lower health literacy and socioeconomic status may also influence the underutilisation of care and the persistence of unhealthy habits such as smoking . The urban-rural divide ultimately results in a higher disease burden in remote communities, characterized by delayed diagnoses and higher hospitalization and mortality rates . In patients with chronic respiratory diseases, e-health interventions have been shown to reduce emergency department attendance [ 17 – 19 ], hospitalisation rates , the number of exacerbations and to increase pharmacological treatment compliance and physical activity rates . The use of e-health interventions may, therefore, help to reduce health disparities in remote populations living with these diseases [ 20 – 22 ]. Indeed, there is evidence that telehealth interventions are non-inferior for improving COPD self-management in rural areas . However, beyond the effectiveness of e-health interventions, whether implementation is successful is likely context-specific, and a comprehensive understanding of factors influencing this process is paramount. The aim of this qualitative evidence synthesis (QES) was, therefore, to analyse the qualitative and mixed-methods literature on e-health interventions in people with chronic respiratory diseases in remote settings and thereby identify barriers and facilitators for implementation. The QES was structured according to Cochrane’s EPOC guidance and the ENTREQ reporting guidelines (checklist: Supplementary Table 1) . The PerSPE(c)TiF framework was used to define the research question (Supplementary Table 2) . In brief, the aim of this QES was to explore any factors related to the successful implementation of e-health technologies in remote settings worldwide, for diagnosis and follow-up of chronic respiratory diseases from the perspective of patients, healthcare workers and other stakeholders. Citations were imported to Endnote version 20.3 on 21.4.2023, and duplicates were removed, followed by title/abstract screening and full-text review based on predefined inclusion and exclusion criteria (Table 1 ). Articles were screened by two authors (ES and EB) independently. In case of disagreement, the article was included in the full-text review. All qualitative and mixed-methods studies published in English describing diagnosis, monitoring, or treatment of patients with chronic respiratory diseases, defined according to the criteria of the American Lung Association , in remote and rural settings using e-health were included. We chose to include abstracts as well as full-text articles in order to obtain the most comprehensive data, as the chosen methodology of thematic synthesis is appropriate for relatively thin data, keeping the level of confidence in the data in mind when interpreting these. With regard to COVID-19, studies on diagnosis, monitoring or long-COVID were included, while studies on short-term treatment were not, as findings from the former were expected to be relevant for this QES. Table 1 Inclusion and exclusion criteria Criteria for selection Inclusion criteria Exclusion criteria Type of article Full-text articles or abstracts of primary research Literature review, systematic review, QES Methodology Any qualitative or mixed-methods approach Quantitative research without qualitative components Study population Patients of any age suffering from or suspected of chronic respiratory diseases including multi-morbidity patients, and/or their families; any stakeholder working on chronic respiratory disease e-health interventions including but not limited to healthcare workers, administrative personnel, policy makers, technology manufacturers, researchers. Patients with conditions other than chronic respiratory conditions; stakeholders not working on chronic respiratory disease e-health interventions Type of setting Remote or rural settings (no specific geographic restrictions) Non-remote settings Type of Intervention Any e-health intervention directed at the management of chronic respiratory disease patients (diagnostic process, follow up, monitoring and treatment) Interventions without e-health components; e-health interventions not directed at chronic respiratory disease patients; multi-disease e-health interventions where findings for subgroup of chronic respiratory disease patients are not described Timing No time limit Language English Non-English language Type of outcomes Any qualitative outcomes related to factors influencing the use of e-health, including but not limited to: - User attitudes and satisfaction with the technologies - Factors for prolonged engagement - Factors hindering e-health implementation Quantitative outcomes not accompanied by qualitative findings The assessment of the methodological limitations was done according to the Critical Appraisal Skills Programme (CASP) tool for qualitative research to evaluate the methodological rigour of selected studies . The CASP assessment is presented in Supplementary Table 4. The findings were also assessed for confidence according to GRADE CERQual method (Supplementary Table 5). CERQual takes into account four different factors: methodological limitations, relating to the study design or conduct , coherence, assessing the fit between the primary study findings and review findings , adequacy, describing the overall richness and quantity of the data and relevance, describing the extent to which the data is applicable and relevant to the review question . After assessing each individual component, the overall confidence was judged by one author (ES) as high, moderate, or low. The RETREAT framework , taking into account the review question, epistemology, timeframe, resources, expertise, audience and types of data, guided the choice of a thematic synthesis approach (Supplementary Table 6). The selected articles were uploaded to Atlas.ti software . Articles were coded sentence-by-sentence based on units of meaning, applied to both first and second-order constructs present in the results and discussion sections of papers, to capture both primary opinions and authors’ interpretations . First-order constructs (quotes from participants in the original study) are presented in italics, and second-order constructs are presented in normal font. Descriptive themes were generated grouping similar codes into a hierarchical tree structure. Thematic synthesis was combined with an a priori ‘best fit’ framework approach . The themes were discussed between the authors (ES, FB, and EB) and existing frameworks were sought to guide the analysis and presentation of the data, ultimately identifying the Digital Health Equity Framework (DHEF) . The DHEF draws on the leading health disparities framework. It includes key determinants of health at different levels and incorporates a digital environment domain which enabled us to classify and interpret factors related to implementation of e-health in remote locations. In order to identify themes, deductive coding was used applying the DHEF and an inductive coding process was utilised to capture novel insights. Aligning with the DHEF, themes were arranged according to their relevance to determinants of health at different levels: individual (e.g., patient or healthcare provider attitude towards technology), interpersonal (e.g., patient-tech-healthcare provider relationship), community (e.g., healthcare infrastructure), society (e.g., tech policies and data standards). An additional level was added relating to the intervention itself (e.g., technology characteristics, design quality and product presentation). Within the data, barriers and facilitators were identified, highlighting those unique to remote populations. Table 2 – overview of selected articles First author, year Country, location Setting Type of publica-tion Participants Medical condition E-health intervention Study population CERQual assess-ment of confidence in the evidence Age % Female Languages spoken Alexander, 2021 USA, North Carolina McDowell, Mitchell, and Yancey counties, designated as Health Professional Shortage Areas for primary care and predominately rural Qualitative research Patients ( n = 17) COPD Telehealth Mean age 61 years +-SD 9.1 41 English High Alwashmi, 2019 Canada Newfoundland and Labrador – area with substantial remote and rural population Qualitative research Nurses, physicians, pharmacists ( n = 30) COPD Mobile health interventions Mean age 39 years NR NR High Anticona, 2015 Peru Rural areas in the Highlands and the Amazon basin Qualitative research Physicians ( n = 30) Multiple, including chronic respiratory diseases Technological innovations, including telehealth and mobile health Age 26–30 years 43 Spanish Low Boyd, 2007 Australia, Victoria Five different rural Victorian towns Mixed methods Patients, developers, community members (8 focus groups of 4–10 participants) Asthma Multimedia education program NR NR NR Moderate Brown, 2017 USA, North Dakota Oakes, located in rural Dickey County Mixed methods Patients ( n = 18) Asthma Telehealth for asthma education NR NR NR Moderate Chaiyachati, 2013 South Africa, KwaZulu-Natal Umzinyathi, a rural district of KwaZulu-Natal Mixed methods Mobile healthcare workers ( n = 5) Multidrug resistant TB Mobile phone application Median age 32 years (range 27–46) 20 English Moderate Chen, 2012 USA, West Virginia Rural Appalachia Abstract Patients ( n = 5) Lung cancer Nurse-interpreted telementoring intervention with patient ducation Mean age 66 years +- 6.4 60 NR Moderate Concotelli, 2012 USA, New Mexico NR Abstract Patients CF Telehealth adolescent support group NR NR NR Low Cox, 2022 Australia, Victoria 33% of participants “from a rural location” Abstract Patients ( n = 20) Chronic respiratory diseases Telerehabilitation NR NR NR Low de Batlle, 2021 Spain Lleida, a large rural area of over 4300 km 2 Mixed methods Patients ( n = 76) COPD Mobile health-enabled integrated care model Mean age 82 years 46 NR Moderate De San Miguel, 2013 Australia, Western Australia Metropolitan area in Western Australia Mixed methods Patients ( n = 80) COPD Home-based telehealth monitoring Mean age 73 years, range 54–88) 52 English Moderate Demchenko, 2015 Canada, Saskatchewan People living more than 100 km from Saskatoon Abstract Patients ( n = 46) Lung cancer Nurse-clinical administered telehealth clinic NR NR NR Low Douglas, 2013 Canada, Alberta First Nations communities in Alberta Mixed methods Education instructors, phycisians, academics Asthma Self-management education curriculum NR NR NR Moderate Ellington, 2021 Uganda, Jinja district Two federally funded Ugandan primary care health facilities, one peri-urban and one rural Qualitative research Health care workers ( n = 31) Lower respiratory disease Mobile health tool N = 10 < 30 years; n = 11 30–40 years; n = 4 > 40 years 68 English and local languages Moderate Godden, 2011 UK, Scotland Five participants “worked exclusively in remote and rural areas, while the others provided services across the region for urban, rural and remote patients” Mixed methods Phycisians, nurses, respiratory care professionals ( n = 20) COPD, Asthma, Lung cancer, OSAS Telehealth NR 65 NR High Goodridge, 2011 Canada, Alberta “This health region encompasses an area of more than 40,000 km2, has population densities ranging from 1.1–2.0 persons per km2 and is designated as having no metropolitan influence by Statistics Canada.” Qualitative research Patients ( n = 7) COPD, Bronchiectasis Not specified Mean age 75 years (range 57–88) 71 English Low Guthrie, 2020 USA, Texas “Northeast Texas, a 35-county area with a population close to 1.5 million, over half living in a rural area” Abstract Patients CF Telemedicine NR 55 NR Low Hatem, 2022 USA, Massachusetts NR Abstract Patients COPD, COVID, interstitial lung disease Telehealth-supported home based pulmonary rehabilitation program Mean age 71 years 5 NR Low Johnson, 2021 USA, South Carolina Four of nine schools were classified as rural Mixed methods Nurses, phycisians, respiratory therapist, program coordinator ( n = 26) Asthma Mobile health application 25-34y n = 4; 35-44y n = 7; 45-54y n = 10; 55-64y n = 4; 65 + n = 1 96 NR Moderate Khan, 2017 Canada Northern communities in Saskatchewan Mixed methods Phycisians TB, various conditions Multiple NR NR NR Low Kok, 2023 Uganda, various districts Rural communities, with a distance of at least 7 km to a health facility Mixed methods Community health workers ( n = 24) COVID Telehealth Mean age 38 years 90 Luganda, Lusoga, Runyankole Moderate Latycheva, 2013 Canada Five First Nations and Inuit communities from across Canada Mixed methods Patients, parents, grandparents, community members, teachers Asthma Web-based asthma education materials N = 14 6 to 12 years; n = 18 12 to 18 years; age of adults participants NR NR English High Locke, 2019 USA, Rural patients, as defined by the US Census Bureau Mixed methods Patients COPD, Asthma Video telehealth inhaler training program Mean age 69.2 years 0 NR Moderate Lundell, 2020 Sweden, Västerbotten county A large and sparsely populated area with long distances to health care facilities Qualitative research Patients ( n = 13) COPD Home telemonitoring system NR 62 Swedish Moderate MacGeorge, 2021 USA, South Carolina NR Abstract Nurses, teachers, administrators Asthma Telehealth-delivered school-based program NR NR NR Low Mair, 1999 UK NR Mixed methods Phycisians, Patients COPD Telecommunications technology Mean age 62 years (range 52–72) 67 NR Low Mathur, 2019 India, Assam state Five rural blocks from Darrang and Kamrup district of the Assam state Qualitative research Patients ( n = 40) TB Outbound automated calls Mean age 34 years, range 18–60 20 Assamese Moderate Mc Veigh, 2019 USA, Idaho Large veteran hospital in Boise, Idaho, providing services to veterans located in mountainous regions of the county Qualitative research Nurses, phycisians, social workers ( n = 16) Non-malignant respiratory disease Telemedicine/digital platform for palliative care NR NR English Moderate McGee, 2020 USA, various states Patients residing in rural/geographically isolated areas Mixed methods Patients ( n = 49), informal caregivers ( n = 49) Chronic lower respiratory disease Telehealth services Patients: mean age 706 years 6.1 English Moderate Mendez, 2021 Chile, Santiago Participants from any Chilean region Mixed methods Patients, physiotherapists ( n = 22) COPD Mobile phone application Median 37 (IQR 33–39 y) 50 Spanish High Musiimenta, 2020 Uganda, Southwestern TB clinic within Mbarara Regional Referral Hospital in rural, southwestern Uganda Mixed methods Patients ( n = 35), social supporters ( n = 15) TB Mobile health intervention Median 32 (patients); 37 (social supporters) 43 (patients); 60 (social supporters) Runyankole Moderate Ng, 2014 Australia, South Australia Port Augusta and Whyalla (300 and 381 km from Adelaide respectively) and Tennant Creek (500 km from Alice Springs) Abstract Patients COPD, CF, OSAS Telehealth consultations NR NR NR Low Otty, 2023 Australia, Queensland Regional tertiary public hospital in northern Australia, with a significant number of geographically dispersed rural and remote communities Qualitative research Patients, informal caregivers ( n = 19) Lung cancer Telehealth consultations Median 64 (IQR 52–82 y) 48 NR Moderate Petitte, 2014 USA, West Virginia Patients’ homes in rural Appalachia Mixed methods Patients ( n = 10) Lung cancer Home telemonitoring intervention Median 66 (IQR 58–73) 50 NR Low Ratchakit, 2020 Thailand, Chiang Rai TB clinic at Chiangrai Prachanukroh Hospital, a tertiary-care hospital located in Thailand’s northernmost province Mixed methods Patients ( n = 80) TB Mobile health intervention Mean 50 ± 16 (intervention arm) 40 Thai Moderate Raza, 2009 USA, Michigan/Wisconsin Tertiary care hospital in Milwaukee and small rural hospitals in Iron Mountain (MI) and Appleton (WI) for veterans located in northern Wisconsin and the Upper Peninsula of Michigan Mixed methods Patients ( n = 314) Pulmonary specialist diagnostic consult Telehealth consultations NR NR NR Moderate Roberts, 2012 UK, Scotland Sheltered housing in Oban and in a community hall on the Isle of Luing Qualitative research Patients, phycisians, nurses, nurse specialists, housing warden, project managers (n = NR) COPD Home and community telehealth monitors NR NR NR High Ruseckaite, 2021 Australia, Victoria NR Abstract Patients, informal caregivers ( n = 15) CF Telehealth consultations Mean 40.2 (patients); 41.8 (caregivers) NR NR Low Venter, 2012 New Zealand Rural communities in Turangi and Taupo areas Mixed methods Patients ( n = 20) COPD Home telemonitoring intervention NR NR NR Moderate Wilson, 2022 USA, Midwest states Rural areas across five Midwest states Abstract Phycisians, nurse practicioners, phycisian assistants ( n = 10) COPD Telemedicine NR NR NR Low Young, 2012 USA, Wisconsin Family Health Center of Marshfield, serving 254 rural municipalities in north-central Wisconsin Mixed methods Patients ( n = 98) Asthma Telepharmacy intervention Mean 44.6 (SD 15.8) 75 English High CF cystic fibrosis, COPD chronic obstructive pulmonary disease, NR not reported, OSAS obstructive sleep apnea syndrome, TB tuberculosis We included 11 primary qualitative studies, 21 mixed-methods studies, and 9 abstracts. The studies were published between 1999 and 2023. The geographical location varied; fifteen studies took place in the USA, six in Canada, six in Australia, three in Uganda, three in the UK, two in India and one in Peru, South Africa, Spain, Sweden, New Zealand, Chile and Thailand each (see Table 2 for details on the setting). Sixteen studies (39%) reported the language spoken by the participants, which was English in nine studies, Spanish in two studies, and Assamese, Swedish, Runyankole and Thai in one study each. One study reported that interviews and focus groups were done in English and ‘local languages’, and one study was done in Luganda, Lusoga and Runyankole. None of the studies reported the proportion of participants whose mother tongue was not English. Five studies explicitly mentioned the inclusion of Indigenous, First Nations or Aboriginal participants , two of which reported involvement of these groups in the study design itself, either in the form of an advisory group or by partnering of the researchers with First Nations leaders . Twelve studies included a mix of different chronic respiratory diseases, ten of the studies focused on COPD, seven on asthma, four on tuberculosis, four on lung cancer, three on cystic fibrosis and one on COVID-19 (Table 2 ). The results of the CASP assessment of methodological limitations of the included studies are shown in Supplementary Table 4. According to the CERQual assessment of the findings, there were seven studies that inspired high confidence in their findings, while twenty instilled moderate confidence. The remaining fourteen studies elicited low confidence in their results (Table 2 and Supplementary Table 5). The reasons for low confidence ratings were diverse; notably, for several studies only abstracts were published without corresponding full articles, impacting the ability to assess the methodology and the richness and thickness of the findings. Table 3 Overview of facilitators and barriers for e-health interventions associated with each level Level Facilitators Barriers Individual • Patient willingness to become more autonomous, increase health literacy and self-management quality • Usability as educational tool • Patient familiarity with technology • Patient and HCP interest in learning new technologies • Scheduling flexibility and reduction of in-person appointments • No need for companion • Sense of reassurance with constant monitoring/access • Personalized care • Reduced travel costs, overall affordability • Cost-effectiveness • May not be helpful unless integrated with behavioral change interventions • Patient age and poor digital literacy • Patient and HCP lack of knowledge about existing resources • Patient low literacy levels • Patient and HCP low interest in new technologies, technology perceived as challenging, skepticism • Preference for in-person consultation and fear of loss of human contact • Sense of hyperconnectivity • Increased workload and fear of not being adequately compensated (HCP) • Negative attitude towards tech devices (e.g., smartphones) • Steep set-up phase • Cost of device, lack of insurance Interpersonal • HCP can manage more patients • Reduced risk of infectious diseases transmission • Improved constant monitoring • Clinical team building and creation of multidisciplinary teams • Greater family involvement • Reduced non-verbal communication • HCP feeling constantly available “on demand” • Sense of false security in the patient • Deterioration of relationships within pre-existing teams due to increased responsibilities Community • Community engagement • Use of role models • Cultural appropriateness • Availability of the translation in local languages • Focus on multiple conditions • Poor networks • Lack of pre-existing health infrastructure • Focus on one disease at a time only Society • Lack of concern about personal health data privacy • Use of HIPAA compliant technologies • Trust in tech developer • Governmental and local leaders’ involvement • Private-public partnerships • Perceived as cost-saving • COVID-19 experience • General availability and diffusion of tech devices • Risk of health data leakage • Lack of global privacy standards • Concerns around cost-effectiveness • Maintenance of the infrastructure • High training demand Technology and device • Ease of set-up and use • Easy interventions and advice • Translation in local language • Availability of tech support • Large fonts, audio support (e.g., for visually impaired) and recorded materials • Appealing graphic features, fun approach, customization • Portable, light devices • Content and language complexity • Excessive length of interactive sessions • Unavailability in local language • Non-communicability between different software • Bulky, heavy devices • Difficult to clean HCP healthcare provider, HIPAA Health Insurance Portability and Accountability Act E-health requires a different form of patient participation than traditional consults. This participatory nature could positively influence self-efficacy and health literacy. Many healthcare providers (HCPs) and patients felt that e-health facilitated autonomy and increased the quality of self-management. E-health was often used as a tool for education and was seen to have advantages compared to traditional in-person education, allowing for more detailed instruction over an extended period . For patients, being able to review information at their convenience was important. Additionally, using e-health, they gained a better comprehension of their own physiological reactions and general health status , leading to improved self-management of disease . Nonetheless, numerous sources also reported negative attitudes towards the use of e-health technologies. Patients’ and HCPs’ negative feelings arose primarily from a sense of hyperconnectivity, increased workload , and scepticism about the reliability of devices and efficacy of the interventions . Many patients and HCPs reported a sense of threat over the risk of losing human contact and face-to-face clinical interactions [ 35 – 37 , 49 , 67 ]. In one study, this was described as feeling “chained” to the digital environment: The importance of accessibility features, such as large fonts loud voices , audio components for the visually impaired and text for the hearing impaired , was emphasized as their absence hindered utilization for patients with corresponding disabilities. For e-health equipment utilizing visual displays like computer or mobile applications, a visually appealing graphic user interface was favoured across several studies . Graphics were found to engage users and bolster long-term adherence . Reminders and notifications were explicitly preferred and linked to improved medication adherence . Some studies highlighted the preference for recording and reflecting on the given advice . This QES identified various factors relating to the effective implementation of e-health interventions for chronic respiratory diseases in remote locations. In general, e-health interventions were well received but also carried a number of concerns and limitations that could reduce their effectiveness. Moreover, e-health was deemed insufficient as a standalone intervention and should be integrated into existing care practices [ 35 – 37 , 58 , 64 , 72 ]. E-health was identified by many patients living in remote communities and HCPs as a tool with the potential to overcome the obstacle of distance to health facilities, reducing commutes and related costs, and expanding access to care. Increased self-efficacy and health literacy were additional benefits at the individual level. Concerns and barriers were noted across all levels. At the community level, concerns related to lack of telecommunications and healthcare infrastructure— hence, while e-health was perceived as a solution to certain resource limitations, other resource limitations undermined its potential. The main concerns at the interpersonal level related to the reduction of in-person interactions and fear of losing personal connections. Additionally, HCPs identified additional burdens for the staff, negative attitudes towards technology, and poorly planned implementation processes as barriers to successful adoption. Our findings are in line with previously published reviews , but some specifically relevant issues are worth highlighting. Adapting e-health interventions to the local setting was seen as especially relevant in remote and rural environments . Some studies suggested that integrating aspects important to the community, such as symbolism , traditional medical practices and habits specific to the location with ‘Western’ medical information was appreciated, leading to higher adoption. By contrast to cultural factors, considerations about the availability of e-health or applications in the preferred language of the patient/community were discussed in very few papers, highlighting an important gap in the evidence base. In addition to the setting, the target population is also key to consider at an early stage since the feasibility of e-health interventions may vary between patient groups. For example, patients in poor health with advanced disease might be physically unable to self-measure or undergo pulmonary rehabilitation without in-person assistance . Another aspect of this was described in a meta-ethnography on telemedicine in COPD: while patients with high disease burden experienced an increased need for telemedicine, patients in a stable period or at an early phase of the illness perceived the disease as becoming too prominent with telemedicine, and a need to be more detached . Familiarity with technology in general and training needs may also differ between groups, as we identified longer learning times for patients with lower education levels and advanced age . It is worth noting that the term ‘e-health’ includes a wide variety of interventions, and although the choice for a specific e-health modality was not the focus of this QES, many papers discussed their reasoning behind this. Individual preferences varied: some users preferred mobile-phone-based interventions and apps , others preferred video interaction over phone calls , some wanted live interactions with immediate feedback , others preferred non-live communication done at the convenience of the patient , some liked group-based interventions , others wanted graphical aspects rather than text or audio and yet others expressed the desire to combine different modalities . While some studies described the preference for combining modalities, such as video consults with devices for measuring physical parameters, others cautioned against it, warning that multiple electronic systems from different manufacturers may not communicate . However, while individual preferences emerged, none of the studies focused on comparing different types of e-health interventions, and assessing the advantages and disadvantages of different modalities is beyond the scope of this paper. This study has a number of limitations. First, there is a risk of over- or underestimating e-health acceptability and feasibility barriers due to selection bias in the primary research, preferentially including individuals with explicit views or motivations towards e-health. Second, it is challenging to generalize from studies undertaken from a wide range of contexts—across countries, populations, languages, continents etc—given the impact of setting- and population-specific factors; nevertheless, we found themes common across settings. Third, many studies focused on the initial phases of the implementation, potentially magnifying perceptions related to technology novelty while overlooking factors influencing longer-term use and adherence. Moreover, geographical representation poses a limitation, with an overrepresentation of Global North viewpoints. Certain populations (e.g. non-English speakers) were also underrepresented in the included papers, which may further limit generalizability of our findings. Additionally, while we aimed to include various patient populations, there was a higher number of studies on COPD and asthma compared to cystic fibrosis and other chronic diseases, potentially limiting the generalizability of findings. Lastly, there’s the possibility of publication bias, wherein less impactful qualitative research might not have been published , potentially leading to an incomplete representation of viewpoints. We recognize that e-health is a rapidly evolving field, and our findings will need ongoing validation against emerging evidence, technological advances, and changes in digital policy as they become available. Despite these limitations, this synthesis may have some implications for both practice and research. Our findings suggest that a functional infrastructure that can respond to changing demands is a prerequisite for the effective use of e-health. When considering a new intervention, all stakeholders should be involved in the design and implementation process. Informing patients about the content and goal, benefits and limitations of the intervention is essential to avoid miscommunication and to support willingness to engage . Even though customization may be more labour- and cost-intensive when compared to the use of generic interventions, attention should be paid to local and cultural aspects relating to language, beliefs and attitudes, as failure to do so may result in a lack of use in the longer term. General digital literacy was found to be an important factor relating to the use of specific e-health interventions and providing education to increase this could increase the adoption and use of e-health. Some level of governmental involvement is required to ensure compatibility of systems, adequate resources and equitable distribution of devices. Interesting areas for future research, beyond the scope of this synthesis, include comparing various e-health modalities and focusing on a specific disease under the umbrella of chronic respiratory disease, as well as comparing our findings with the use of e-health in non-respiratory diseases. Further research should include underrepresented populations and consider new technological and societal developments. This synthesis identified several factors that can affect the successful implementation of e-health in remote and rural locations for chronic respiratory disease patients. These factors can be used to inform the design and implementation of future e-health interventions. Intervention objectives, target population, geographical location, telecommunication and health care infrastructure capacity, local culture, language, and norms, and available human resources should be carefully considered to optimise the interventions for the best outcomes. Relevant stakeholders must be consulted throughout the process to produce an appropriate and feasible intervention. | Review | biomedical | en | 0.999997 |
PMC11699755 | Schistosomiasis, one of the most devastating neglected tropical diseases, poses a persistent public health and economic challenge in the developing world. 1 , 2 , 3 It has recently spread from developing countries to European nations due to human migration and climate change. 4 , 5 The disease causes significant illness and death, 6 promotes the transmission of human immunodeficiency virus, 7 and can lead to bladder cancer. 8 Currently, there is no effective vaccine against schistosomes. The only available treatment is praziquantel (PZQ), a chemotherapy that has been in use for over 40 years. 9 However, relying solely on PZQ-based control programs is unlikely to achieve disease control goals, as PZQ-treated patients, especially children, quickly become reinfected. 10 , 11 Additionally, concerns about drug resistance in schistosomes, particularly in mass drug administration programs, are growing. 12 , 13 Freshwater snails serve as obligate intermediate hosts for the digenetic trematodes Schistosoma spp., the causative agents of schistosomiasis. This is because the life cycle of schistosomes involves asexual and sexual developmental stages within a snail intermediate host and a mammalian definitive host, respectively. Snail control, alone or in combination with other strategies, has proven to be the most effective means of reducing schistosomiasis prevalence in endemic areas. 14 , 15 However, the widely used molluscicide niclosamide has harmful effects on the aquatic ecosystem, as it is toxic to other aquatic animals. 16 , 17 Given the critical role of snails in the aquatic ecosystem, an ideal biocontrol strategy should aim to disrupt parasite life cycles without eliminating the intermediate snail hosts. Field evidence supports this strategy, as the introduction of schistosome-resistant Biomphalaria tenagophila snails to endemic areas in Brazil has resulted in reduced disease transmission. 18 Biomphalaria glabrata — Schistosoma mansoni has been used as a model system for studying the compatibility between snails and schistosomes, particularly snail resistance to schistosomes, since the mid-20th century. 19 These studies have primarily focused on immunological responses, with significant progress made in recent omic-based research. 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 It is well established that immunological responses have genetic bases. 30 , 31 Previous studies have shown that snail resistance or susceptibility to schistosomes has a strong genetic component. 32 , 33 , 34 , 35 , 36 Therefore, genetic mapping of schistosome resistance or susceptibility should offer valuable insights into these mechanisms and help elucidate the underlying immunological responses. This knowledge could potentially aid in developing biocontrol programs, which have shown promise in controlling vector-borne diseases through clustered regularly interspaced short palindromic repeats (CRISPR)-mediated gene drive technologies. 37 , 38 , 39 , 40 A significant study on the genetic analysis of compatibility between snails and schistosomes using the B. glabrata — S. mansoni model was published by Charles Richards in 1970. 32 Richards laid the foundation for understanding snail resistance to schistosomes through extensive classical crosses between snails with different resistance phenotypes. Genetic mapping using various mapping populations (most of which are pre-existing laboratory strains) and genotyping assays led to the identification of multiple resistant loci, located on different chromosomes, 41 , 42 , 43 , 44 , 45 , 46 yielding valuable insights into the resistance mechanisms while also leaving unanswered questions (see details in the discussion section). The success of genetic mapping relies heavily on the strategic design of mapping populations and genotyping technologies. Typically, classic mapping populations involve F2 offspring and backcrosses derived from crossing two inbred parents. Recombinant inbred (RI) lines or RILs, derived from the F2 population, offer distinct advantages for genetic mapping. RILs are created by crossing two parental strains with contrasting phenotypes followed by successive generations of inbreeding (selfing or full-sib mating). Meiotic crossover events result in a mosaic parental genome in each RI line, and subsequent inbreeding increases recombination events and leads to a rapid reduction in heterozygosity. 47 , 48 , 49 , 50 , 51 RILs have commonly been used in plant genetics and breeding but have seldom been utilized in animal genetics, particularly for non-model organisms. This is mainly due to the labor-intensive, expensive, and time-consuming process of generating animal RILs, as well as the significant challenges associated with their maintenance. We produced RILs from a cross between two well-defined homozygous lines of B. glabrata , the iM line and iBS90. 45 To dissect this genetic resource, we employed whole-genome sequencing (WGS), a high-throughput genotyping assay, to scan every single nucleotide across the genomes of 46 individual RIL snails, representing 46 phenotyped RILs. This approach, designated as RIL-WGS, allowed us to reveal a significant number of single-nucleotide polymorphisms (SNPs) and bin markers for subsequent genome-wide association study (GWAS) and quantitative trait loci (QTLs) analysis. As a result, we identified a small genomic region and genes within that region involved in anti-schistosome defense. Two important biological characteristics of the B. glabrata – S. mansoni system contributed to the successful genetic design of the snail RILs. Firstly, the wild-type pigmentation follows Mendelian inheritance patterns, allowing us to confirm successful crosses. Secondly, B. glabrata is hermaphroditic, enabling both crossing and selfing. The breeding of snail RILs originated from an effort that spanned over 20 years at The University of New Mexico. During this period, the iM line and iBS90 were developed through 81 and 41 generations of selfing from a single M line and BS90 snail, respectively. 45 Both lines were confirmed to be homozygous with contrasting resistance phenotypes and were used as parental snails to generate RILs. A total of 338 pairs, randomly generated from different F2 intercrosses, served as founders for subsequent selfing . Over more than 3.5 years, 137 RIL lines were obtained and tested for their phenotype. Among them, 118 RILs displayed a clear phenotype (42 resistant and 76 susceptible), while 19 had ambiguous phenotypes as only a portion of the snails in each line shed few cercariae. From the 118 RILs with clear phenotypes, we randomly selected 46 to represent 46 RI lines, with half ( n = 23) being either resistant or susceptible to schistosomes. Figure 1 Breeding scheme of RIL snails and testing of their resistant or susceptible phenotype Homozygous iM line (albino and susceptible to schistosomes) and homozygous iBS90 (pigmented and resistant to schistosomes) snails were used as parent snails. Single iM line and iBS90 snails were placed in a 1 L plastic cup and allowed to produce F1 progeny. Since pigmentation is a dominant Mendelian trait, three possible outcomes were expected in F1 snails: albino F1 snails produced by selfing the iM line snail and pigmented F1 snails generated either from selfing the iBS90 snail or from a cross between the iM line and iBS90 snails. Albino F1 snails were discarded, while pigmented F1 snails were retained. These pigmented F1 snails were placed individually in plastic cups to produce F2 snails. To distinguish the two types of pigmented F1 snails (from selfing or crossing), we examined the colors of the F2 snails. If all F2 snails were pigmented, it suggested they originated from a single F1 snail through selfing of the parental iBS90, and they were discarded. If the F2 snails showed a mix of albino and pigmented individuals, it indicated that their parental F1 snail was produced from a cross between the iM line and iBS90 snails. The F2 snails were then retained for subsequent breeding. An albino F2 snail and a pigmented F2 snail from these F2 snails were randomly paired and placed in a plastic cup to produce offspring (an F1 population). From this F1 population, individual F1 snails were kept in plastic cups and allowed to self for 10 generations; in each generation, one snail was selected to produce the next generation through selfing. As a result, the RILs were obtained. Each RI line was tested for the resistance phenotype after miracidia exposure and cercarial shedding, as described in the STAR Methods section. All photographs presented in this figure and in the graphical abstract were prepared by S.-M.Z. Since the susceptible iM line and the resistant iBS90 were used as the parental snails to generate the RIL population, the Illumina reads used to assemble the genomes of the two lines 45 were retrieved for the current study. To ensure data comparability, the same quality control criteria described in the STAR Methods section were applied to all raw reads generated by 150 × 2 paired-end Illumina sequencing from the iM line, iBS90, and RILs. This resulted in 109.86, 118.34, and 664.4 Gb of clean reads from the iM line, iBS90, and RIL snails, respectively. Using our recently published chromosome-level assembly of B. glabrata as the reference genome, 52 the mapping rates of the iM line, iBS90, and RIL snails to the reference genome were 99%, 97%, and 96%, respectively. The sequence coverages for the iM line, iBS90, and the RIL population were 125X, 132X, and 16X, respectively ( Table S1 ). A total of 9,079,154 SNPs were identified between the iM line and iBS90 parental snails. Among these, 273,307 SNPs were found in coding regions (including upstream −5kb and downstream +2kb), with 119,241 SNPs resulting in nonsynonymous substitutions. These SNPs were used to genotype the RIL population using CLC genomics workbench. The resulting sequence variation data were exported as variant call format files, which were then combined using the Bcftools software package. The mean depth of SNP coverage for the iM line, iBS90, and RIL snails was 125X, 111X, and 14.20 ± 0.38X, respectively ( Table S2 ). The distribution of SNP coverages is shown in Figure 2 A. After filtering out markers with high missing genotype (>20%), low coverage (<5), and low minor allele frequency (<10%), a total of 7,330,259 SNP markers were retained for downstream analysis of the association between phenotype and genome-wide SNPs. The distribution of these high-quality SNPs across the 18 chromosomes shows a general correlation with genomic sizes (r = 0.71) . The longest chromosome (chr 1) has the highest number of SNPs, while chromosome 6 has the lowest. The average SNP density across the 18 chromosomes is 9.11 ± 0.47/kb, with the highest density on chromosome 18 (11.97/kb) and the lowest on chromosome 3 (5.04/kb) . Figure 2 SNP analysis (A) Distribution of mean coverage (depth) for the 9,079,154 SNPs identified in the 46 RIL snails. (B) Number of SNPs across the 18 chromosomes. (C) Density of SNPs across the 18 chromosomes. Data are represented as mean ± SEM. GWAS was performed on the RIL population using 7,330,259 SNP markers. A total of 120,698 SNPs were identified at a significant level of p ≤ 1 × 10 −5 . Among these, 837 SNPs were found to have significant associations ( p ≤ 5 × 10 −8 ) and were distributed across six chromosomes (chr 4, 5, 12, 15, 16, and 18). However, most of the SNPs (99.28%, 830/836) were located on chromosome 5 . Figure 3 GWAS (A) Manhattan plot showing genomic regions associated with snail resistance in the RIL population. Fisher’s exact test (−log10( p value)) was used to investigate the association between the resistant phenotype and SNPs ( n = 7,330,259). The bold dark dashed line and gray dashed line indicate the genome-wide significance levels at p = 5 × 10 −8 and p = 1 × 10 −5 , respectively. (B) Genetic divergence test (F ST ) between resistant and susceptible snails in 10-kb windows ( n = 51,284) for variants across the 18 chromosomes. The bold dark dashed line indicates significant genome-wide F ST at p ≤ 0.05 for each chromosome. (C) Sliding window-based F ST analysis on chromosome 5. Individual variants are represented by gray circles (displaying only those with F ST > 0.01, n = 74,499), while mean values are shown using sliding windows of 10-kb, marked by red lines. The blue bracket indicates a ∼3 Mb genomic region (positions: 39,634,500 nt–42,686,436 nt) with the highest divergence. Data are represented as mean ± SEM. Fixation index F ST analysis of the 7,330,259 SNP markers was performed using a 10-kb window analysis on susceptible and resistant RIL groups. This approach allowed us to identify regions that showed differences between the two groups. A jackknife procedure was used to test whether F ST values were statistically different from zero. 53 We used a significance level of p ≤ 0.05, with a weighted F ST value of 0.3061 from the genome-wide distribution, to define high F ST outliers. Out of the 51,284 10-kb windows (F ST > 0), a total of 2,568 10-kb windows showed significant divergence between the two phenotypes. Among these, 2,563 10-kb windows were located on chromosome 5, two on chromosome 12, and three on chromosome 16 . From the genome-wide 7,330,259 SNP markers, 470,229 SNPs on chromosome 5 were extracted and filtered to exclude SNPs with a significant deviation from the 1:1 segregation ratio ( p < 0.01) and low homozygous genotypes (≤40). This resulted in 74,499 SNPs at F ST > 0.01, including 42,473 SNPs at F ST > 0.3061 and 373 SNPs with the highest F ST value (>0.433) in a small genomic region (39.6–42.6 Mb) ( Table S5 ). Similar filtering criteria were applied to chromosomes 12 and 16, resulting in 16 significant SNPs on chromosome 12 and 5 significant SNPs on chromosome 16 ( Table S6 ). Overall, this analysis revealed a ∼3 Mb region (position: 39,634,500 nucleotides [nt]–42,686,436 nt) on chromosome 5 that exhibited the highest divergence in 10-kb windows between the susceptible and resistant RIL groups . The average F ST value of the specific 3 Mb region (0.41 ± 0.004) was significantly greater than that of the neighboring 3 Mb region to the left (0.29 ± 0.004), as determined by a pooled t test . We designated this genomic region or QTL as the B. glabrata schistosome resistance region 1 (BgSRR1). Further analysis was conducted using bin marker-based genetic mapping. 54 , 55 To identify genomic intervals without recombination events in RIL populations, a 10-kb sliding window with Binmarker v.2.3 was used to generate a total of 2,190 bin markers from the 7,330,259 SNPs across the 18 chromosomes ( Table S7 ). The length of the bins was found to be correlated with the number of SNPs per bin (r = 0.91) . On average, the length of a bin was 353,427 bp, and each bin contained an average of 3,347 SNPs . Analysis of the distribution of the genome-wide recombination pattern revealed that the centromeric regions of most chromosomes had significantly fewer recombination events . Figure 4 Bin marker analysis (A) Scatterplot of bin markers, with the x axis indicating the length of the bin markers and the y axis indicating the number of SNPs per bin. Red dashed lines represent the linear trend line. Subplots on the top and right display histograms of bin length and the number of SNPs in each bin marker. Data are represented as mean ± SEM. (B) Distribution of bin markers across the 18 chromosomes. (C) Distribution of genome-wide recombination breakpoints on each of the 18 chromosomes. The red bar shows the relative number of observed recombination crossover sites in the RIL population. To further refine our analysis, we filtered 2,190 bin markers by excluding those with a significant deviation ( p < 0.001) from the 1:1 segregation ratio. The remaining markers were then used to construct a linkage map and conduct QTL analysis. A total of 2,121 bin markers were used to construct the genetic map, resulting in a map distance of 1,311.4 cM, with an average distance of 0.62 cM between adjacent markers . The number of bin markers varied across chromosomes, ranging from 81 on chromosome 8 to 182 on chromosome 11. The largest marker gap was observed on chromosome 1 with a length of 15.47 cM, followed by chromosomes 4, 8, and 12, each with gaps of approximately 13 cM ( Table S8 ). Figure 5 Recombination bin map of the 46 RILs The colors red, green, and blue represent the genotypes of the iM line (AA), the iBS90 (BB), and the heterozygous genotype (AB) and missing data, respectively. P1 and P2 refer to the iM line and iBS90, respectively. QTL analysis indicated that snail resistance is controlled by a major QTL on chromosome 5, located between bin marker BgChr5_39634500:1:1 and BgChr5_39675885:1157240:1117, with a significant logarithm of odds (LOD) score of 6.04 . The bin marker on the left contains only one SNP at position 39,634,500 nt on chromosome 5, while the bin marker on the right encompasses 1,117 SNPs and spans a physical length of 1,157,240 bp. This QTL has an additive effect of 0.35 and accounts for 46.2% of the phenotypic variance. The 95% confidence interval for this QTL ranges from 37.5 cM to 39.5 cM, corresponding to a physical position from 39.6 to 41.9 Mb on chromosome 5, further confirming the 3 Mb region identified as the region of highest F ST divergence . Figure 6 QTL profiling of schistosome resistance across the 18 chromosomes of the snail RILs A major QTL was detected on chromosome 5 between bin markers BgChr5_39634500:1:1 and BgChr5_39675885:1157240:1117, with a significant LOD score of 6.04. A total of 118 protein-coding genes were identified in the BgSRR1, and detailed information about all 118 genes can be found in Table S9 . Among the 118 genes, 73 have homologs with known functions, which are listed in Table 1 (see the discussion section for more information). Table 1 A list of genes in BgSRR1 that encode proteins with homologs No Gene product No Gene product No Gene product 1 cis -aconitate decarboxylase 26 FGGY carbohydrate kinase domain-containing protein 51 trichohyalin-like isoform X1 2 thiosulfate sulfurtransferase 27 ran-binding protein 3 52 splicing factor 3B subunit 5 3 trichohyalin 28 glutathione peroxidase 1 53 TATA box-binding protein-like protein 1 4 WASH complex subunit strumpellin 29 DNA-directed RNA polymerases I, II, and III subunit RPABC1 54 acyl-protein thioesterase 1-like isoform X2 5 exportin-2 30 phosphatidylinositol-glycan-specific phospholipase D 55 NIPA-like protein 2 6 CDK5 and ABL1 enzyme substrate 1-like isoform X1 31 bifunctional polynucleotide phosphatase/kinase 56 transmembrane protein 59 7 apolipophorins-like isoform X1 32 serine-rich adhesin for platelets-like isoform X1 57 D-amino-acid oxidase (2) 8 transmembrane protein 8A-like isoform X1 33 annexin A4 58 kinesin-like protein KIF2A 9 calcium homeostasis endoplasmic reticulum protein 34 protein phosphatase 1 regulatory subunit 7 59 tachykinin-like peptides receptor 99D 10 MAP kinase-interacting serine/threonine-protein kinase 1 35 orexin receptor type 2 (2) 60 BgFReDn19 (4) 11 DNA repair endonuclease XPF-like isoform X1 36 adipocyte plasma membrane-associated protein (4) 61 protein FAM166C A 12 superkiller viralicidic activity 2-like 2 isoform X1 37 E3 ubiquitin-protein ligase HECTD3 62 transcription elongation factor A N-terminal and central domain-containing protein 2-like isoform X1 13 derriere protein 38 ankyrin repeat domain-containing protein 13C (2) 63 mediator of RNA polymerase II transcription subunit 26 14 mRNA export factor 39 ethylmalonyl-CoA decarboxylase-like isoform X2 64 SCL-interrupting locus protein 15 ATP synthase subunit delta mitochondrial 40 transmembrane protein 65 (2) 65 cytidine monophosphate (UMP-CMP) kinase 1 cytosolic 16 diisopropyl-fluorophosphatase 41 zinc-finger protein 451 66 choline transporter-like protein 1 17 acyl-CoA synthetase YngI 42 mediator of DNA damage checkpoint protein 1 67 high-affinity cAMP-specific 3p 5′-cyclic phosphodiesterase 7A 18 DNA-directed RNA polymerase I subunit RPA1 43 defense protein 3 (3) 68 mitochondrial fission regulator 2 19 N-acetyltransferase ESCO1 44 ferric-chelate reductase 1 69 pikachurin 20 ATP-dependent RNA helicase DDX43 45 DNA excision repair protein ERCC-6-like 2 70 histone H2A 21 chromatin modification-related protein EAF7 46 TBC1 domain family member 2B (3) 71 THAP domain-containing protein 6 22 stromal membrane-associated protein 1-like isoform X1 47 leucine-rich repeat-containing protein 72 galectin-4 23 zinc-finger SWIM domain-containing protein 5-like isoform X1 48 protein phosphatase 1 regulatory subunit 7 73 eyes absent 4 24 GTP-binding protein Di-Ras2 49 peroxidasin – – 25 HMG box-containing protein 4 50 regulator of telomere elongation helicase 1 isoform X1 – – Note: The number in parentheses at the end of a gene name is the total number of the genes from the same gene family (with the same gene name) in the BgSRR1. Gene Ontology (GO) analysis shows enriched biological processes, molecular functions, cellular components, and pathways . Three significant Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with double-stranded RNA binding, protein heterodimerization activity, and basal transcription factors, along with 68 enriched GO categories, were revealed. These GO categories comprise 21 biological processes, 15 cellular components, and 32 molecular functions ( Table S10 ). The most highly enriched categories include double-stranded RNA binding for molecular function, nucleotide excision repair for biological processes, and three cellular components (WASH complex, endodeoxyribonuclease complex, and histone deacetylase complex). The functional linkage network analysis revealed the top 10 GO enrichments, including GO:0005634 nucleus, GO:0034654 nucleobase-containing compound biosynthetic process, GO:0019438 aromatic compound biosynthetic process, GO:0018130 heterocycle biosynthetic process, GO:1901362 organic cyclic compound biosynthetic process, GO:0097659 nucleic acid-templated transcription, GO:0006351 transcription DNA-templated, GO:0032774 RNA biosynthetic process, GO:0006139 nucleobase-containing compound metabolic process, and GO:0044271 cellular nitrogen compound biosynthetic process . Figure 7 GO and KEGG pathway analyses (A–D) show biological processes, molecular functions, cellular components, and the top 20 pathways, respectively. (E) The hierarchical clustering tree summarizes the correlation among the top 20 significant pathways. Pathways with many shared genes are clustered together. Larger dots indicate more significant p values. (F) The interactive network plot shows the relationship between the top 10 enriched pathways. Two pathways (nodes) are connected if they share 20% (default) or more genes. Darker nodes represent more significantly enriched gene sets. Larger nodes represent larger gene sets. Thicker edges represent more overlapping genes. A thorough understanding of the fundamental mechanisms that control the traits of interest is crucial for the development of genetically modified organisms for biomedical research. Extensive genetic analyses have been conducted on anti-parasite traits in disease vectors such as mosquitoes, 56 , 57 , 58 , 59 , 60 leading to active studies on genomic modifications for both basic and applied research. 38 , 39 , 40 However, limited progress has been made with schistosomiasis vector snails, hindering our ability to pursue similar innovative work for schistosomiasis control. Different from the relevant genetic studies conducted on vector snails or other mollusks, the current study is based on our long-term efforts to develop genetic resources (the homozygous iM line and iBS90, F2 segregating population, and RILs) and genomic resources (scaffold- and chromosome-level assembled genomes) for the schistosomiasis model snail B. glabrata , with the main objective of deciphering the mechanisms underlying snail resistance to schistosomes. 45 , 52 The development of RIL snails represents one of our efforts in this direction, despite the painstaking nature of the work involved. RIL snails were produced using an advanced design. The RILs were obtained through two crosses: a parental outcross and an F2 intercross, followed by 10 generations of selfing . This design differs from the standard RIL design, which involves 6–7 generations of inbreeding starting with F2 offspring resulting from a single outcross (parental cross). 49 , 51 , 61 The increased intercrossing, along with a greater number of generations of inbreeding (selfing), should further enhance mapping resolution and reduce the size of QTL by accumulating additional meiotic crossover events. Indeed, the resistance QTL size was reduced from approximately 8 Mb using the F2 population to about 3 Mb, as revealed by the current RIL population. Importantly, our current findings from two genetic analyses, GWAS and bin marker-based QTL, unequivocally confirm the 3 Mb resistance locus BgSRR1 and its genomic location on chromosome 5, which are also consistent with our previous F2-ddRADseq mapping 45 (see further discussion in the following). We carefully determined the resistance phenotype of each RI line, as it is important for genetic mapping. In our genetic studies, resistance or susceptibility was defined based on cercarial shedding rather than snail infection. Cercarial shedding directly contributes to disease transmission and human infection. After exposure to schistosome miracidia, many snails become infected or are penetrated by the miracidia, but not all infected snails shed cercariae. Some parasites experience impeded development but still survive in the snail host for a long time, resulting in no cercariae being released from these hosts. This phenomenon was observed in our recent work, which showed that some resistant snails (without shedding cercariae) possessed a varying number of schistosome reads from DNA extracted from the entire snail body. 45 The laboratory-based finding was confirmed by field observations. A large-scale polymerase chain reaction (PCR)-based surveillance program in coastal Kenya demonstrated that the rates of schistosomes present in snail hosts, as detected by PCR, were significantly higher than those observed through cercarial shedding (28%–54% vs. 0.14%–3.4%). 62 If a snail does not shed cercariae, it plays no role in disease transmission, regardless of whether it is infected. As our goal is to apply our findings to field applications, the focus of our investigations is on the phenotype of cercarial shedding rather than on infections. We employed WGS as a genotyping assay to sequence the genomes of individual RIL snails instead of using pooled DNA samples from multiple snails with the same phenotype (Pool-seq). For each RIL, deep genome sequencing (∼16X coverage) was conducted on a single RIL snail to represent the corresponding RIL for genetic mapping, as all individuals within the same RIL are nearly genetically identical. 49 , 50 , 51 Although this approach is more costly and labor intensive compared to Pool-seq, it provides data that can be used for accurate analyses or re-analyses. As a result, we were able to identify a large number of SNPs ( n = 7,330,259) and bin markers ( n = 2,190) across the 46 RIL genomes. The genetic mapping conducted with the current linkage map, which has denser markers compared to the F2-based linkage map (0.62 cM vs. 1.73 cM), has revealed a smaller QTL size. Subsequent GWAS and QTL analysis both indicated that the snail B. glabrata has a ∼3 Mb BgSRR1 on chromosome 5. This genomic region shows a peak F ST value across chromosome 5 . Linkage mapping analysis of this region reveals only three recombination points, identifying a large haplotype block spanning approximately 1.16 MB and containing 1,117 SNPs. BgSRR1 identified by the current RIL-WGS approach agrees with our previous findings using the F2-ddRADseq analysis (i.e., the resistance locus on chromosome 5). 45 In addition to chromosome 5, SNPs linked to resistance were also detected on chromosomes 4, 12, 15, 16, and 18, despite the limited number of SNPs. These chromosomes, especially chromosomes 12 and 16, deserve attention in future studies. We cannot exclude the possibility that loci on these chromosomes may have an effect or a minor effect on resistance. To compare our current findings with previous reports, we mapped QTLs reported by other laboratories 41 , 42 , 43 , 44 , 46 to the 18 chromosomes based on our chromosome-level assembly of B. glabrata . 52 Surprisingly, the chromosomes containing QTLs identified by other research groups differ from chromosome 5 and from those with a limited number of significant SNPs (i.e., chromosomes 4, 12, 15, 16, and 18) . Please note that the chromosome numbers (i.e., the order from 1 to 18) in Figure 8 from Zhong et al. 52 are not exactly the same as the linkage group numbers in Figure 7 of the paper published by Bu. et al.. 45 Therefore, the genes identified by other groups, including a cluster of genes encoding transmembrane proteins, 41 , 42 , 43 , 44 , 46 are not present in our BgSRR1. The reason for the discrepancy between our findings—both previous and current—and those reported by others is still unknown. Figure 8 Distribution of QTLs reported on the 18 chromosomes based on the chromosome-level assembled genome of iM line B. glabrata The previously reported QTLs, including OPM-0423, 41 GRC, 42 RADres and SOD, 43 PTC2, 44 , 46 and qRS2.1 and qRS5.1, 45 are indicated by solid red bars. The pink star marks the chromosomal location of BgSRR1. The RAPD marker OPZ-11 41 is not indicated due to its repetitive sequence. Although the GO analysis of the protein-coding genes in BgSRR1 was conducted, caution is warranted in interpreting the findings because about one-third of the genes in the region lack homologs with known functions and could not be included in the GO analysis. Nonetheless, the analysis offers useful insights into the mechanisms of schistosome resistance in snails. Some previously unrecognized pathways may be involved in the defense responses. For example, the enriched GO categories include many genes and pathways related to metabolism. Immunometabolism has recently emerged as a dynamic field in immunology but has not yet been explored in snail immunology or host-parasite interactions. 63 , 64 , 65 Focusing on genes with known functions, we are excited to find that BgSRR1 possesses genes known to be involved in cellular immunity in snails. The presence of the mitogen-activated protein kinase (MAPK) gene in BgSRR1 is supported by earlier studies suggesting that MAPK-based signal transduction plays a critical role in hemocyte-mediated encapsulation and H 2 O 2 production, leading to the killing of intramolluscan schistosomes. 66 , 67 , 68 Interestingly, two genes from the peroxidase gene family, glutathione peroxidase and peroxidase , have been identified in BgSRR1. Peroxidases are antioxidative enzymes that scavenge H 2 O 2 and inhibit apoptosis. 69 An earlier study also revealed that a thioredoxin peroxidase or peroxiredoxin gene was highly expressed in resistant B. glabrata snails compared to susceptible ones in response to schistosome infection. 70 Additionally, BgSRR1 contains a gene encoding the enzyme thiosulfate sulfurtransferase, which may also have antioxidative properties. 71 These findings suggest that BgSRR1 is involved in regulating cell-mediated immunity, particularly in relation to redox balance. BgSRR1, however, does not contain genes previously reported to play a significant role in humoral immunity in B. glabrata , such as biomphalysin , 72 fibrinogen-related proteins ( FREPs ), 73 , 74 , 75 , 76 , 77 macrophage migration inhibitory factor genes, 78 and genes encoding proteins associated with the Toll-like receptor pathway. 79 , 80 Notably, we have identified other humoral immune genes in BgSRR1, including apolipophorin , 81 defense proteins , 82 RNA helicase , 83 and E3 ubiquitin protein ligase genes, 84 although the roles of these genes in schistosome resistance have not been reported in snails. It is worth mentioning that BgSRR1 contains four ficolin genes (only fibrinogen [FBG]) but none of the FREPs . Ficolins, which are important players in innate immunity, have been extensively studied in the mosquito- Plasmodium model, 85 , 86 , 87 but not in the snail-schistosome system. We have observed genes in BgSRR1 that encode enzymes involved in immune cell metabolism. For example, the enzyme cis -aconitate decarboxylase, encoded by immune response gene 1 , produces itaconate, an intermediate metabolite from the tricarboxylic acid cycle in immune cells. 88 Recent studies have demonstrated that itaconate is an important immunometabolite that regulates host defense and inflammation. 89 , 90 The potential role of immunometabolism in defense is also suggested in the GO analyses discussed earlier. Future investigations into the functions of these genes in BgSRR1, especially those not yet studied in B. glabrata , may reveal unexpected aspects of snail-parasite interactions, provide valuable insights into snail defenses, and help identify key resistant genes. In conclusion, our approach (RIL-WGS), combined with our RIL genetic resource, powerful genome-wide genotyping, GWAS, and bin marker-assisted QTL analysis, has enabled the identification of the BgSRR1 on chromosome 5 of B. glabrata , an important molluscan vector of human schistosomiasis. The identification of BgSRR1 and the genes conferring schistosome resistance has the potential to advance our understanding of host-parasite interactions and facilitate the development of snail-targeted biocontrol strategies for schistosomiasis, a parasitic disease that infects 251 million people worldwide. 91 While our current findings are robust and supported by a well-developed genetic resource, reliable phenotype and genotype data, and multiple genetic analyses, it is important to acknowledge their limitations, which are common in genetic studies. Firstly, the use of RILs limits the ability to capture information regarding dominance due to their high homozygosity. 92 In fact, our RIL-WGS approach did not detect the presence of a susceptibility-associated QTL under the dominance effect on chromosome 2. 45 Secondly, it should be recognized that our findings are based on a well-developed laboratory system. Therefore, further evaluation and testing of our findings in other systems, particularly in field settings, are necessary. REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins CTAB solution Teknova Lot no: C219009G1801 Chloroform: Isoamyl alcohol Sigma-Aldrich Lot no: 1003577830 Proteinase K TermoScientific Lot no: 10198999 RNase A TermoScientific Lot no: 2653498 Isopropyl alcohol Honeywell Lot no: CZ999 200 proof pure ethanol KOPTEC Lot no: A08232309D Deposited data Illumina sequence data National Center for Biotechnology Information (NCBI) ( https://www.ncbi.nlm.nih.gov ) BioProject ID: PRJNA1133633. BioSample accession numbers: SAMN42382410-SAMN42382455. Experimental models: Organisms/strains iM line of Biomphalaria glabrata University of New Mexico, USA Si-Ming Zhang iBS90 of Biomphalaria glabrata University of New Mexico, USA Si-Ming Zhang RILs of Biomphalaria glabrata University of New Mexico, USA Si-Ming Zhang PR1 strain of Schistosoma mansoni Biomedical Research Institute in Maryland, USA Margaret Mentink-Kane Software and algorithms Trimmomatic 3.9 Bolger et al. 93 https://github.com/timflutre/trimmomatic CLC Genomics Workbench 23 QIAGEN Aarhus, Denmark https://digitalinsights.qiagen.com/ bcftools Danecek et al. 94 https://samtools.github.io/bcftools/bcftools.html vcftools Danecek et al. 95 https://vcftools.github.io/index.html PLINK 1.9 Purcell et al. 96 https://www.cog-genomics.org/plink/ JMP 14 SAS Institute Inc., Cary, NC https://www.jmp.com/en_us/home.html Binmarkers-v2 Qin et al. 97 https://github.com/lileiting/Binmarkers-v2 IciMapping Meng et al. 98 http://www.isbreeding.net ShinyGO Ge et al. 99 https://bioinformatics.sdstate.edu/go/ Other A scaffold-level assembled genome of iM line and iBS90 of Biomphalaria glabrata Bu et al. 45 https://doi.org/10.1038/s42003-022-03844-5 A chromosome-level assembled genome sequence of Biomphalaria glabrata Zhong et al. 52 https://doi.org/10.1038/s42003-022-03844-5 The snail Biomphalaria glabrata , a major intermediate host of human schistosomiasis in Neotropical countries, was used for this study. The efforts to generate B. glabrata recombinant inbred lines (RILs) are described in the results section. Breeding, cultivation, and storage of the RIL snails were conducted at the Center for Evolutionary and Theoretical Immunology (CETI), University of New Mexico (UNM), United States. The generation of recombinant inbred (RI) lines (RILs) is also described in the results section. The PR1 strain of Schistosoma mansoni used to infect the snails was collected from the Biomedical Research Institute in Maryland, USA ( https://www.afbr-bri.org ). To determine the phenotype of the RI lines, 6–8 juvenile snails (0.3–0.6 mm shell diameter) were randomly chosen from each RI line for infection. The snails were individually placed into the wells of a 24-well cell culture plate (one snail per well) and 20 schistosome miracidia were added to each well. The snails were fully submerged in water overnight to ensure complete exposure to the miracidia. Afterward, the exposed snails were transferred to large tanks for continued culturing until cercarial shedding was performed. Examination of the phenotype began at 45 days post-exposure (dpe). The exposed snails were placed individually in the wells of a 24-well plate and exposed to light for 0.5 h (hr). Snails that shed cercariae were considered susceptible to schistosomes, while those that did not shed cercariae were transferred to the aquatic tank and cultured for later examination of shedding. If a snail did not shed cercariae at 60 dpe, it was classified as a resistant snail. This procedure allowed us to determine the phenotype of each RI line. Once the phenotype was determined, the remaining snails from each RI line (those not exposed to schistosomes) were preserved in liquid nitrogen for DNA extraction. Only the RI lines that exhibited the same phenotype in all tested individuals were selected for genetic mapping. A single snail thawed from liquid nitrogen was placed into a 1.5 mL tube and ground in 750 μL of CTAB buffer. 100 After homogenization, 20 μL of proteinase K (20 μg/μL) was added to the homogenate and incubated at 60°C for 1 h (hr). Next, 750 μL of chloroform: isoamyl alcohol (24 : 1) was added and rocked for 0.5 h. Following centrifugation, the supernatant was transferred to a new tube. To degrade and remove RNA, 10 μL of RNase (10 μg/μL) was added to the new tube and incubated at 37°C for 0.5 h. An equal volume (750 μL) of chloroform: isoamyl alcohol was added to the solution and rocked at room temperature for 10 min (min). Genomic DNA was precipitated using isopropyl alcohol, washed with 70% ethanol, and dissolved in nuclease-free water. The genomic DNA was qualified and quantified using agarose gel electrophoresis (1%) and the Qubit 2.0 DNA HS Assay (ThermoFisher), respectively. For library preparation, the KAPA Hyper Prep kit (Roche) was used. In brief, the genomic DNA was sheared into 500 bp fragments using the Covaris LE220-plus. After ligating the adapters, the fragments were amplified by PCR. The quantity and quality of the libraries were assessed using the Qubit 2.0 DNA HS Assay, the Tapestation High Sensitivity D1000 Assay (Agilent Technologies), and the QuantStudio 5 System (Applied Biosystems). Finally, the libraries were sequenced using an Illumina NovaSeq S4. The raw Illumina reads for the two parental lines (iM line and iBS90) were retrieved from GenBank . 45 It is important to note that all Illumina data, including those from the two parental snails and the RIL snails were generated from the same Illumina platform (Admera Health; www.admerahealth.com ) and the same quality control was applied to all samples. All raw reads were trimmed and cleaned using Trimmomatic v0.39 93 with the following parameters: ‘ILLUMINACLIP: TruSeq3-PE-2.fa: 2:30:10 HEADCROP:7 LEADING: 25 TRAILING: 25 SLIDINGWINDOW: 4:25 MINLEN: 36’. The clean reads from each RIL sample were individually aligned to the reference genome of B. glabrata 52 using the Map Reads to Reference tool in the QIAGEN CLC Genomics Workbench 23 (Qiagen Genomics, Denmark) with the default parameters. SNP calling for the iM line and iBS90 was conducted according to the methods described previously. 45 The Identify Known Mutations from Mappings tool of the CLC workbench was used to genotype the RILs individually at SNP loci that were polymorphic in the two parental snails. Subsequently, the SNPs identified from the CLC were exported individually to VCF files and then merged into a single VCF file using bcftools. 94 Finally, the resulting VCF file was filtered using vcftools 95 with the following parameters: ‘--minDP 5; --maxDP 100; --maf 0.1; --max-missing 0.8'. Single SNP genotype and phenotype association analyses were conducted using PLINK software. 96 To identify significant associations, we applied the widely accepted threshold of p < 5 × 10 −8, 101 which is derived from a Bonferroni correction for all independent SNPs in the genome. To measure the divergence between susceptible and resistant populations, we calculated the fixation index (F ST ) using VCF tools. We used a sliding window of 10 kb, with an increment of 5 kb, to perform this calculation. Significant high F ST outliers were identified based on the 95th quantile from the genome-wide distribution and jackknife procedure. Outlier analysis was conducted using the Jackknife Distances in SAS JMP 14. This involved calculating pairwise distances between data points, resampling the data by removing one point at a time, and analyzing the variance of the resulting distances. Points with unusually high variance were flagged as potential outliers and further validated. To identify genomic intervals in a mapping population with no recombination events, we used the Binmarker-v2.3 tool ( https://github.com/lileiting/Binmarkers-v2 ). 97 This tool employs a sliding window approach of 10 kb to generate genetic bin markers. Missed genotypes were imputed and miscoded genotypes were corrected using strict criteria: a genotype that differed from surrounding genotypes, no missing data in surrounding genotypes, and identical surrounding genotypes. Next, markers with 100% identical markers were merged together. These bin markers were then organized based on the physical position of the chromosome. A change in genotyping within any sample was considered a recombination breakpoint. SNPs between recombination breakpoints were classified as bin markers, indicating that no recombination occurred within that bin. Bin markers showing significant deviation ( p < 0.001) from the 1:1 segregation ratio were excluded from constructing the linkage map. Heterozygous genotypes were treated as missing data and imputed using the "maxmarginal" method implemented in the R/qtl package. Linkage map construction and QTL analysis were conducted using QTL IciMapping version 4.2.53. 98 Simple interval mapping and inclusive composite interval mapping were employed to detect potential QTLs associated with snail resistance or susceptibility to schistosome parasites. A significant threshold of the logarithm of odds (LOD) (LOD = 4.0) based on 1,000 permutation tests was applied. The coding genes were further verified manually by BLAST searching against NCBI databases. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analyses were performed using the web-based tool ShinyGO 0.80 ( http://bioinformatics.sdstate.edu/go/ ). 99 A flowchart showing bioinformatic and genetic analyses is provided in Figure S1 . PLINK was employed for genome-wide association studies (GWASs) with a stringent p -value threshold of 5 × 10 −8 to identify significant associations. Vcftools was used to calculate fixation index (F ST ) statistics, and outlier analysis was performed using Jackknife Distances in SAS JMP 14, with a p -value threshold of <0.05 for significance. A permutation test with 1,000 iterations and a type I error rate of 0.05 was used to establish the significance threshold for QTL LOD scores. Functional enrichment analysis, including GO and KEGG pathways, was conducted with an E-value threshold of < 1e-5. Significant GO term enrichment was assessed using Fisher’s exact test, applying a p -value threshold of <0.05. These analyses identified genetic variants associated with snail resistance and explored their functional implications. | Review | biomedical | en | 0.999995 |
PMC11699760 | Xylazine—often referred to as “tranq” or “ anastesia de caballo ” in Spanish—is a α2-adrenergic receptor agonist, used for sedation in veterinary contexts [ 1 – 3 ]. Unlike other α2 agonists, such as clonidine or dexmedetomidine, xylazine has not been approved for human consumption in the United States (US) or other jurisdictions . Xylazine appeared in the illicit drug market of Puerto Rico in the early 2000s where it was used to augment the effects of heroin . It subsequently emerged as an additive to illicit fentanyl in mainland US in the late 2010s and early 2020s . The prevalence of xylazine among fatal overdoses has risen exponentially in subsequent years [ 7 – 9 ], however its causal role in overdose events remains a matter of scientific discussion . Qualitative data suggest that xylazine may increase the complexity and difficulty of life-saving treatment during overdoses . The arrival of xylazine-fentanyl co-use to the North American overdose crisis has been highly notable, and xylazine has been identified as an emerging threat by the government of the US , Chile , by the Organization of American States , and more recently by Mexico . However, the scope of the problem in the Mexican context deserves further study, as robust epidemiological data are not yet available. Preliminary evidence suggest that xylazine may be found in high concentration among fentanyl samples in Tijuana . Additionally, little information is available in the literature about the clinical management of xylazine-involved overdoses, especially in community harm reduction spaces. Here we present a series of clinical cases of xylazine-fentanyl overdose, which exemplify the clinical approach developed, and the lessons learned, by an overdose response team at Prevencasa, a community harm reduction clinic in Tijuana, Baja California, Mexico. The patients whose cases are featured here provided written consent. Prevencasa serves a large population of people who inject and smoke illicit opioids, many of whom have been deported from the US. The clinic has more than a decade of experience responding to overdose events in the surrounding neighborhood of Zona Norte (which concentrates a large number of illicit drug sales points and open-air drug use). Although overdose events were initially sporadic, they became increasingly common with the arrival of illicit fentanyl to Tijuana . In response, the clinic trained an overdose response team, with designated clinical members (usually one physician, one nurse, and one or more harm reduction staff members) equipped with naloxone and other supplies. All team members were trained in how to assess for the clinical signs and symptoms of opioid overdose, as well as how to administer naloxone administration. MD and RN team members were additionally trained in how to rapidly assess key vital signs, especially using pulse oximetry, and in the administration of oxygen and other life-saving measures. A 61-year-old man with opioid and methamphetamine use disorder, well-known to the clinic, who uses a wheelchair due to a complete right lower limb amputation, was found lying face first on the ground, across the street from the harm reduction clinic, bleeding from a head laceration . His respiratory rate was imperceptible, he was cyanotic, and his pupils were pinpoint. A nurse administered a 4.0 mg intranasal dose of naloxone, and the patient quickly resumed visibly respirating, however, he did not regain consciousness. Pulse oximetry showed the patient saturating at 96%, with a pulse of 95 bpm, and auscultation revealed normal breath sounds. The patient remained unarousable for approximately 20 min, during which time the clinical team monitored his oxygenation status, breath sounds, and pulses. He subsequently awoke suddenly in a delirious state and became agitated, endorsing distressing withdrawal symptoms. He pulled himself into his wheelchair and wheeled himself into traffic in a nearby roadway, exposing himself to considerable physical risk. There he once again lost consciousness, falling backwards onto the curb. Several community members placed him back into his wheelchair, and brought him into the clinic, where he accepted post-overdose monitoring. His vitals remained stable for the next two hours. Urine screening revealed positivity for xylazine, fentanyl, and methamphetamine. The patient reported a two-decade history of opioid use disorder, and endorsed currently both smoking and injecting ‘China White’ (a fentanyl powder formulation that increasingly contains xylazine in Tijuana) mixed with methamphetamine every two hours. The patient provided a drug sample to the on-site drug checking program, as well as a urine sample. Both tested positive for xylazine, using two different brands of immunoassay qualitative testing strips, as well as fentanyl. The patient received counseling about the risks of xylazine and fentanyl. A 28-year-old man with long-standing methamphetamine use disorder and no other known medical history was found unconscious, in a supine position, on the sidewalk, a few meters from the harm reduction clinic. Community members on scene reported that they observed him smoking a substance from a glass pipe prior to losing consciousness. He was not arousable, and pulse oximetry showed an oxygen saturation of 81% and a heart rate of 92 beats per minute. His blood pressure and blood glucose were within normal limits. He responded to verbal and physical stimuli only by opening his eyes but was not verbally responsive. On several occasions he attempted to sit up but was too sedated to do so. The overdose response team delivered oxygen through nasal cannula via a portable oxygenation concentrator. The patient’s oxygenation saturation immediately responded by rising to 100%. The patient was brought into the clinic for vital sign monitoring and supplemental oxygen. The response team opted against naloxone administration given that his vital signs stabilized with supplemental oxygen. After 40 min the patient awoke calmly, was alert and oriented to self, place, and date, and was informed about the process of his care, as well as the possible exposure to xylazine. He endorsed having used the street opioid mixture “China White” in a recreational fashion, although typically he only consumes stimulants, and it is unusual for him to consume opioids. He denied having used benzodiazepines, alcohol or other psychoactive substances. The patient declined urine or substance testing or any further medical care and left the clinic. He was seen one week later for follow-up care and was found in stable condition and received counseling. A 36-year-old man with long-standing opioid use disorder was found sitting on a curb, leaning back onto a car, very close to the harm reduction clinic, his eyes were covered by a hat. He was sitting among a group of various community members who were smoking and/or injecting illicit opioids. He did not respond to verbal cues, which prompted a member of the overdose response team to attempt to arouse him with touch, to which he also did not respond. The overdose response team member noted that the patient’s extremities were stiff, cold, and diaphoretic. The rest of the response team was summoned, the patient was laid flat on the ground, and was noted to be cyanotic, with blue lips and nails. Pulse oximetry revealed an oxygen saturation of 20%. A dose of intramuscular naloxone of 0.4 mg was administered, and within the next 2 min, oxygen saturation reached 99%, although the patient remained unresponsive to verbal and physical stimulus. The patient was placed on his side in recovery position, vital signs were assessed for the next 12 min, at which time he began to move his arms and legs in an agitated fashion, with his eyes remaining closed. Shortly thereafter he opened his eyes, was fully oriented, and was able to follow verbal guidance. He accepted post-overdose care in the clinic and offered a sample from the syringe containing the substance he had consumed prior to overdose, still loaded with 0.2 ml of prepared “China White”. The sample tested positive for fentanyl and xylazine. The patient’s urine tested positive for fentanyl, xylazine and methamphetamine. The patient received counseling about the risks of xylazine when combined with fentanyl. These three clinical cases highlight a range of typical scenarios of xylazine-involved overdose seen by the overdose response team at the Prevencasa community harm reduction clinic in Tijuana, Mexico. In the early months of 2024, due to the arrival of xylazine, a very notable shift in the clinical characteristics of overdose events was observed, which required the overdose response team to adapt its practices. Patients who would have previously quickly regained consciousness after a 4.0 mg dose of intranasal naloxone began to exhibit 10–30-minute periods of unconsciousness, despite adequate oxygenation as measured with pulse oximetry. Patients increasingly began to awaken in a confused or delirious state, exhibiting aggression, and often would expose themselves to physically dangerous circumstances (e.g. walking in traffic, laying on roadways) appearing to be unaware of their surroundings, significantly complicating field management. During this time, routine urine toxicology was offered to patients, as was direct testing of substances and drug wrappers, all of which showed high rates of xylazine positivity, using several brands of lateral flow immunoassay testing strips . In response, the clinical team developed a set of strategies to manage xylazine-involved overdoses (Table 1 ). One valuable lesson was the insight to carefully titrate—and at times even reduce or withhold entirely—the dose of naloxone provided to overdose patients. We hypothesized that local sales points decreased the concentration of fentanyl provided to consumers, replacing it with xylazine. Therefore, 4.0 mg intranasal doses of naloxone seemed to induce more problematic withdrawal symptoms in patients than prior to the arrival of xylazine, as seen in case 1. Affected individuals increasingly would awaken expressing concern about physically uncomfortable withdrawal symptoms, which often prompted an immediate search for repeat illicit opioid consumption, and complicated post-overdose clinical monitoring. Instead, the use of smaller intramuscular doses (such as in case 3), titrated to adequate oxygenation—not consciousness—provides more stable patient care and better outcomes. In urgent overdose response situations, there is a pressing temptation to administer additional naloxone to unconscious patients. However, the non-opioid sedation period induced by xylazine requires re-centering the focus on oxygenation and respiratory status, not consciousness. The team has therefore increased its use of field pulse oximetry as the key clinical parameter of interest, re-doubled efforts to focus on field airway management, including proper positioning, and the use of mobile oxygen tanks. In some instances, perhaps when xylazine is the predominant component driving sedation, adequate oxygenation status can be achieved without naloxone, and supplemental oxygen and airway management are the key techniques employed (such as in case 2 above). The assessment of respiratory status has therefore become a key priority for xylazine overdose management. Although community naloxone distribution has become common, and overdoses involving only opioids are often managed in practice by individuals who lack medical training, the prolonged period of sedation with potential airway compromise induced by xylazine may require specialized knowledge to manage. Community-based training in airway management has been suggested as a key response tactic to xylazine among harm reduction practitioners to solve this challenge . We have also provided xylazine testing strips to patients, and syringe exchange clients, and provided education to the local population about the risks of xylazine. Xylazine testing strips are a relatively new introduction to the market, and are currently approved for forensic purposes, including directly testing substances. However they have been demonstrated, in practice, to work on urine samples . One concern is that the thresholds used in commercially available xylazine strips may be too low, resulting in a risk of false negative results . However, strips can also be given to participants to directly test their own drug supply before consumption. Many individuals seeking illicit opioids do not wish to consume xylazine—especially once they are aware of its associated health risks . Providing participants with xylazine testing strips—coupled with adequate counseling from medical providers—can therefore empower them to shift their demand towards products with fewer health risks. This can also serve as a segue into a helpful conversation between patient and provider about harm reduction in the midst of active substance use, that can often lead to engagement in opioid substitution treatment. Collectively these strategies have helped the clinical team manage xylazine-fentanyl overdoses in the field and educate the patient population about evolving health risks associated with shifts in the illicit drug supply. However, more research is needed about the clinical management of xylazine-involved overdoses, especially in community clinics relevant broadly to harm reduction contexts. The techniques we propose here should be validated with more formal clinical studies. Beyond the current response to xylazine-fentanyl combinations , many of these techniques may also apply broadly to the evolving polysubstance crisis unfolding in North America , which increasingly also involves nitazenes, novel synthetic benzodiazepines, and other synthetic sedatives mixed with fentanyls. Across these drug categories, naloxone-resistant sedation and field airway management may be core issues. Table 1 Strategies to manage xylazine-fentanyl overdose in the field Strategy Description Field Pulse Oximetry Portable, battery-powered pulse oximetry can be used in the field to monitor oxygenation status in patients who have not regained consciousness. Titrate Naloxone to Oxygenation Status Naloxone administration, either intramuscular or intranasal, can be titrated to oxygenation status, not level of consciousness, to avoid inducing unnecessary opioid withdrawal symptoms after the xylazine sedation period passes. Portable Oxygen and Airway Management Portable oxygen tanks, and proper positioning of patients (laying on their side, jaw thrust, visualize airway) can assist in improving oxygenation after naloxone administration, assess for vomit and gastric contents, employ portable suction as needed. Scene Safety Patients with prolonged periods of sedation are vulnerable to traffic, assault, and other physical risks. Response team staff members can help secure the scene. Distribute Xylazine Testing Supplies Testing strips for xylazine presence using immunoassay technology are inexpensive (about $1 per strip) and easy to use. They can be distributed to participants to assist them in screening their drugs. In practice many patients show positivity in urine samples, although the strips have not yet been FDA approved for this purpose, and false negatives may occur. Communnity Education Many patients prefer to avoid xylazine once they are aware of it, understand the health risks, and are equipped with testing strips to detect it. Providers can counsel patients about the risks of xylazine and suggest safer options. This can help encourage patients to switch to methadone or buprenorphine treatment. | Study | biomedical | en | 0.999997 |
PMC11699763 | Ceramic restorations merge between excellent biocompatibility and optimal optical and material properties, satisfying both patient and clinical demands [ 7 – 9 ], which is indicated for veneers, inlays, onlays, crowns, and FPDs [ 7 – 11 ]. Glass-ceramics, including lithium disilicate and zirconia-reinforced lithium silicate, are aesthetically appealing restorative materials fabricated with computer-aided design (CAD) computer-aided manufacturing (CAM) technologies. They were designed to replace metal-alloy frameworks, providing benefits in optical, physical, and biological properties . The durability of ceramic restorations primarily depends on the quality of bonding and the proper application technique. Cementation enhances fracture resistance by filling the irregularities on the etched fitting surface of the restoration, thereby enhancing bonding strength and preventing crack propagation especially the glass ceramics . Maintaining fracture resistance is essential for ensuring the effectiveness of ceramic restorations. It is influenced by preparation design, ceramic material properties, restoration thickness, cementation technique, functional load, and internal ceramic defects . Preparation designs, including cavity depth, width of the isthmus, the degree of taper, and the internal line angles, can influence fracture resistance. Additionally, the aging of the ceramic restoration/tooth complex may affect the failure rate . The marginal fit of restoration is a critical element that affects the longevity of overlay restorations. Poor adaptation can create gaps between the restoration and tooth, leading to microleakage, plaque buildup, and potential recurrence of decay . It’s worth mentioning that only a limited number of studies have investigated the impact of the design of preparation on the characteristics of overlay restorations, with most of these focusing mainly on the fracture resistance of the restorations [ 1 , 3 , 19 – 22 ]. Most of the previous pertinent research has assessed inlays and/or onlays that provide partial cusp coverage . An information gap exists concerning the impact of preparation configuration on the marginal fit of ceramic overlays. Therefore, this research aimed to examine the marginal gap and fracture resistance of Advanced Zirconia-reinforced Lithium disilicate (ALD) overlays with various preparation designs. The null hypothesis for this study stated that there is no significant difference in marginal fit (before or after thermal aging) or fracture resistance among the studied groups. The sample size was estimated assuming a 5% alpha error and 80% study power. Based on the difference between independent means of previous study , the minimum sample size was calculated to be 7 samples per group, increased to 9 samples to make up for processing errors. Total sample = number per group x number of groups = 9 × 3 = 27 samples. Group (O) a traditional overlay preparation with anatomical occlusal reduction 1.5 mm. Group (OS) an overlay preparation with anatomical occlusal reduction 1.5 mm and 1.0 mm shoulder finish line circumferentially. Group (OG) an overlay with anatomical occlusal reduction 1.5 mm and central groove preparation with a pulpal depth of 1.0 mm and a width of 2.0 mm. The typodont teeth that had been prepared were subjected to scanning using an extraoral scanner (InEos X5; Dentsply Sirona, USA), to design three dies corresponding to the tooth preparations. Print out of twenty-seven dies made from 3D printing resin (Model Resin, Formlabs, Somerville, MA), which possess a comparable modulus of elasticity to dentin (modulus = 10 GPa). A laboratory printer (FormLab 2, Formlabs) was used . Each resin die was subsequently embedded in a copper metallic mold filled with auto-polymerizing acrylic resin (Acrostone, Egypt), exposing only the crown and 2 mm apical to the cementoenamel junction, simulating the bone level. A computer-aided design (CAD) digital software (inLab CAD SW 22.0.0; Dentsply Sirona) was used to design the restorations with a standardized morphology [Fig. 1 ] . Twenty-seven overlays were fabricated using CAM technology (inLab MCXL, Dentsply Sirona) using Advanced Zirconia-reinforced lithium disilicate CAD/CAM blocks (Dentsply Sirona, Tessera ™). A 9.5% hydrofluoric acid (Porcelain Etch ® , Bisco, USA) was applied on the dry intaglio surface of the restorations for 20 s, rinsed with a copious amount of water for 60 s, and air dried. Silane (Porcelain Primer; Bisco, Schaumburg, IL, USA) was applied on the etched restorations surface and air dried after 60 s. The overlays were then cemented to the prepared dies with a dual-cure resin cement following the manufacturer’s instructions (BisCem; Bisco, Schaumburg, IL, USA). Cement was auto-mixed and applied on both the intaglio surface of the restoration, and the prepared die surface, each restoration loaded with cement was gently seated on its corresponding prepared die, initially with finger pressure followed by application of a constant static load of 200 g , by a specially designed device to ensure seating of the restoration for 60 s before photopolymerization. Then specimens were tack-cured for 2 s and excess cement was eliminated using scalpel, subsequently, a light curing procedure was applied for 20 s on each surface. Finally, the tooth-restoration interface was polished with polishing disks. The specimens were stored in water at room temperature before being tested. Evaluation of marginal fit was conducted following the methodology outlined by Holmes et al., The fit accuracy of the overlays on their corresponding prepared teeth was measured twice before and after the thermal aging procedure. Six measurements were taken for each: mesiobuccal, midbuccal, distobuccal, mesiolingual, midlingual, and distolingual. The measurements were performed at a magnification of X20 and X110 using a stereomicroscope connected to a digital camera [ Fig. 2 ] . The measurements were determined, and a mean value was calculated and performed by a single-blind examiner for each overlay. Thermal aging was performed with a custom-built device for a total of 5000 cycles representing a 6-month clinical service, between 5 and 55 degrees Celsius in water baths with dwell times of 15 Sects. . A universal testing machine (5ST, Tinius oslen, England) was used to evaluate the fracture resistance of the specimens. A stylus with a 6 mm diameter custom-made stainless-steel ball was used to apply the load on the central fossa on the occlusal surface of the overlay along the long axis. A rubber sheet was placed under the sphere indenter to serve as a cushion and distribute forces evenly at the occlusal surface. The crosshead speed was 1 mm/min until fracture [Fig. 4 ]. The software program (version 10.2.4.0; Horizon) automatically recorded the maximum loads for each specimen in newtons (N). The failure mode for each specimen was classified according to the structures involved in the fracture according to Burke’s classification (Table 1 ) . Fig. 4 Schematic representation of the fracture resistance test setup. A universal testing machine (5ST, Tinius Olsen, England) applied load using a custom-made 6 mm diameter stainless-steel ball on the central fossa of the overlay along the long axis. A rubber sheet was placed beneath the sphere indenter to cushion and evenly distribute forces across the occlusal surface. The load was applied at a crosshead speed of 1 mm/min until fracture occurred Normality was checked using Shapiro Wilk test and Q-Q plots. Marginal fit and fracture resistance were not normally distributed thus both were presented mainly using median, minimum, maximum in addition to mean and standard deviation. The Kruskal Wallis test was used to analyze data between groups, followed by Dunn’s post hoc test with Bonferroni correction. Differences in marginal fit before and after thermal aging were analyzed using Wilcoxon Sign Rank test. All tests were two-tailed, with the significance level established at a p-value of ≤ 0.05. Data was analyzed using IBM SPSS, version 23 for Windows, Armonk, NY, USA. For marginal gap, the greatest mean marginal gap prior to thermal aging was observed in group O with a median gap value of 76.97 μm followed by group OG with a median gap value of 52.94 μm and the group OS with a median gap value of 35.57 μm. The marginal gap significantly increased after thermal aging in all three groups ( p < 0.05) following the same pattern as before thermal aging where the maximum marginal discrepancy was noted in group O with a median gap 114.84 μm succeeded by group OG with a median gap value 103.01 μm and the minimum marginal gap was noted in group OS with a median gap value 93.50 μm [Fig. 5 ]. For fracture resistance, the O group had the highest median fracture load value 1809.08 N followed by Os group with median 1632.77 N. The OG group had the lowest median fracture load values of 1379.63 N [Fig. 6 ]. Fig. 6 Waterfall chart showing a comparison of fracture resistance of the three studied groups. Group O had the highest median fracture load value of 1809.08 N, followed by Group OS, with a median fracture load value of 1632.77 N. The OG Group had the lowest median fracture load value, 1379.63 N Among the fracture modes , there is no statistically significant difference between the three studied groups ( p = 0.027). In the (O) group ( n = 9), 3/9 (33.3%) had mode II of fracture, 2/9 (22.2%) had mode III of fracture, and 4/9 (44.4%) had mode V of fracture. In group (OS) ( n = 9), 3/9 (33.3%) had mode II of fracture, 1/9 (11.1%) had mode III of fracture, and 5/9 (55.6%) had mode V of fracture. In group (OG) ( n = 9), 1/9 (11.1%) had mode II of fracture, 3/9 (33.3%) had mode III of fracture, and 5/9 (55.6%) had mode V of fracture, [ Figs. 7 and 8 , and 9 ] indicates the frequency of various modes of failure within the groups. This research sought to evaluate and compare the marginal fit and fracture resistance of three different overlay designs using advanced zirconia-reinforced lithium disilicate CAD/CAM material. Both null hypotheses were rejected, as the preparation designs used significantly affected fracture resistance and marginal fit of studied groups. This outcome is consistent with prior research findings that demonstrated a substantial influence of preparation design on the marginal fit and the fracture resistance of restorations . In this study, the marginal fit and fracture resistance of the advanced zirconia-reinforced lithium disilicate (ALD) CAD/ CAM material for three different overlay preparation designs on molars were compared. This study selected advanced zirconia-reinforced lithium disilicate (ALD) over other materials, such as 5Y-Zirconia and novel fully crystallized lithium disilicate, due to its superior combination of mechanical strength and aesthetic properties. While 5Y-Z offers enhanced translucency, its reduced fracture toughness and absence of transformation toughening make it more prone to failure under stress. ALD, by contrast, provides a favorable balance between strength and translucency, making it particularly suitable for posterior restorations. Moreover, novel fully crystallized lithium disilicate materials were not fully stabilized or commercially available when the study was initiated. ALD also simplifies bonding protocols by eliminating the need for sandblasting, which can compromise surface integrity, thereby ensuring both durability and efficiency . Diligent efforts were made to replicate the clinical intraoral environment in this study. A single scanner system was used to standardize the intraoral scanning process, minimizing variations and ensuring more accurate results. Each preparation design was duplicated to produce standardized dies using 3D printing resin (Model Resin, Formlabs, Somerville, MA) possessing an elasticity modulus similar to that of dentin , for evaluation of ceramic overlays’ marginal adaptation and fracture resistance. The stimulation of the clinical environment was also taken into account in various aspects of the research, including tooth preparation, impression creation, procedure for fabrication, and cementation of restorations. In this research, 3D-printed resin dies (Model Resin, Formlabs) were chosen over natural teeth due to their ability to provide consistent geometry across all specimens, eliminating variations in size and preparation design inherent to natural teeth. This approach also minimizes the challenges and variability associated with collecting, storing, hand-prepping, and handling natural teeth. Resin dies have a tensile strength of 61.0 MPa, which falls within the range of dentin tensile strength (44.4–97.8 MPa), making them a suitable substitute . Studies have demonstrated that crowns cemented to resin die yield fracture strength results comparable to those cemented on natural teeth. For instance, one study concluded that resin dies closely mimic the fracture behavior of crowns on dentin. Furthermore, another study reported that zirconia crowns fractured on resin dies exhibited similar strength values to those fractured on enamel dies . They did not replicate the natural tooth structure for cement adhesion. However, adhesion was not a primary focus of our research. Based on Holmes et al., the term “marginal gap” denotes the vertical gap between the cervical edge of restoration and the prepared tooth surface. In simpler terms, the marginal gap pertains to the surface area of the cement, which is subjected to the oral environment and is susceptible to degradation . In this study the marginal gap was evaluated using the direct-view technique using a stereomicroscope. Some earlier research has also utilized this method . The marginal fit was evaluated in this study before and after thermal aging. The findings indicated that the marginal gap for all groups was below 120 μm before and after thermal aging, falling within the range considered clinically acceptable as determined by the Consensus among most authors suggests . Comparing data on marginal adaptation from various studies poses challenges and potential inaccuracies due to several factors, including variations in preparation design, measurement techniques, the number and location of measurement points, the type of resin cement employed, and the method used to fabricate the restoration. Advanced 4- or 5-axis milling machines, which can enhance the fit between the prepared tooth structure and the restoration’s intaglio surface, also contribute to these variations. However, the precision and reliability of the outcomes improve when more measurement points are included and consistently assessed at the same locations across samples. This approach reduces variability, ensuring that the results accurately reflect the restoration’s adaptation quality. Our study adopted this approach to ensure accurate and reliable results. Therefore, carefully considering these variations is essential when making in vitro comparisons of data . In this research, the restorations were adhered under 200 g applied weight to standardize the pressure applied . However, to ensure consistency in the cementation procedure, all restorations were placed by the same operator. Based on the findings of this research, group OS revealed the minimal marginal gap after cementation with a median gap value of 35.57 μm, succeeded by group OG with a median gap value of 52.94 μm and group O with a median gap value of 76.97 μm. Therefore, the preparation design of group OS (overlay preparation with 1.5 mm anatomical reduction of the occlusal surface and 1.0 mm circumferential shoulder finish line) has more retentive form and more defined margins, resulting in a reduction of the marginal gap size. These results aligned with the findings of Yang et al., who assessed how tooth preparation design influences the marginal adaptation of composite resin CAD-CAM Onlays by comparing two different preparation designs. They noted that the conventional design, which provided more excellent retention, resulted in greater adaptation. However, this result contradicted the findings of Falahchai et al., and Kim et al., Who determined that preparation designs lacking retention would offer superior adaptation to those with retention features. The variation can be ascribed to the different materials employed for restoration fabrication, variations in preparation designs, cementation techniques, and distinct techniques for gap measurement. In this research, there was a reduction in marginal fit (increase in micro gap) in all groups after exposure to thermal aging compared to the initial measurements. This observation aligned with the outcomes reported in earlier studies . They reported that the thermal aging affected all groups’ marginal fit and showed decreasing marginal adaptation. This finding was in agreement with the outcomes obtained in the current study. In group O, the statistical significance of the change in marginal gaps was noticeably lower compared to group OG and group OS. This may be due to the nature of 3D printing resin material; the temperature variations cause the resin to undergo expansion and contraction, particularly in thin margins . In the current study, the fracture resistance was evaluated for all groups after thermal aging. The force required to cause a fracture in the group (O) was 1809.08 N. However, it was 1632.77 N for group (OS). The lower value was recorded in group (OG) 1379.63 N. Studies showed that the occlusal forces generated during chewing and biting usually reach approximately 100 N, with a maximum bite force in habitual occlusion of up to 320 N . These values are considerably lower than the fracture resistance observed in our results, suggesting that partial restorations can comfortably endure occlusal forces without risk of failure. The durability of all-ceramic restorations is impacted by various factors, such as the ceramic material’s microstructure and fatigue, fabrication methods, preparation design, and bonding technique . The results of this study showed that group O (teeth with 1.5 mm anatomical occlusal reduction that received occlusal veneers) and group OS (teeth with 1.5 mm anatomical occlusal reduction and 1.0 mm circumferential shoulder finish line preparation) exhibited the highest fracture resistance of the studied groups under the imposed forces , This observation was consistent with the results obtained from Clausen et al., Who evaluated occlusal overlays and the influencing of ceramic material and preparation design. They reported that all-ceramic full coverage restorations bonded exclusively to enamel exhibited a tendency towards increased fracture resistance compared to those bonded to dentin with a finishing line located in enamel. Falahchai et al. revealed that teeth with less extensive restorations and sound marginal ridges demonstrated a lower incidence of fractures, reporting that increasing the reduction of tooth structure in the central area, such as with a mesio-occluso-distal preparation, leads to compromising the strength of the remaining tooth structure. Alternatively, the intracoronal extension of an overlay may produce a wedging effect. Hence, this clarifies why the fracture resistance outcomes of group (OS) exceeded those of group (OG) (characterized by anatomical occlusal reduction with a central groove), where both the central portion of the tooth and marginal ridges were eliminated. Also, preparation designs that emphasize retention have complex shapes with sharp inner edges, making them more prone to developing predetermined weak points. These geometric changes can lead to localized stress concentrations . This finding aligned with Channarong et al. , Who evaluated the resistance of bonded ceramic overlay restorations to fractures as influenced by different preparation designs. They found that the more natural tooth structure that remains, the more favorable the long-term prognosis for that tooth. Therefore, the conservation of natural tooth structure is crucial for the overall lifespan of the tooth. Also, they demonstrated that overlay restorations utilizing the adhesive system could reinforce defective teeth to a fracture resistance level similar to that of intact teeth. In contrast, the margin type and axial wall length did not affect the final fracture resistance. However, these results contradicted Alberto et al.‘s findings . They reported that the fracture resistance of the full-coverage crown restorations was notably greater than that of the occlusal-cover restorations. Also reported that the effect of the amount of the preparation that was 2.0 mm and 1.5 mm thick needed significantly more fatigue cycles to fail (17 − 15 times fatigue cycles) compared to crowns with a thickness of 1.0 mm. Also, the study has shown that self-adhesive resin cement exhibits weaker mechanical and bonding properties compared to conventional resin-luting cement . However, previous research has demonstrated that a 1 mm thin layer of lithium disilicate ceramic bonded to enamel exhibits a lower risk of fracture compared to bulker restorations (1.5–2 mm as recommended) bonded to dentin, primarily because of reduced mechanical complications . This confirms that other factors like ceramic thickness and type, type of cementation, tooth architecture, and study design can influence fracture resistance in addition to preparation design. Previous research indicated that any loss of tooth structure, whether caused by caries or cavity preparation, reduces the fracture resistance of restorations. The literature suggests that more conservative preparation designs with lower retention features tend to enhance fracture resistance . Building on the previous findings, this explains the results observed in our study. Group O, which featured the most conservative preparation design with no retentive form, demonstrated the highest fracture resistance. In contrast, Group OG, which included a central groove and internal angles, showed increased stress concentration in these areas, leading to reduced fracture resistance. According to the study’s results, various preparation designs exhibited distinct. frequencies of modes of failure. Nevertheless, these differences were not significant. More than half of the restoration fractures noted in this study involved damage to both the die and the restorative material. This observation is consistent with the results documented in previous studies by Johnson et al., And Alberto et al., . All tested groups showed marginal fit results within clinically acceptable parameters. All preparation designs utilized in this study for creating overlay restorations from ALD ceramics demonstrated acceptable fracture resistance. Higher fracture resistance was shown with intact marginal ridges and less extensive preparations. Modification of tooth preparation significantly impacted the magnitude of the fracture resistance and the marginal gap observed around ALD overlays. Thermal aging affected the marginal gaps of the three groups following the same pattern as before thermal aging. | Other | biomedical | en | 0.999996 |
PMC11699785 | The incidence of anterior cruciate ligament (ACL) tears in the United States has been reported at 6.8 per 100,000 annually, making it the most common knee ligament injury . Failure rates amongst primary ACL reconstruction (ACLR) procedures have been estimated to range from 3.2 to 11.1% . Several factors contribute to postoperative failure, including traumatic reinjury, tunnel malposition, and biological failure . Beyond revisions, secondary injuries such as meniscus tears and contralateral injuries are reasons for overall reoperation after primary ACLR . The presence of concomitant meniscal tears, such as ramp lesions with ACL injuries, has been reported to range from 9 to 40% and is associated with a 7.7% rate of secondary meniscectomy . Because of this, it is important for surgeons to have a guide for predicting risk profiles for secondary injury or revision after ACLR. In recent years, the use of artificial intelligence (AI) has become popular in orthopedic research . Specifically, machine learning (ML) models can learn complex patterns and associations between variables and outcomes from large datasets . These relationships can be used to generate predictive models incorporating patient demographics, injury characteristics, and surgical techniques, which can be used in the clinical setting . Machine learning models can be classified into classical machine learning (Random Forest, Gradient Boosted Regression Model (GBM) etc.) and deep learning with neural networks (NN) (Artificial Neural Networks, Multi-Layer Perceptron etc.). Classical machine learning models tend to be faster and require fewer resources, however, they require manual feature selection, whereas NN models are able to automatically learn features from raw data . ML research has been performed in various orthopedic domains, such as hip arthroplasty, hip arthroscopy, and spinal cord injuries [ 10 – 12 ]. Therefore, the application of ML in predicting objective outcomes following ACLR offers great potential to be used to identify and manage patient expectations, tailor rehabilitation regimens to maximize functional recovery, and to identify optimal candidates for specific surgical interventions. While the use of AI in ACL literature is promising, surgeons must familiarize themselves with the overall results, advantages, and disadvantages of ML models . To date, no review has provided a comprehensive summary of the utilization of ML models in predicting postoperative outcomes after ACLR. Therefore, this systematic review aims to summarize the statistical performance of machine learning in predicting revision, secondary injury, or reoperations in ACLR, and to provide a general overview of findings from these models. It was hypothesized that ML models would be superior in predicting these outcomes compared to standard logistic regression models. This systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) and Revised Assessment of Multiple Systematic Reviews (R-AMSTAR) guidelines for coordinating and reporting systematic reviews . Three online databases (PubMed, Medical Literature Analysis and Retrieval System Online (MEDLINE), Excerpta Medica dataBASE (EMBASE)) were searched from database inception to February 6, 2024, to identify literature on the use of ML to predict revision, secondary injury, or reoperation in ACLR. The search strategy is described in Supplementary Table 1 . Inclusion criteria included the following: (1) studies examining machine learning models to predict objective outcome measures (e.g. revision, reoperation, secondary injury) following ACLR, or external validity of established databases using machine learning models to predict the aforementioned outcomes, (2) simulation-based or laboratory studies and (3) studies written in English. Exclusion criteria included (1) systematic reviews or meta-analyses, (2) text-book chapters, (3) conference abstracts, (4) biomechanical studies, (5) levels of evidence V (i.e. case reports), (6) case series with less than five patients, and (7) cadaveric/animal studies. References of included studies and of pertinent review papers were manually searched to ensure all means of study identification were exhausted. If multiple papers reported the same outcomes using identical patient cohorts, only the article with the largest sample size or latest follow-up period was included. If multiple papers presented overlapping but non-identical cohorts, all articles were included as the extent of patient overlap was unable to be determined. The inter-reviewer agreement was evaluated using a kappa (κ) statistic for screening. A priori classification was defined according to the following criteria: a κ of 0.91–0.99 was almost perfect agreement; a κ of 0.71–0.90 was considerable agreement; a k of 0.61–0.70 was high agreement; a κ of 0.41–0.60 was moderate agreement; a κ of 0.21–0.40 was fair agreement and a κ or ICC value of 0.20 or less was no agreement . The Methodological Index for Non-Randomized Studies (MINORS) criteria were used for methodological quality assessment . Based on the MINORS criteria, non-comparative studies could get a maximum score of 16. For non-comparative studies, classification was a priori based on a previous systematic review: 0–4 indicated very low-quality evidence, 5–7 indicated low-quality evidence, 8–12 indicated fair-quality evidence, and scores ≥ 13 indicated high-quality evidence . Two review authors independently extracted and summarized data from included articles using a Google Sheets (Google LLC, Mountain View, CA, USA) spreadsheet. Demographic data such as number of patients, mean age, patient sex, and follow-up times were recorded. Machine learning specifics included the primary outcome of interest (e.g. revision, secondary injury, or reoperation), statistical software used, models assessed, training and test splits, and the handling of missing data. Adherence to the Transparent Reporting of a multivariable prediction model for Individual Prognosis or Diagnosis (TRIPOD) guidelines was also assessed . Discrimination, or classification accuracy, was assessed using area under the receiver operating curve (AUC) and concordance. AUC values range from 0 to 1, with increasing values representing increased discriminatory capacity . Concordance is another representation of AUC, ranging from 0.5 to 1, with increasing scores indicating a model that more accurately identifies the most true positive results and least false negative results . Calibration was assessed using calibration slope, intercept, and error. The calibration intercept is the tendency of a model to overestimate results, with scores approaching 0 indicating less frequent overprediction or underprediction . Calibration slope identifies if predictions are precise or extreme, with scores closer to 1 indicating better model predictions across the range of possible outcomes . Brier scores combine both discrimination and calibration, with values ranging from 0 to 1, with lower scores indicating higher accuracy . Factors deemed highly predictive for revision, secondary injury or reoperation were also extracted. The level of evidence of each paper was reported according to the authors’ statement or, if unstated, was reported using the Oxford Centre for Evidence-Based Medicine (OCEBM) guidelines . The initial search resulted in 780 studies, of which 304 were duplicates. Of the 476 remaining, 20 were selected for full-text screening after abstract and title screening. Nine full-text articles satisfied the eligibility criteria and were included in the final analysis . There was a high level of agreement during title and abstract screening (κ = 0.892, 95%CI 0.799–0.986) and perfect agreement at the full-text stages (κ = 1.00). Five of nine (55.6%) studies reported following the TRIPOD guidelines . Two studies examined the external validity of a previous machine-learning model developed using source data from the Norwegian Knee Ligament Registry (NKLR) database . The other seven studies had their own internal validation [ 24 – 27 , 29 , 31 , 32 ]. Primary model development was performed using the NLKR, DKLR, and Rochester Epidemiology Project databases in three , two , and three studies , respectively. One study each used data from the American College of Surgeons National Surgical Quality Improvement Program database , Shanghai Sixth People’s Hospital , and the STABILITY I trial . This review included nine studies comprising 125,427 patients with a mean follow-up of 5.82 (range of means: 0.08–12.3) years. Of the eight studies that reported on patient sex, 51,511 female patients (41.5%) were included. The average age of patients at surgery was 26.73 (range of means: 19–32) years. A detailed description of study characteristics and demographics can be seen in Table 1 . Table 1 Study characteristics Author (Year) Study Design (Level of Evidence) MINORS SCORE (/16) Purpose Source data if external validation study Database/Institution TRIPOD Followed? Number of patients/knees Mean age at surgery Female (%) Follow-up of Outcome (years) Martin Retrospective Cohort (III) 12 Assess sample size effect on accuracy NR NKLR + DKLR Yes 62,955 MEDIAN 26 ( IQR: 20–36) [Missing data 1870] 26,446 7.6 (4.5) Martin Retrospective Cohort (III) 12 External Validation of Cox Lasso NKLR DKLR Yes 10,922 29 (11) 4916 8.4 (4.3) Martin Case-control (III) 12 Determine if machine learning analysis of NKLR can identify the most important risk factors associated with subsequent revision of primary ACL reconstruction NA NKLR NR 24,935 28 (11) 10,916 1, 2, 5 years Johnson Retrospective Cohort (III) 12 Machine learning to predict ACL re-operation NA Rochester Epidemiology Project Yes 1400 27 NR 9 years (min 2 years) Lopez Retrospective Comparative Prognostic (IV) 11 Machine learning (ML) models to predict outcomes following ACLR NR American College of Surgeons National Surgical Quality Improvement Program database NR 21,636 31.8 (10.5) 7638 (35.3%) 30 days Ye Case-control (III) 12 Machine learning to determine objective and subjective clinical outcomes of ACLR and to determine the most important predictors NA Shanghai Sixth People’s Hospital NR 432 26.8 (8.4) 112 (25.9%) 6 years (3.1) Martin Retrospective Cohort (III) 11 To assess the external validity of the NKLR model using STABILITY 1 RCT NKLR NKLR + STABILITY 1 trial NR 591 19.0 (3.2) 304 (51.4) 1, 2 Jurgensmeier Retrospective Cohort (III) 11 Machine learning to determine risk of secondary meniscal injury post primary ACLR NA Rochester Epidemiology Project (REP) Yes 1187 25 (18–34) 502 (42.3%) 12.3 (6.6–17.6) Lu Retrospective Cohort (III) 12 Machine learning to compare risk and timing of secondary meniscal injury between nonoperative, delayed ACLR, and early ACLR patients NA Rochester Epidemiology Project (REP) Yes 1369 28 (18–37) 677 (40.7) min 2 year MINORS: methodological index for non-randomized studies, TRIPOD: transparent reporting of a multivariable prediction model for individual prognosis or diagnosis, NKLR: norwegian knee ligament registry, DKLR: danish knee ligament registry, RCT: randomized controlled trial, NR: not reported, NA: not applicable, ACL: anterior cruciate ligament Primary outcomes were revision ACLR, secondary meniscus injuries, graft failure, and all-cause reoperation in five [ 26 , 28 – 31 ], two , one , and one study , respectively. The most common model was Random Forest/Random Survival Forest, used in six studies (66.6%). The second most used model was the Cox Lasso model, assessed in four studies [ 28 – 31 ] (44.4%). Two studies used neural networks, MLPClassifier (Multi-Layer Perceptron) and Artificial Neural Network (ANN) (22.2%), respectively. With respect to model evaluation, seven (77.8%) studies used calibration [ 24 , 25 , 27 – 31 ], five (55.6%) used concordance (including C-statistic AUROC) [ 27 – 31 ], four (44.4%) used AUC (including Discrimination - AUROC) [ 24 – 26 , 32 ], and two (22.2%) used Brier Score . Five (55.6%) studies used a 75/25 training/test split [ 24 , 28 – 31 ], two (22.2%) used 0.632 bootstrapping with 1000 resampled datasets , one (11.1%) used an 80/20 training/test split , and one (11.1%) used a 90/10 training/test split . Seven studies reported on their handling of missing data [ 24 – 27 , 29 – 31 ], of which five (71.4%) studies used multiple imputation . One external validation study included patients with data for features used in all five models from the source study . Another study excluded patients with missing data . A full description of the Machine Learning Methods can be seen in Table 2 . Table 2 Study methods Author (year) Primary Outcome Statistical Software and ML algorithms Models Model Evaluation Training/Test Split Missing Data Method Martin Revision R (Version 4.1.11 R Core Team) Cox lasso Random survival forest Gradient boosting Super learner Concordance - Harrell C-index Calibration 75/25 Multiple imputation Martin Revision R (Version 3.6.1) Cox Lasso Concordance - Harrell C-index Calibration NR (external validation study - original model 75/25) Patients included if they had data for five predictive models from original model Martin Revision R (Version 3.6.1) Cox Lasso Survival Random Forest Generalized Additive Model (GAM) Gradient Boosted Regression Model (GBM) Calibration Concordance 75/25 Multiple imputation Johnson All-cause re-operation SciPy version 1.6.2 MLPClassifier GaussianNB LogisticRegression KNeighborsClassifier BaggingClassifier RandomForestClassifier AdaBoostClassifier GradientBoostingClassifier XGBClassifier AUC Calibration AUPRC F1 Recall Accuracy Precision 75/25 Multiple imputation Lopez ACLR post-op outcomes (revision included) TensorFlow Python open-source coding platform (Google Brain, Alphabet Inc., Mountain View, CA) Artificial Neural Network ML Logistic Regression AUC Accuracy 80/20 Excluded Ye Graft failure SPSS (Version 25.0; IBM Corp) Logistic Regression Gaussian Naïve Bayes Random Forest XGBoost Isotonic XGBoost Sigmoid XGBoost AUC Accuracy F1 90/10 NR Martin Revision R Cox Lasso Concordance - Harrell’s C-index Calibration 75/25 NA Jurgensmeier Secondary meniscus tear R 4.1.2 using RStudio version 1.4.1717 (RStudio, Boston, MA) SVM Random Forest XGBoost Elastic Net Discrimination - AUROC Calibration Brier score 0.632 bootstrapping with 1000 resampled datasets Multiple imputation Lu Secondary meniscus tear R 4.1.2 using RStudio version 1.2.5001 (RStudio, Boston, MA). Random Survival Forests C-statistic (AUROC) (Concordance) Calibration Brier Score 0.632 bootstrapping with 1000 resampled datasets Multiple imputation ML: machine learning, AUC: area under the curve, AUROC: area under the receiver operating curve, AUPRC: area under the precision-recall graph, ACLR: anterior cruciate ligament reconstruction, NR: not reported, MA: Massachusetts, CA: California, NR: not reported, NA: not applicable Various features were assessed when evaluating machine learning models. Age and body mass index (BMI) were examined in eight [ 24 , 25 , 27 – 32 ] (88.9%) and six [ 24 – 27 , 29 , 32 ] (66.7%) studies, respectively. Both sex [ 24 – 27 , 32 ] and time between injury and ACLR [ 28 – 32 ] were considered in five (55.6%) studies. Femur fixation method [ 28 – 31 ] and KOOS QOL [ 28 – 31 ] were assessed in four studies each (44.4%), while graft choice , sports participation , and meniscal injury were examined in three (33.3%) studies each. Other features selected can be seen in Table 3 . Table 3 Model Complete Set Author (year) Feature Selection AUC Calibration Intercept Calibration Slope Brier Score Concordance (95 CI) Calibration Error Martin Age at surgery Yrs. injury to surgery KOOS QOL Graft: hamstring Age at injury Femur fix: susp/cort. Graft: QT/BQT KOOS Sport Men. injury: none Activity: pivoting Graft: other Fix. comb: susp/interference Surgery on same knee KOOS All low NR NR NR NR 1 year: Cox Lasso 0.59 (0.56–0.61) RSF: 0.67 (0.64–0.69) GB: 0.67 (0.65–0.70) SL: 0.67 (0.65–0.69) 2 year: Cox Lasso 0.58 (0.56–0.61) RSF: 0.67 (0.64–0.69) GB: 0.67 (0.64–0.69) SL: 0.67 (0.64–0.69) 5 year: Cox Lasso 0.58 (0.56–0.61) RSF: 0.67 (0.65–0.69) GB: 0.67 (0.64–0.69) SL: 0.67 (0.64–0.69) 1 year: Cox Lasso 7.19, n.s RSF: 5.54, n.s GB: 7.48, n.s SL: 8.67, p = 0.034 2 year: Cox Lasso 8.17, p = 0.043 RSF: 6.42, n.s GB: 4.53, n.s SL: 4.10, n.s 5 year: Cox Lasso: 11.37, p = 0.01 RSF: 9.27, p = 0.026 GB: 11.07, p = 0.011 SL: 11.82, p = 0.008 Martin Patient age at primary surgery KOOS QoL score at primary surgery Graft choice Femur fixation method Years between injury and ACLR NR NR NR NR 1 year: Cox Lasso: 0.678 2 years: Cox Lasso: 0.676 5 years: Cox Lasso : 0.678 1 year: Cox Lasso: 22.24, p < 0.001 2 years: Cox Lasso: 11.82, p = 0.008 5 years: Cox Lasso : 13.98, p = 0.003 Martin Age at surgery Fixation combination Tibia fixation Femur fixation BMI KOOS Sport at surgery KOOS QOL at surgery Years from injury to surgery Age at injury Hospital type Further injury Meniscus injury Injured side NR NR NR NR 1 year Cox Lasso: 0.686 Random Forest: 0.672 GAM 0.687 GBM 0.669 2 year Cox Lasso 0.684 Random Forest: 0.670 GAM 0.685 GBM: 0.666 5 year: Cox Lasso: 0.683 Random Forest: 0.670 GAM: 0.684 GBM: 0.665 1 year Cox Lasso: 4.89, n.s Random Forest: 3.12, n.s GAM 4.79, n.s GBM 4.98, n.s 2 year Cox Lasso 11.35, p = 0.01 Random forest: 11.66, p = 0.009 GAM 11.19, p = 0.011 GBM: 3.76, n.s 5 year: Cox Lasso: 6.19, n.s Random Forest: 3.71, n.s GAM: 6.98, n.s GBM: 0.38, n.s Johnson Age Sex BMI Occupation Sport participation Injury mechanism Occurrence of reoperation after ACLR MLPClassifier: AUC = 0.61 GaussianNB: AUC = 0.58 LogisticRegression: AUC = 0.70 KNeighborsClassifier: AUC = 0.68 BaggingClassifier: AUC = 0.75 RandomForestClassifier: AUC = 0.76 AdaBoostClassifier: AUC = 0.73 GradientBoostingClassifier: AUC = 0.75 XGBClassifier: AUC = 0.77 NR NR NR NR NR Lopez Sex Race BMI (Calculated From The Recorded Height And Weight) American Society Of Anesthesiologists (ASA) Classification History Of Smoking Diabetes Hypertension Requiring Medication Wound Infection Use Of Steroids For A Chronic Condition Bleeding disorders were Abstracted Perioperative Data Such As Anesthesia Type (General, Spinal, IV Sedation, Regional, Other) Surgery Setting (Inpatient Vs Outpatient) Operative Time (Prolonged Operative Time Defined As > 120 min) ANN: Reoperation: 0.842 ACLR-related Readmission: 0.872 Logistic Regression: Reoperation: 0.601 ACLR-related Readmission: 0.606 NR NR NR NR NR Ye Age Sex BMI Time From Injury To Surgery Participation In Competitive Sports Preoperative Lysholm And IKDC Scores Posterior Tibial Slope High-Grade Knee Laxity Graft Diameters Of Anteromedial And Posterolateral Bundles Medial And Lateral Meniscal Resection Follow-Up Period Meniscal Reinjury After ACLR Graft Failure: XGBoost (excellent): AUC = 0.944 (0.001), Accuracy = 0.986 (0.012) Residual Laxity: Random Forest (excellent): AUC = 0.920 (0.014), Accuracy = 0.914 (0.024) NR NR NR NR NR Martin Patient Age At Primary Surgery Knee Injury And Osteoarthritis Outcome Score Quality Of Life Subscale (KOOS-QOL) Score At Primary Surgery Graft Choice Femur Fixation Method Time Between Injury And ACLR NR NR NR NR 1 year: Original Norwegian Algorithm Performance: 0.686 (0.652–0.721) STABILITY data: HT = HT, HT + LET = BPTB: 0.713 (0.634–0.791) HT = HT, HT + LET = Unknown: 0.609 (0.528–0.691) All patients = HT: 0.674 (0.597–07.51) 2 year: Original Norwegian Algorithm Performance: 0.684 (0.650–0.718) STABILITY data: HT = HT, HT + LET = BPTB: 0.713 (0.637–0.789) HT = HT, HT + LET = Unknown = 0.608 (0.530–0.688) All patients = HT: 0.673 (0.598–0.747) 1 year: Original Norwegian Algorithm Performance: 4.9 n.s. STABILITY data: HT = HT, HT + LET = BPTB: 2.6 n.s. HT = HT, HT + LET = Unknown: 10.6 p < 0.01 All patients = LT: 8.7 p < 0.01 2 year: Original Norwegian Algorithm Performance: 11.3 p = 0.01 STABILITY data: HT = HT, HT + LET = BPTB: 11.7 p < 0.01 HT = HT, HT + LET = Unknown: 8.9 p < 0.01 All patients = LT: 10.2 p < 0.01 Jurgensmeier Age Sex Body mass index Sport participation Diagnosis of hypermobility or malalignment Radiographic findings Management SVM: Apparent 0.782 (0.779–0.785), Internal Validation 0.738 (0.736–0.739) Random Forest: Apparent 0.997 (0.994–0.999), Internal Validation 0.790 (0.785–0.795) XGBoost: Apparent 0.814 (0.813–0.816), Internal Validation 0.758 (0.755–0.761) Elastic Net: Apparent 0.673 (0.61–0.736), Internal Validation 0.646 (0.643–0.648) SVM: 0.0161 Random Forest: 0.006 XGBoost: 0.007 Elastic Net: 0.0165 SVM: 1.091 (1.086–1.096) Random Forest: 0.9608 XGBoost: 0.9569 Elastic Net: 0.8926 SVM: 0.14 (0.13–0.15) Random Forest: 0.10 (0.09–0.12) XGBoost: 0.12 (0.11–0.14) Elastic Net: 0.18 (0.17–0.20) NR NR Lu Age Sex Body mass index Activity level Occupation Comorbid diagnosis Radiographic findings Management ACLR: 0.80 (0.76–0.83) Non-op: 0.66 (0.58–0.74) ACLR: 0.0051 (− 0.014–0.024) Non-op: 0.0048 (− 0.059–0.069) ACLR: 0.97 (0.89–1.05) Non-op: 0.97 (0.65–1.30) ACLR: 0.106 (0.029–0.183 Non-op: 0.111 (0.034–0.188) NR NR ACLR: anterior cruciate ligament reconstruction, AUC: area under the curve, CI: confidence interval, KOOS: knee osteoarthritis and outcome score, QOL: quality of life, QT: quadriceps tendon, BQT: quadriceps tendon with a bone -block, GB: gradient boosted regression model, RSF: random survival forest, SVM: support vector machine, HT: hamstrings tendon, BPTB: bone-patellar tendon-bone, LET: lateral extra-articular tenodesis, SL: super learner, GAM: generalized additive model, NR: not reported, n.s: not significant, non-op: non-operative Five studies reported an AUC for their chosen models [ 24 – 27 , 32 ]. Overall, AUC for the strongest-performing models in each study ranged from 0.77 to 0.997, indicating that these models ranged from fair to excellent discrimination. The best-performing model was Random Forest (AUC = 0.997) when used to predict secondary meniscus injury . One study found that XGBoost was the best model for predicting graft failure (AUC = 0.944) . When Artificial Neural Network (ANN) was compared with logistic regression, ANN was superior, with an AUC of 0.842 (good discrimination) compared to 0.601 (poor discrimination) for Logistic Regression (LR) . In another study, Random Forest was slightly superior to LR with AUCs of 0.77 and 0.70, respectively . Four studies reported on concordance [ 28 – 31 ]. Overall, concordance for the best-performing models in each study ranged from 0.67 to 0.71, indicating that these models ranged from poor to fair discrimination. The best-performing model was Cox Lasso (Concordance: 0.71) . One study assessed the STABILITY trial and found that when STABILITY patients with hamstring tendon (HT) autografts in addition to lateral extra-articular tenodesis (LET) were coded as receiving a bone-patellar tendon-bone graft (BPTB) from the NKLR data, this subgroup achieved the highest concordance, with scores of 0.713 (range: 0.634–0.791) and 0.713 (range: 0.64–0.79) at one and two years, respectively . In the same study, the original Norwegian Algorithm reported concordance of 0.686 (range: 0.65–0.72) and 0.684 (range: 0.65–0.72) at one and two years, respectively . One study reported predicting revision concordance for several models over different time intervals. At the one-year interval, the Cox Lasso model had the lowest concordance, with a score of 0.59 (range: 0.56–0.61) and 0.58 (range: 0.56–0.61) at two and five years. The RSF, GB, and SL models all showed higher concordance scores of 0.67 (ranges, RSF: 0.64–0.69, GB: 0.65–0.70, SL: 0.65–0.69), maintaining their scores at two and five years . Another study reported concordance for the Cox Lasso model of 0.678 at one year, 0.676 at two years, and 0.678 at five years . One study found that the GAM model had the highest concordance across all time points (1-year: 0.687, 2-year: 0.685, 5-year: 0.684) . This was followed by Cox Lasso (1-year: 0.686, 2-year: 0.684, 5-year: 0.683), Random Forest (1-year: 0.672, 2-year: 0.670, 5-year: 0.670), and GBM (1-year: 0.669, 2-year: 0.666, 5-year: 0.665) . Two studies reported on Brier Scores . Random Forest was the most accurate model in a study reporting on secondary meniscal injuries, and the studies had Brier scores ranging from 0.10 to 0.18, indicating low deviation of predictions and actual outcomes. One study found that Random Forest was the most accurate, with a Brier score of 0.10 (range: 0.09–0.12) at a mean follow-up of 12.3 (6.6–17.6) years, with key variables being time to return to sport, visual analog scale (VAS) pain score at injury, and time to surgery . Another study predicting secondary meniscal injuries reported a Brier score of 0.106 (range: 0.029–0.183) at a minimum two-year follow-up using the Random Survival Forest model, with key variables being time to return to sport, VAS pain score at injury, and hypermobility . Two studies looked at calibration intercept and slope . The best-performing models for calibration interval reported scores ranging from 0.0051 to 0.006. The random survival forest model performed best when predicting secondary meniscal injury after ACLR with a score of 0.0051 (− 0.014 to 0.024) at a minimum two-year follow-up . The positive intercept indicates that included ML models tend to underestimate the risk; however, the confidence interval suggests that the systemic underprediction is not statistically significant. The best-performing models for calibration slope reported slopes from 0.96 to 0.97, with Random Survival Forest reporting the highest score (0.97) at a minimum two-year follow-up in a study predicting second meniscal injury . XGBoost was similar with a slope of 0.957 (0.952–0.962) at a mean follow-up of 12.3 (6.6–17.6) years in a study predicting second meniscal injuries . These values suggest that the models used tend to slightly overestimate risk, placing too much importance on predicting features. Overall, both studies revealed that the calibration intercept and slope were most accurate using the predictive features of time to return to sport and VAS pain score. Calibration error was measured in four studies [ 28 – 31 ]. One study reported calibration errors for various models at one-, two-, and five-year marks . At one year, the Cox Lasso, Random Survival Forest (RSF), and Gradient Boosting (GB) models all had non-significant calibration errors, whereas the Super Learner (SL) model demonstrated a calibration error of 8.67 ( p = 0.034). At two years, the Cox Lasso model showed a significant calibration error of 8.17 ( p = 0.043). At five years, calibration errors were significant in all models: 11.37 ( p = 0.01) for Cox Lasso, 9.27 ( p = 0.026) for RSF, 11.07 ( p = 0.011) for GB, and 11.82 ( p = 0.008) for SL. One study reported significant calibration errors for the Cox Lasso model, with errors of 22.24 ( p < 0.001) at one year, 11.82 ( p = 0.008) at two years, and 13.98 ( p = 0.003) at five years . One study found significant miscalibration at two years, with calibration errors of 11.35 ( p = 0.01) for Cox Lasso, 11.66 ( p = 0.009) for Random Forest, and 11.19 ( p = 0.011) for Generalized Additive Model (GAM). None of the models showed significant calibration errors at five years. Another study externally validated the original Norwegian Algorithm using the STABILITY trial. They found that the subgroup (HT + LET patients coded as having BPTB grafts) with the highest concordance had a significant calibration error of 11.7 ( p < 0.01) at two years . The original Norwegian Algorithm also showed a significant calibration error of 11.3 ( p = 0.01) at two years. Other subgroups analyzed showed evidence of miscalibration at one and two years, respectively ( p < 0.01). Complete data from the model set can be seen in Table 3 . Only one study reported data on multiple imputation analyses . The concordance data from this study was not significantly different ( p < 0.05) from the original set. The specific data can be seen in Table 4 . However, the calibration data revealed an increased statistically significant calibration error in the multiple imputation cohort. At one year, two of the four models showed miscalibration ( p > 0.05), and at two and five years, all models showed significant miscalibration ( p < 0.05). The calibration error at one year ranged from 4.17 to 8.35. At two years, it ranged from 8.34 to 8.98; at five years, it ranged from 8.30 to 14.05. Table 4 Multiple Imputation Data Multiple Imputation Data Set Author Concordance (95 CI) Calibration Martin 1 year: Cox Lasso 0.59 (0.56–0.61) RSF: 0.66 (0.64–0.69) GB: 0.68 (0.65–0.70) SL: 0.67 (0.65–0.70) 2 year: Cox Lasso 0.59 (0.56–0.61) RSF: 0.67 (0.65–0.70) GB: 0.67 (0.65–0.70) SL: 0.67 (0.65–0.70) 5 year: Cox Lasso 0.58 (0.56–0.61) RSF: 0.67 (0.65–0.70) GB: 0.67 (0.65–0.69) SL: 0.67 (0.65–0.70) 1 year: Cox Lasso 8.35, p = 0.039 RSF:4.17, p = 0.244 GB: 7.57, p = 0.056 SL: 7.99, p = 0.046 2 year: Cox Lasso 8.81, p = 0.032 RSF: 8.96, p = 0.030 GB: 8.98, p = 0.030 SL: 8.34, p = 0.039 5 year: Cox Lasso: 8.30, p = 0.040 RSF: 8.95, p = 0.030 GB: 11.53, p = 0.009 SL: 14.05, p = 0.003 Original Data Set Martin 1 year: Cox Lasso 0.59 (0.56–0.61) RSF: 0.67 (0.64–0.69) GB: 0.67 (0.65–0.70) SL: 0.67 (0.65–0.69) 2 year: Cox Lasso 0.58 (0.56–0.61) RSF: 0.67 (0.64–0.69) GB: 0.67 (0.64–0.69) SL: 0.67 (0.64–0.69) 5 year: Cox Lasso 0.58 (0.56–0.61) RSF: 0.67 (0.65–0.69) GB: 0.67 (0.64–0.69) SL: 0.67 (0.64–0.69) 1 year: Cox Lasso 7.19, n.s RSF: 5.54, n.s GB: 7.48, n.s SL: 8.67, p = 0.034 2 year: Cox Lasso 8.17, p = 0.043 RSF: 6.42, n.s GB: 4.53, n.s SL: 4.10, n.s 5 year: Cox Lasso: 11.37, p = 0.01 RSF: 9.27, p = 0.026 GB: 11.07, p = 0.011 SL: 11.82, p = 0.008 KOOS: knee osteoarthritis and outcome score, CI: confidence interval, GB: gradient boosted regression model, RSF: random survival forest, SL: super learner, GAM: generalized additive model, n.s: not significant Various features were considered most important predictive features (top three) by the assessed models. Years from injury to surgery were considered most important by four models (Random Forest, SL, GBM, GAM). Graft choice was considered most important by three models (Cox Lasso, GBM, GAM). Three models considered age at surgery most important (Random Forest, SL, GBM). Femur fixation was considered most important by three models (Cox Lasso, GBM, and GAM). A comprehensive list of the importance of the other features can be seen in Table 5 . Table 5 Model performance Author (year) Factors predicting outcomes (in order of importance) Martin Random forest : age at surgery age at injury years from injury to surgery KOOS QOL Cox model (lasso) : femur fix - susp/cort graft qt/BQT fix comb: interfer/susp Graft other Femur fix interf. Grand boosted regression : age at surgery years from injury to surgery graft age at injury KOOS QOL Super learner : age at surgery years from injury to surgery KOOS QOL Graft hamstring age at surgery Martin NA - External validation study Martin Cox-Lasso : graft choice femoral fixation KOOS QoL at time of surgery Years from injury to surgery Age at the time of surgery Random Forest : age at time of injury tibial fixation device fixation device combination GAM : Graft Years from injury to surgery Femur fixation other KOOS QOL at surgery GBM : Age at surgery Years from injury to surgery Femur fixation KOOS QOL at surgery Lopez Surgery Setting Operative Time BMI Age Race Johnson Systemic Inflammatory Disease Distal Tear Location Concomitant MCL Repair VAS Proximal Tear Location Ye Medial Meniscal Resection Participation In Competitive Sports Posterior Tibial Slope Graft Diameter Of PLB Male Gender Martin NA - External Validation Study Jurgensmeier Time to RTS VAS Pain Score at injury Time to surgery Age at injury Tear location Lu Time To RTS VAS At Injury Consultation Hypermobility Involvement In Noncontact Sports African American Race RTS: return to sport, VAS: visual analogue scale, PLB: posterolateral bundle, MCL: medial collateral ligament, BMI: body mass index, KOOS: knee osteoarthritis and outcome score, QOL: quality of life, GBM: gradient boosted regression model, GAM: generalized additive model, NA: not applicable The primary finding of this systematic review was that existing machine learning models to predict secondary injury or surgery after ACLR are variable in terms of discriminatory performance. Overall, Random Forest models were the most effective at predicting outcomes when using AUC, Brier, Calibration slopes and intercepts. Cox-Lasso was the most effective model when using concordance. Of the four studies reporting on AUC, values were relatively high, ranging from 0.77 to 0.997. However, of the four studies reporting on concordance, the mean values of all studies were closer to 0.5 than 1.0. Furthermore, there was variability when evaluating calibration. While the two studies reporting on Brier scores, calibration slope, and intercept reported minimal evidence of miscalibration in highest performing models, the four studies reporting on calibration error found significant evidence of miscalibration at either two and five-year follow-ups amongst 10 of 14 models assessed. Factors deemed important for secondary ACLR or injury (e.g. secondary meniscus injury, graft failure) were also variable from model to model and study to study. Machine learning has become incredibly popular in developing models to predict postoperative outcomes, and there is immense potential benefit in using these analyses to generate prediction models and calculators. However, this review demonstrates that there is still room for improvement in model performance. One recent study of 104 patients reported AUC values for several factors predictive of revision, ranging from 0.756 to 0.813 . These values fall in the range of the predictive models that reported on AUC included in this review. Some models in this review had AUCs over 0.95 for predicting secondary meniscus injuries and revision, suggesting strong discriminatory power . However, it is notable that other studies providing C-statistics reported relatively low discrimination, with mean values all being under 0.75. The findings in this review align with a recent systematic review on ML models in various orthopaedic sub-specialties . They found that in spine surgery, hip arthroscopy, total joint arthroplasty, and shoulder arthroplasty, the C-statistics ranged from 0.65 to 0.92, 0.51–0.94, 0.63–0.89, and 0.70–0.95, respectively. While some models from this review had low C-statistics (e.g., closer to 0.5), others had values closer to 1 . They also noted the lack of external validation and inconsistent adherence to predictive modeling guidelines. Therefore, it is possible that existing models investigating revision and secondary injury risk may be missing key important factors. External validation studies are essential to assess the generalizability of machine learning models. One included study attempted to externally validate the revision prediction model from the original NKLR dataset using the Danish Knee Ligament Registry (DKLR) . Concordance was similar between populations (DKLR: 0.68; NKLR: 0.68–0.69); however, there was significant evidence of miscalibration at one, two, and five years when evaluating the DKLR group. Furthermore, compared to the NKLR dataset, calibration error at one and five years was greater (4.89 versus 22.24 and 6.19 and 13.98 respectively) . The other external validation study assessing the STABILITY trial using the NKLR model reported a concordance of 0.71; however, it found significant evidence of miscalibration at two years. While two of six studies demonstrated strong calibration, these models have not been externally validated like that of the NKLR database. Having models demonstrate strong calibration and concordance at the two-year mark is incredibly important as one in 17 (6%) of ACLR patients will suffer a second ACL injury within two years of the index operation. Furthermore, rates of secondary ACL injury (e.g. ipsilateral or contralateral) at five, ten, and fifteen years have been reported to be 12%, 27%, and 31% . Continuous evaluation of established and novel machine learning algorithms is incredibly important for prediction calculators to translate effectively into clinical practice. Factors that were not included in the current review that may be important when considering secondary injury risk include concomitant lateral extra-articular tenodesis (LET) procedures, meniscus status, medial collateral ligament (MCL) injuries, and elevated posterior tibial slope (PTS) (or effects of bone morphology). The STABILITY I study, a large multicenter randomized controlled trial (RCT) comparing ACLR with and without LET, found that at 24 months postoperative, the LET group had a rupture rate of 4.1% compared to 11.2% in the non-LET group ( p = 0.004) . A secondary analysis from this trial demonstrated that younger age, greater posterior tibial slope, high-grade knee laxity, and earlier return to sport all contributed to increased odds of rupture. Larger hamstring autograft diameter was protective in reducing the odds of knee laxity in the form of asymmetric pivot shift . Several of these factors were not a part of the risk calculators developed from machine learning algorithms. Specifically, some studies suggest that an increased PTS may place strain on the ACL, increasing the risk of failure . Certain groups have proposed a threshold of 12 degrees, and have advocated for the use of slope-reducing osteotomies to reduce the PTS, especially in revision settings . Machine learning analyses offer immense potential in terms of predictive capacity, however it is clear that there is much room for improvement, especially in the field of predicting revision or secondary knee injury after ACLR. With the risk of revision still being an issue, this review advocates for including factors such as the inclusion of LET procedures, graft diameter, meniscus status, and elevated posterior tibial slope in developing these models. Furthermore, future studies are encouraged to continue to attempt to externally validate existing and novel models to assess generalizability. Demonstrating strong concordance or AUC and little evidence of miscalibration both in the short-term and long-term is essential in order to implement risk-calculators in the clinical setting. There are a few limitations to this review. First, there were only two inclusions that served as external validation studies, which limit the generalizability of the reported findings. Second, only 55% of studies reported adhering to the TRIPOD guidelines for diagnostic studies, indicating high variability in the quality of individual datasets and reporting of results. This limitation is also noted in a recent systematic review on ML models in orthopaedic trauma, which reported a TRIPOD statement adherence of 62% , highlighting the need for better adherence to reporting guidelines. Third, there were limited amounts of comparisons with traditional multivariate logistic regression analyses, preventing the ability to make conclusive statements about the superiority or inferiority of machine learning models when the two methods are compared. Only two studies in this review included NN models, which is another source of weakness. NN modelling would allow for the inclusion of image data and, thus, the creation of multimodal models that incorporate images and clinical variables. In this review, the NNs did not perform better than classical models, which may be because NNs require more resources to create and larger datasets to avoid overfitting. Ultimately, current ACLR prediction models mainly incorporate classical ML, as opposed to multimodel prediction models. Multicenter collaboration based on high-quality prospective databases and registries, with agreement between investigators on feature inclusion, is needed for high-quality ML prediction algorithms. Only nine studies were included in this review, all of which were level IV evidence, preventing the ability to perform a meta-analysis and pool machine learning performance statistics. The average quality of the included studies was fair, which limits the reliability of the findings and highlights the need for further high-quality research in this domain. Finally, it is important to note that these predictive models are preliminary and have not been assessed in a prospective cohort of patients. Future adequately powered longitudinal studies testing these models are needed to ascertain their external validity. Machine learning models designed to predict the risk of revision or secondary knee injury demonstrate variable discriminatory performance when evaluated with AUC or concordance metrics. Furthermore, there is variable calibration, with several models demonstrating evidence of miscalibration at two or five-year marks. A key limitation of this study is the lack of external validation of existing models, which restricts their generalizability. Future efforts should focus on validating current models in addition to developing and integrating multimodal neural networks to improve predictive accuracy and reliability. Further comparisons with traditional multivariate logistic regression analysis are also needed to validate the benefit of more advanced models. | Study | biomedical | en | 0.999996 |
PMC11699795 | It was reported that, after relining procedures for ill-fitting dentures, patients expressed a greater level of comfort with their new dentures . On the other hand, as a result of multiple laboratory procedures, material dimension changes, polymerization shrinkage, and residual monomer that may cause pain and mucosal reactions, many edentulous patients are not completely pleased with their dentures and maintain complaining from discomfort, pain, and inadequate retention while using the denture . Patients with compromised medical history that contra-indicate the use of dental implants require to have more valid and reliable treatment options for constructing more satisfactory complete dentures, as a result, providing patients with alternative treatment options and different manufacturing techniques is one of the main goals of merging modern innovations into dental clinics . Digital dentistry plays an extremely important role in removable dentures fabrication recently, it always requires a very accurate impression in order to produce retentive and well adapted denture. To digitally fabricate a prosthesis, a digital impression is obtained either from intraoral scanning of the residual ridge or extra-oral scanning of the master cast and send to the dental laboratory to create a digital model on which the denture is designed and manufactured either by additive or subtractive method . Computer-aided design (CAD) computer-aided manufacturing (CAM) systems prove to reduce complicated laboratory steps, time and effort consumed by the laboratory technicians, the edentulous patients and the dentists . Patient’s data is recorded and stored for future use if duplicate dentures are needed . Digital dentures showed satisfying clinical outcomes with a smoother, more regular surface than conventionally processed ones [ 9 – 14 ]. The digitally fabricated complete denture from resin discs that were produced under elevated temperature and pressure to overcome the problem of dimension changes of heat-polymerized acrylic resin . Complete denture retention and adaptation are greatly affected by many factors as method of fabrication, denture extension, shape and compressibility of the supporting tissues, the thickness of the denture base, the material from which denture constructed. The digital superimposition of scanned denture fitting surface with either the scanned oral tissues or a cast is a recently introduced approach to evaluate and compare the adaptation of the constructed dentures . Patients with significant skeletal problems (class II or III) or who went through severe osseous surgery or any hereditary or acquired osseous abnormality find it challenging to place the denture properly on the supporting ridge. If the patient suffers from xerostomia, excessive flabby ridges, any pathology of the oral mucosa, or any inflammatory changes, candidiasis, hyperplasia, neurological disorders, and malignancies If the denture creates a major speech problem, poor esthetics, or an unsatisfactory jaw relationship. Tissue preparation: Before making the relining impression, patients were advised to remove their previous dentures from their mouth for at least three to four days and massage their soft tissues two to three times per day to simulate blood flow and improve the recovery of any small ulcers or inflammations with the application of proper medicaments. Denture preparation: Occlusion must be balanced before the relining process to guarantee even occlusal contact and that the denture will not move or tilt when the patient is asked to close his mouth during impression material setting. To prevent contact with the reflected tissues, denture borders were lowered by around 2 mm, except the denture posterior border (post dam). The denture tissue surface was relieved by about 1–2 mm to create even space for impression material. Border molding with putty consistency rubber base impression material (Speedex addition silicon, Coltene, Switzerland) was done, then the denture fitting surface was loaded with light body rubber base impression material, which should flow evenly to cover the entire fitting surface and the borders of the denture with a thin continuous layer. The denture was then inserted inside the patient's mouth, and border molding was completed manually and functionally. Before boxing and pouring the relining impression, the maxillary denture was scanned from all surfaces, fitting (impression surface), polished, and occlusal surfaces using a desktop scanner (3Shape E2, Copenhagen, Denmark.) the scanned images were exported to the computer software as standard tessellation language file (STL) . STL file was imported into a dental CAD/CAM milling machine software program (Exocad DentalCAD; exocad, GmbH, Germany) to fabricate a new maxillary complete denture after finalizing the denture design digitally from PMMA discs of 25 mm length (Yamahatchi dental MGF, Japan) . After milling, finishing, denture characterization were completed using three shades of composite. (Visio,lign, Bredent, GmbH, Germany) . Fig. 2 Scanned maxillary denture with relining impression Fig. 3 CAD/CAM milled denture Fig. 4 A Finished milled denture, B Finished relined denture Denture processing was completed by flasking the denture into a metal flask, immersed it into boiling water for wax elimination, packing the heat cure acrylic resin (Acrostone, Egypt), curing the resin by long cycle in a hot water bath at 72 ◦C for 6.5 h, then deflasking, finished and polished was done . A pilot study was conducted measuring retention and adaptation of 5 samples of the relined dentures group and the CAD/CAM milled dentures group. The Cohen’s d effect size was measured from the pilot study. Based on the Cohen’s d effect size with assuming a type I error of 0.05 and a study power of 0.9, the sample size required to detect a significant difference between the two groups was increased to include 12 samples. A (19-gauge) orthodontic wire in the form of a hook was secured on the center of the palate at a point of intersection of the lines connecting the hamular notches, the right and left canines with the midline by self-cured acrylic resin material (Acrostone, Egypt), then the denture placed on the supporting tissues intraorally . Fig. 5 Denture with the hook seated intraoral The patient was in an upright position. A digital force gauge (Force Gauge HF- 50N) was linked to the hook and a downward pulled force was applied slowly in a vertical direction opposite to that of the denture path of insertion until the denture was displaced, the force value recorded at the time denture displacement was registered. After repeating this procedure three times, the average was determined . Fig. 6 Retention measurement After scanning of the denture fitting surface, the STL file was imported to a three-D measurement program (Geomagic Control X, 3D Systems, United States) software to flip the fitting surface of the dentures to resemble the intra-oral tissue surface to build and superimpose STL files using first initial alignment and then best-fit alignment . For every measurement point, the root mean square (RMS) (mm) was computed considering both positive and negative-values, The adaptation deviations of the repaired dentures were calculated as done by the previous study . Fig. 7 A Color map of group A adaptation. B Color map of group B adaptation. Pressure from the denture fitting surface to the oral tissues is indicated by yellow to red colors. The denture fitting surface and oral tissues are separated by a blue color. The green color denotes that the oral tissues and the denture fitting surface are in contact Statistical analysis for retention values and data was tested for normality using the Shapiro–Wilk test. Between-group comparisons were conducted using an independent t test, while within-group comparisons were conducted using paired t test. The significance level was set at p = 0.05. statistical analysis was completed by SPSS software (version 25). While statistical analysis for adaptation, data descriptive statistics consisted of mean, standard deviation, median, minimum, and maximum values. Data was tested for normality using the Shapiro–Wilk test and an independent t test was used for between-group comparison. The significance level was set at 0.05. Statistical analysis was completed by SPSS software (version 25). Retention values were significantly higher after two weeks than at the insertion time ( p < 0.001) within both groups. Group B showed significantly higher retention than group A at the insertion time ( p = 0.007) and 2 weeks later ( p < 0.001). As showed in Table 1 Fig. 8 . Table 1 Descriptive statistics, the results of independent t test in the rows for between-group comparison and the results of paired t test in the columns for within group comparison at different time intervals Group A Group B p- value Insertion Time Mean (SD) 5.0 (0.2) 5.2 (0.2) 0.007* Median (Range) 5.1 (4.7—5.3) 5.3 (5.0—5.4) After 2 weeks Mean (SD) 5.5 (0.1) 6.1 (0.2) < 0.001* Median (Range) 5.4 (5.3—5.7) 6.1 (5.8—6.4) p- value < 0.001* < 0.001* Fig. 8 Bar chart representing the retention values of both groups at denture insertion time and two weeks after Group A showed significantly higher RMS (less adaptation) than group B ( p < 0.001) with a mean difference 0.47 and 95%CI: (0.46,0.48). as showed in Table 2 Fig. 9 . Table 2 Comparison between the results of independent t test of RMS values of the two groups Descriptives Milled Group Relined Group p- value Mean 1.22 0.74 < 0.001* SD 0.01 0.01 Median 1.22 0.75 Min 1.20 0.72 Max 1.24 0.76 Fig. 9 Bar chart representing the mean RMS in both groups The success of any prosthesis in terms of speech, function, appearance, and denture retention is thought to be greatly influenced by patient happiness. The primary factor considered when assessing the clinical performance of complete dentures is their ability to remain in the mouth during function. Wearers of CDs always have one basic complain: insufficient retention . The current in-vivo study was conducted to assess the degree of patients’ satisfaction and compare the adaptation and retention gained from relining loose maxillary denture conventionally using flasking procedure versus a newly CAD/CAM milled maxillary denture obtained from scanning the relining impression to fabricate complete denture digitally. The current study findings stated that all patients were satisfied with both of their dentures regarding retention, speech and chewing ability (occlusion) this is mainly because the digital dentures were milled from the scanned old denture with the same teeth setting and relationship, so the occlusion and speech weren’t changed. On the contrary, Kang YJ et al. did a cross-over single-blinded randomized in-vivo study to compare between the digitally fabricated complete dentures versus conventionally fabricated complete dentures. It was found that masticatory efficiency and speech with the digitally fabricated complete dentures were less favorable than the conventionally fabricated complete dentures, however, comparable results were obtained for denture adaptation and patient satisfaction between the two denture groups. Saponaro et al. had studied and compared patient satisfaction of digitally constructed dentures and it was shown that, 3 (6.25%) of the 48 participants reported having bad esthetic from their CDs, while 1 of the 48 individuals (2.08%) suffered from altered phonetics. On the second visit, 24.44% of the digital dentures were not placed because of poor retention, an improper centric relationship, poor aesthetics, and altered phonetics . Group B (relined dentures) showed a statistically- significant higher retention values at all follow up periods and also more denture adaptation compared to group A (milled dentures), this may be explained by the fact that relining impression was taken under patient’s biting force, so it is muco-compressive impression recorded the areas of the peripheral soft tissues under compression. The same outcome was revealed from an in-vivo study comparing digital and conventional impression technique in edentulous maxillae and it was revealed that conventional impression-taking is more precise than digital impressions in recording the peripheral soft tissues responsible for creating strong peripheral seal areas to ensure retentive removable prostheses . Over-extended borders of digitally constructed maxillary dentures was found to be another reason that could explain their reduced retentive values compared to conventionally constructed dentures as shown in a study done to compare the digitally and the conventionally constructed complete denture, it was claimed that over-extended borders of the digital dentures were obvious at the time of denture insertion caused denture displacement and reducing the retention of digital denture, however, after two weeks and three months follow up periods, no significant differences were found between both denture groups . However, in this study overextended denture borders was minimal and not considered the main cause of reduced retention of digitally constructed dentures. The scanner employed in this study was able to accurately scan the borders of the existing dentures, occlusal surface, and the relining impression surface (fitting surface) of the old denture with precise capturing of complete denture contours and soft tissue details . however, the fitting surface of the milled denture revealed a highly smooth texture that hinders improved appropriate retention, border seal, and physical adaptability to the underlying tissues. Another study evaluated denture retention of digitally constructed dentures (milled and printed). Retention values of both denture groups after 2 weeks follow up period were significantly improved from baseline, the reason for this might be related to the settling of the denture base, functional adaptation, and neuromuscular coordination of the patients . It should be known that there is no intraoral scanner could produce a true precise functional impression with proper borders extension and shape. Nonetheless, without functional molding of the vestibular mucosa and slight compression on the highly-resilient posterior palatal seal areas, an acceptable border and palatal seal wouldn’t be achieved, which consequently, affect denture retention negatively . With the same concept, a study was done to compare the accuracy of scanning edentulous maxillary arch using different types of intraoral scanners versus scanning the conventional impression and the referring casts (laboratory scanners) and the results showed that laboratory scanning revealed more accurate results . Other adverse outcomes reported with digital complete dentures regarding differences in VDO measurements, poor esthetics, bad teeth setting, and impaired speech . In the present study, patients had their existing loose dentures constructed from 2 years or less with proper VDO and correct centric relation to avoid teeth wear, low VDO and to ensure proper occlusion for the digitally constructed denture. Moreover, many other studies confirmed that milling burs size, shape, and sharpness played a significant role in the shape of the final milled prothesis regarding its surface roughness and surface adaptation which limits the accurate reproduction of denture fitting surface details that will negatively affect the intimate adaptation with oral mucosa . However, in pilot cohort research done by Bidra et al., it was found that while patients expressed great satisfaction with phonetics and esthetics, just 50% of them were dissatisfied with the retention, stability, or adaptability of their digitally produced dentures . In contrast, Kattadiyil et al. reported no discernible change in phonetics or esthetics and discovered a markedly higher retention rate for milled maxillary complete dentures when compared with conventional ones made for the same patient. Patients who had digital dentures scored better overall, and for denture base contour, fit, stability, retention, and extension. This was explained by the fact that, in contrast to traditional ones, there was no polymerization shrinkage throughout the fabrication process, which resulted in greater fit and retention [ 42 – 44 ]. The idea of incorporating relining impression of loose old denture with scanning, designing and fabricating complete dentures digitally is reliable and could result in successful denture. although this digital manufacturing method could not improve denture retention and adaptation compared to the conventional relining procedure, but it has the advantages of avoiding material discrepancies during flasking and curing, in addition to avoiding complicated laboratory steps and long processing time. This study has been tried to replace conventional laboratory steps of denture relining with more digital, easy, and time saving ones, but some limitations were observed; considering that intraoral scanners are reliable devices for imaging oral tissues with great accuracy, however, recording the tissues with slight pressure is not achieved till now. Future studies are recommended to investigate the possibility of substituting the conventional clinical steps of denture construction with digital ones to convert prolonged clinical appointments to be less time consuming and more reliable with analysis related to the functional effectiveness of the resultant dentures should be considered. | Other | biomedical | en | 0.999998 |
PMC11699797 | Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is an acquired multi-system disease characterized by persistent fatigue and exertional intolerance with a disproportionate worsening after physical or cognitive exertion referred to as post-exertional malaise (PEM). Furthermore, it is accompanied by a variety of other symptoms that are related to immunological, cognitive, and autonomic dysfunction. The worldwide prevalence was estimated at 0.89% in a pre-pandemic meta-analysis, though estimates vary according to the case definitions used. 1 While there is evidence for a genetic predisposition to ME/CFS, 2 it is most often triggered by viral infections. 3 Since the COVID-19 pandemic, SARS-CoV-2 has become the leading viral trigger for ME/CFS. 4 Given the high incidence of COVID-19, the prevalence of ME/CFS is expected to strongly increase. The diagnosis of ME/CFS is based on established diagnostic criteria, with the most frequently used being the Canadian Consensus Criteria (CCC) and the Institute of Medicine (IOM) criteria. These require the presence of key symptoms including lingering fatigue, PEM, unrefreshing sleep, cognitive impairment, and/or orthostatic intolerance. 5 ME/CFS diagnosis relies so far on these clinical criteria, although most patients present with objective clinical findings including diminished handgrip strength or autonomic dysfunction. 4 The pathophysiological mechanism of ME/CFS is not yet known, but numerous studies suggest dysregulation of the immune system. Elevated antibodies were found in ME/CFS patients in several studies, most frequently against adrenergic and muscarinic receptors. 6 , 7 Elevated β1 and β2 adrenergic receptor autoantibodies (β1/β2 AR-AB) and M3/M4 acetylcholine receptor autoantibodies (M3/M4 AChR-AB) are likely to modulate the autonomic nervous system function and vasoregulation. 7 , 8 Levels of these autoantibodies were found to be associated with symptom severity and structural alterations in the central nervous system in both post-COVID syndrome (PCS) and ME/CFS. 9 , 10 , 11 , 12 , 13 The β2 AR-AB was the best marker for distinguishing PCS from recovered patients, and levels of β2 AR-AB were associated with both fatigue and vasomotor symptoms in PCS-ME/CFS patients. 10 , 14 Apart from elevated autoantibodies a chronic activation of inflammatory pathways and an alteration in memory B-cells were shown. 15 , 16 The goal of immunoadsorption (IA) is to improve the clinical condition of patients with autoantibody-mediated diseases by selectively removing immunoglobulins from circulation via extracorporeal adsorption from their plasma. 17 , 18 Unlike plasmapheresis, IA employs a high-affinity column that specifically binds and eliminates immunoglobulins including autoantibodies and immune complexes, leaving other plasma components behind. 18 Given the previous evidence for a potential role of autoantibodies in PCS and ME/CFS and the effectiveness of IA in postinfectious ME/CFS, we here aimed to evaluate the effectiveness of IA treatment specifically in post-COVID ME/CFS patients. 19 , 20 Effectiveness was measured by assessing patient-reported outcomes and hand grip strength. We hypothesized that IA would lead to clinical improvement in ME/CFS patients four weeks after treatment, with an additional beneficial effect from repeat IA in responders with relapse. This prospective cohort study, conducted at the outpatient department for immunodeficiencies at the Institute of Medical Immunology, Charité - Universitätsmedizin Berlin, recruited patients between October 2022 and October 2023. Patients received IA treatment and follow-up visits for 12 months. Responders to the first cycle of IA were offered a second cycle. An interim report of the first 10 patients was published in October 2023. 21 This study was being conducted within the National Clinical Studies Group (NKSG), a clinical trial and translational research platform focused on developing therapies for PCS and ME/CFS, funded by the German Ministry of Education and Research (BMBF). 22 Patients were diagnosed and recruited at the outpatient department for immunodeficiencies at the Institute of Medical Immunology at the Charité - Universitätsmedizin Berlin. The diagnosis of ME/CFS was based on the modified Canadian Consensus Criteria (CCC) and a minimum of 14 h of PEM. 23 , 24 Inclusion criteria encompassed elevated β2 AR-AB at the time of screening and SARS-CoV-2 infection at the time of disease onset. All patients had to provide proof of SARS-CoV-2 infection by positive PCR, antigen test, or serology (SARS-CoV-2 nucleocapsid protein antibodies). Five sessions of IA were administered at the Department of Nephrology at Charité - Universitätsmedizin Berlin. IA treatment was conducted in an outpatient setting over a ten-day period, with sessions spaced no more than two days apart. The TheraSorb® – Ig omni 5 adsorber (Miltenyi Biotech B.V. & Co. KG, Bergisch Gladbach, Germany) was used for removal of human lambda and kappa chains containing immunoglobulins IgG (subclasses IgG1-IgG4), IgA, IgM, IgE, and immune complexes as well as free lambda and kappa light chains from the plasma. To ascertain the efficacy of IA, total serum immunoglobulin levels were assessed via immunoturbidimetry before, during, and after treatment. Antibodies against β1, β2, M3, and M4 receptors were determined using ELISA technology by CellTrend GmbH, Luckenwalde, Germany, both before and after treatment. Intra- and inter-assay coefficients of variation for the ELISAs provided by CellTrend were: b1 AR-AB 9.6%/12.0%, b2 AR-AB 4.2%/3.8%, M3 AchR-AB 5.9%/10.1%, and M4 AchR-AB 7.3%/12.5%. Pre- and post-treatment samples were analyzed in the same assay run. The upper normal levels of autoantibodies were determined based on validation studies of a healthy control group and defined as being larger than the 90th percentile of a healthy control group (>14U/l for β2 AR-AB). For patient-reported outcomes, questionnaires were filled out before, during, and after the treatment in monthly intervals and validated by physicians. Patients’ health-related quality of life was assessed using the 36-Item Short-Form Survey (SF-36), with scores ranging from 0 to 100, with 100 indicating no limitations. 26 Response to IA treatment was defined as a minimum increase in the SF-36 physical functioning domain (SF-36 PF) of 10 points from baseline to four weeks post IA, indicating a clinically relevant improvement. 27 Fatigue was evaluated using the Fatigue Severity Scale (FSS), ranging from 9 to 63, with a total score of 36 or more suggesting relevant fatigue. 28 Additionally, disease-related disability was scored according to the Bell score, rating the restriction in daily functioning on a scale from 0 to 100, with 100 indicating no restriction. 29 PEM was evaluated using the DePaul Symptom Questionnaire (PEM-DSQ), ranging from 0 to 20 for both severity and frequency, PEM duration was assessed ranging from 0 to 6, with higher values indicating higher PEM severity. 24 Further cardinal symptoms of both PCS and ME/CFS, including fatigue, muscle pain, immunological symptoms, and cognitive impairment, were scored on a numeric rating scale (NRS) from 1 to 10, with 10 indicating maximum symptom severity (not formally validated). Autonomic dysfunction was evaluated according to the Composite Autonomic Symptom Score (COMPASS31), ranging from 0 to 100, with 100 indicating maximum autonomic dysfunction. 30 Handgrip strength (HGS) of the dominant hand was measured using a digital hand dynamometer (EH101, Deyard, Shenzhen, China) in two separate sessions. Rest time between sessions was 60 min, in which no strenuous physical activity took place. Before starting the measurement, patients were shown two separate demonstrations of how the dynamometer should be used. Patients sat in an upright position facing a standard table during measurements of HGS. The forearm of the dominant hand was placed on the table in full supination holding the dynamometer. Under supervision and verbal motivation, the handle was pulled 10 times with maximum force for three seconds, followed by a five-second relaxation phase. The dynamometer displayed the highest value reached within these three seconds (measurement in kg), this single value was then recorded. The attempt with the highest reading out of ten repetitions was recorded as the maximum strength (Fmax), and the average strength (Fmean) of each session was calculated. 31 Further, the Reactive hyperemia index (RHI), which is a measure for endothelial function, was assessed using a peripheral arterial tonometry device . The technology measures the pulsatile volume changes in the vascular beds of the finger using optical sensors. The subjects were in supine position for a minimum of 15 min before measurements, in a quiet, temperature-controlled room. Occlusion of the brachial artery was performed on the nondominant upper arm using a standard blood pressure cuff. The occlusion pressure was at least 60 mmHg above the systolic blood pressure. Upon release of the cuff, the resulting surge in blood flow causes vessel dilation. Each recording consisted of five minutes of baseline measurement, five minutes of occlusion measurement, and five minutes post-occlusion measurement. The post-occlusion dilation relative to pre-occlusion levels is calculated as the RHI. Endothelial dysfunction was defined as an RHI ≤ 1.67 based on previous cohort studies. 32 Statistical analyses were conducted using R version 4.3.0 and RStudio version 2023.03.1. A linear mixed-effects model (LMM) was employed to assess changes in multiple outcome variables across different time points. The analysis was performed using the lmer function from the lme4 package (version 1.1-35.5), and ggplot2 (version 3.5.0) was utilized for data visualization. For each outcome variable, the LMM included time as a fixed effect and patient number as a random effect to account for within-patient correlation. The mixed model was fitted using restricted maximum likelihood (REML), and statistical significance was evaluated using t-tests with p -values approximated through Satterthwaite's method for degrees of freedom, implemented via the lmerTest package (version 3.1-3). Missing data were accounted for by using all available observations in the model, allowing for the estimation of fixed and random effects without listwise deletion, assuming data are missing at random. For comparisons between groups the non-parametric Whitney-U test was used. Correlation analysis was performed using the nonparametric Spearman coefficient. A two-tailed p -value of <0.05 was considered to provide evidence of a statistically significant result. The β2 AR-AB levels of 402 patients, who met the CCC for post-infectious ME/CFS, were measured between June 2022 and March 2023. Out of these 402 patients, 189 (47%) had elevated β2 AR-AB levels. From this group, patients who planned IA treatment and fulfilled inclusion criteria were offered to participate in this trial and 23 were recruited for the study between September 2022 and October 2023. Three patients withdrew their consent to participate prior to the treatment due to concerns about the burden of study participation. 20 patients completed a six-month follow-up resulting in an 87% retention rate, suggesting that the protocol was generally acceptable to those who proceeded with the treatment. The five IA treatment sessions were completed for all patients within a 10-day period in an outpatient setting. Sessions lasted between 4.5 and 9 h and were followed by a minimum of one day and a maximum of two days of rest. In five out of 20 patients, a Shaldon's catheter was required for vascular access; for the other patients peripheral venous puncture was sufficient. One patient experienced a thrombosis of the internal jugular vein as a side effect of the catheter. Otherwise, no severe side effects of IA treatment were reported. The median disease duration was 22 months (IQR: 15–31) at the time of inclusion. Seven patients were male and 13 were female with a median age of 40 (IQR: 36–51). All patients had a severe degree of disability with a median Bell score of 30 (range 20–40), corresponding to reduction of the functional state to 50% and being usually housebound. Individual patient characteristics are displayed in Table 1 . Table 1 Cohort characteristics and response to treatment as assessed by the SF36 PF. Patient Gender (M/F) Age (years) Time since COVID-19 (months) SF36 PF pre IA SF36 PF post IA (M1) ΔSF36 PF Responder to IA (yes/no) 1 F 36 39 15 70 55 Yes 2 M 41 25 25 70 45 Yes 3 F 56 15 25 65 40 Yes 4 F 37 21 45 85 40 Yes 5 F 52 23 45 80 35 Yes 6 M 59 31 35 65 30 Yes 7 F 51 38 15 35 20 Yes 8 F 31 19 40 60 20 Yes 9 F 59 32 20 35 15 Yes 10 F 50 31 15 30 15 Yes 11 F 36 10 15 25 10 Yes 12 M 36 9 40 50 10 Yes 13 M 46 32 20 30 10 Yes 14 M 57 14 50 60 10 Yes 15 M 33 23 25 30 5 No 16 F 36 15 25 30 5 No 17 F 44 14 5 5 0 No 18 M 33 39 60 60 0 No 19 F 37 15 30 25 −5 No 20 F 39 19 50 45 −5 No F: female; IA: immunoadsorption; M: male; M1: month 1 post immunoadsorption; SF36 PF: Short Form-36 physical functioning. There was a positive correlation between the immune score, which depicts the severity of the lymph node pain, throat pain, and flu-like symptoms, and the levels of antibodies against β1 AR ( r = 0.53, p = 0.017), β2 AR ( r = 0.46, p = 0.040), M3 AChR ( r = 0.60, p = 0.006), and M4 AChR ( r = 0.60, p = 0.006) at baseline. Otherwise, no significant correlation was found between the autoantibody levels and the clinical presentation. There was a mean increase in the SF-36 PF of 17.75 points ( CI : 13.41–26.16, p < 0.001) four weeks post IA. The corresponding Cohen's d was calculated as 1.19, indicating a large effect size. There were, however, four patients with an increase of 10 points, which is considered as a clinically meaningful but small effect. 27 As seen in Fig. 1 , though the SF-36 PF scores tended to decrease between three and six months post IA, at month six post IA a significant mean improvement of 12.81 points ( CI : 4.99–20.61, p = 0.002) remained. Fourteen out of 20 patients (70%) responded to the treatment as defined by an increase of at least 10 points in the SF-36 PF four weeks post IA suggesting a clinically meaningful improvement. Seven patients were non-responders according to this definition; however, two of these showed a delayed response at month 2. Fig. 1 Course of the mean 36-Item Short-Form Survey physical functioning domain (SF36 PF) over the study period. The duration of IA therapy is indicated by the green bar. A higher score indicates less restriction in physical functioning. Error bars represent 95% confidence intervals. Data were analyzed using a linear mixed-effects model fitted by restricted maximum likelihood (REML), with t-tests computed using Satterthwaite's method for degrees of freedom with significance levels indicated as ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. From top to bottom, the panels display the trajectories of the SF36 PF for: a) the whole cohort ( n = 20), b) responders to the treatment defined by a ≥ ten-point increase in the SF36 PF at month 1 post IA ( n = 14), and c) non-responders to the treatment defined by a ≤ ten-point increase in the SF36 PF at month 1 post IA ( n = 7). There was no significant difference in age, symptom severity, disease duration, or level of β2 AR-AB between responders and non-responders. However, among female patients, responders had a significantly higher maximum HGS at baseline ( Mdn = 23.5 kg, IQR: 17.7–25.5 kg) compared to non-responders ( Mdn = 9.8 kg, IQR: 8.53–11.05 kg) ( z = −2.62, p = 0.006, r = 0.73). Serum IgG, IgA, and IgM levels were collected from all patients at baseline, before each treatment, and after treatment. Compared to baseline, IgG, IgA, and IgM were significantly decreased during IA treatment ( p < 0.001) with a mean IgG reduction of 8.66 g/l ( CI : 8.06–9.26 g/l) (79%, CI : 73–84%), mean IgA reduction of 1.3 g/l ( CI : 1.2–1.51 g/l) (68%, CI : 63–78%), and mean IgM reduction of 1.1 g/l ( CI : 0.84–1.34 d/l) (76%, CI : 58–93%) at day five of the treatment. Fig. 2 a–c shows the course of immunoglobulin levels over time. β2 AR-AB decreased in parallel with the immunoglobulins with a mean reduction of 26.57 U/l ( CI : 20.11–33.02 U/l) (77%, CI : 58–95%). Fig. 2 Course of immunoglobulin and autoantibody levels over the study period ( n = 20), the duration of IA therapy is indicated by the green bar. Error bars represent 95% confidence intervals. Data were analyzed using a linear mixed-effects model fitted by restricted maximum likelihood (REML), with t-tests computed using Satterthwaite's method for degrees of freedom with significance levels indicated as ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. From left to right, the panels display the trajectories of: a) immunoglobulin G (IgG), b) immunoglobulin A (IgA), c) immunoglobulin M (IgM), d) β1 adrenergic receptor autoantibodies (β1 AR-AB), e) β2 adrenergic receptor autoantibodies (β2 AR-AB), f) M3 acetylcholine receptor autoantibodies (M3 AchR-AB), and g) M4 acetylcholine receptor autoantibodies (M4 AchR-AB). Patients reported improvements in several key clinical symptoms at four weeks post IA. There were significant improvements in fatigue, as measured by the SF-36 energy/fatigue domain, and in pain, as measured by the SF-36 pain domain, following IA treatment. These improvements remained significant through month six. The maximal change was observed two months after IA, with a mean increase of 19 points ( CI : 11.61–26.39, p < 0.001) and 22.63 points ( CI : 13.28–31.96, p < 0.001) from baseline, respectively, as shown in Fig. 3 . Fig. 3 Clinical symptom progression over the study period ( n = 20). The duration of IA therapy is indicated by the green bar. Error bars represent 95% confidence intervals. Data were analyzed using a linear mixed-effects model fitted by restricted maximum likelihood (REML), with t-tests computed using Satterthwaite's method for degrees of freedom with significance levels indicated as ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. From left to right, the panels display the trajectories of: a) 36-Item Short-Form Survey energy/fatigue domain (SF36 energy/fatigue), where a higher score indicates less fatigue, b) 36-Item Short-Form Survey pain domain (SF36 pain), where a higher score indicates less pain, c) post-exertional malaise (PEM) as assessed by the DePaul Symptom Questionnaire (DSQ-PEM), higher scores indicate more severe PEM, and d) Composite Autonomic Symptom Score (COMPASS31), where a higher score indicates more autonomic symptoms. Furthermore, patients reported a lasting improvement in autonomic symptoms, as shown in Fig. 3 d. Improvements were most noticeable in the orthostatic, secretomotor, and gastrointestinal domains of the COMPASS31, which are shown in Figure S4 in the Supplement , as well as in the total score with a mean decrease of 12.23 points ( CI : 19.06–5.4, p = 0.001) at month one. All patients experienced severe PEM lasting at least 14 h, as specified by the inclusion criteria. Both frequency and severity of post-exertional symptoms significantly decreased after IA according to the DSQ-PEM. The total score is shown in Fig. 3 c, remaining significantly decreased through month six. Additionally, there was a significant mean decrease of 4.9 points ( CI : 1.34–8.46, p = 0.009) in fatigue as assessed by the FSS at month one after IA as shown in Figure S3 in the Supplement . According to the symptom scores (NRS 1–10), there were also improvements in muscle pain, immunological as well as cognitive symptoms, that remained significant through month 6 with their maximum decrease between months two and three after IA as shown in Figure S1 in the Supplement . The improvement in the degree of disability according to the Bell score post IA reached statistical significance only at months two, four, five, and six, with a maximum mean increase of 5.18 points ( CI : 1.24–9.12, p = 0.013) at month two post IA as shown in Figure S2 in the Supplement . To further quantify physical improvement, we measured repeat HGS as an objective marker of muscle fatigue. There were improvements, especially in repeat HGS before and after treatment in female patients as shown in Fig. 4 . The mean HGS during the second measurement was significantly increased six months post IA by a mean of 20% ( CI : 2–39%, p = 0.042) indicating a better recovery of strength within the 60 min of rest time between HGS measurements. In female responders ( n = 9), both mean and maximum HGS were significantly increased as early as four weeks post-IA (not shown). Fig. 4 Course of mean handgrip strength (Fmean) in % in female patients ( n = 13) measured a) initially and then b) repeated after one hour over the study period. The duration of IA therapy is indicated by the green bar. Error bars represent 95% confidence intervals. Data were analyzed using a linear mixed-effects model fitted by restricted maximum likelihood (REML), with t-tests computed using Satterthwaite's method for degrees of freedom with significance levels indicated as ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. As a measure of endothelial dysfunction, the reactive hyperemia index (RHI) was assessed using an EndoPAT device. Six patients, three responders and three non-responders, had a reduced RHI (<1.68). There was no significant change in the RHI at six months post IA. Responders to the first IA treatment were offered a second IA treatment within six to 12 months after completing the initial cycle, when their physical function or symptoms worsened again. The course of physical functioning according to the SF-36 PF in the seven patients receiving a repeat IA is shown in Fig. 5 . Symptoms improved again four weeks after the second IA treatment but then remained at a similar level as after the first IA treatment. Fig. 5 Course of 36-Item Short-Form Survey physical functioning domain (SF-36 PF) over the study period in patients who received a second cycle of IA therapy ( n = 7). The duration of IA therapy is indicated by the green bar. Error bars represent 95% confidence intervals. Data were analyzed using a linear mixed-effects model fitted by restricted maximum likelihood (REML) for each cycle, with t-tests computed using Satterthwaite's method for degrees of freedom with significance levels indicated as ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. There is increasing evidence that autoantibodies, including those targeting β-adrenergic and muscarinic acetylcholine receptors, may contribute to the pathophysiology of PCS and ME/CFS. We here provide evidence from an observational study that depletion of autoantibodies by IA can lead to improvement in overall physical functioning, as well as the severity of several key symptoms, including PEM, fatigue, pain, immunological, cognitive, and autonomic symptoms in a subset of post-COVID ME/CFS patients. The clinical improvements observed after autoantibody removal via IA support the hypothesized involvement of these autoantibodies in the pathophysiology of ME/CFS and PCS in the responders. Meanwhile, a smaller group of patients did not show a response to the treatment. This highlights the potential diverse mechanisms underlying this condition, and indicates that autoantibodies may play a role only in a subgroup of patients. IA has demonstrated clinical improvements in post-infectious ME/CFS patients in two small pre-pandemic trials conducted by our group. 19 , 20 Although in most patients symptoms worsened after a few months, IA can induce longer lasting improvement for more than 12 months in a subset as observed in our previous study in postinfectious ME/CFS. 20 In this study, clinical improvements generally peaked between months two and three, then gradually declined. However, even after six months, statistically significant improvements compared to baseline were still evident. It is important to acknowledge that the observed improvement in SF36 physical functioning does not apply to all patients as there were several non-responders to the treatment and four patients had only a small improvement. Among these, some patients showed improvement only after a delay of two or three months, while others did not demonstrate any noticeable improvement. Thus, it is important to study the efficacy of autoantibody-depleting therapies in larger controlled trials. Further identifying factors predicting individual responses would support the clinical findings. To address this question, we are currently conducting several additional analyses to identify potential biomarkers for treatment response. These analyses include a comprehensive B cell subtyping using CyTOF analysis, the measurement of further autoantibodies, and markers of immune activation, hypoperfusion, microclots, and SARS-CoV-2 persistence. Although the main effect of IA is the depletion of autoantibodies, there is some evidence that memory B cells can be affected by IA. 19 A potentially important finding is that responders had a higher baseline maximum HGS, suggesting they have less severe muscular or mitochondrial impairment. 35 We could not identify any other significant differences in the clinical phenotypes of responders compared to non-responders. However, ME/CFS patients are a heterogeneous group, and there may be comorbid conditions, such as structural neuroanatomic abnormalities, that may have been overlooked in our patient cohort. Growing evidence suggests that SARS-CoV-2 can trigger autoimmune processes, contributing to long-term effects of COVID-19. Dobrowolska et al. summarized recent findings indicating that PCS can develop autoantibodies against a range of antigens, including those specific to the immune and cardiovascular systems, the thyroid, and rheumatoid-specific targets, G-protein coupled receptors, and more. However, the clinical significance of most of these autoantibodies remains unclear. 36 Several studies found elevated autoantibodies against β-adrenergic and muscarinic receptors in PCS patients and demonstrated an association between the levels of these autoantibodies with fatigue, neurological symptoms and pain. 9 , 10 , 11 , 12 , 13 , 37 , 38 Importantly, two recent studies by Chen et al. as well as Santos Guedes de Sa et al. were able to induce similar symptoms in mice by transferring IgG from PCS patients. 39 , 40 Remarkably, Chen et al. showed that patterns of transferred symptoms varied depending on the plasma proteome signature of the patients. IgG from those with neuronal or immune involvement induced pain, while IgG from those with muscular involvement impaired motion in the mice. 39 In the study by Santos Guedes de Sa et al., patients showed a broad pattern of autoantibodies reactive with neuronal and endothelial tissues. IgG from individual patients induced distinct symptoms such as pain, hypersensitivity or loss of coordination in mice. 40 These findings provide further evidence for the diversity of autoantibodies in PCS, possibly driven by prolonged inflammation, infection of various cell types and tissues, and reactivation of Epstein–Barr virus. Our IA study shows that removing autoantibodies generally led to disease improvement in a subset of patients; however, it remains unclear whether the benefits are primarily due to the removal of β2 AR-AB or other specific autoantibodies. Furthermore, decreases of β2 AR-AB and immunoglobulins were seen in all patients, independent of response. The clinical improvement observed after autoantibody removal via IA in a majority of patients supports the hypothesized role of these autoantibodies in the pathophysiology of PCS and ME/CFS in the responders. Although IA has a rapid and high clinical efficacy in a subset of patients, the clinical benefits of IA are in some patients temporary, and the procedure is strenuous. During our trial, one case of internal jugular vein thrombosis occurred as a side effect of the catheter. Although the patient experienced no long-term effects following temporary anticoagulation, the risk of such complications should be considered. Furthermore, it is a highly specialized procedure, available only in select medical centers with the necessary equipment and expertise. B-cell and/or plasma cell depletion thus emerges as a promising treatment option, addressing the urgent need for more effective sustained therapeutic strategies. B cell depletion with the CD20 monoclonal antibody rituximab proved effective in a subset of ME/CFS patients, and its efficacy was found to be associated with the depletion of β2 AR-AB. 41 However, the results from the phase II rituximab trials could not be confirmed in a multicenter trial, which had several limitations. 42 We are currently initiating a trial with inebilizumab in ME/CFS and PCS patients, a monoclonal antibody directed against CD19 on B cells and plasma blasts showing high clinical efficacy in neuromyelitis optica. 43 In this study, only responders to IA will be included in order to select patients with strong evidence of an autoantibody-mediated disease. Further novel treatment approaches are currently being tested in clinical trials for PCS and ME/CFS patients. These include targeting plasma cells by a monoclonal antibody to CD38, enhancing degradation of autoantibodies by Fc receptor inhibition, and neutralizing antibodies against G-protein coupled receptors with the aptamer BC007 . Limitations of this study include the small sample size and non-controlled study design. The study was designed to detect larger effect sizes, smaller effects may not be captured with this sample size. Further studies are therefore required; we and two other sites in Germany are currently conducting randomized controlled trials of IA in PCS and ME/CFS with larger sample sizes. 44 Further, the outcomes are primarily patient-reported. The DSQ-PEM is designed for diagnostic purposes, and not validated for tracking changes in PEM in a clinical trial. While PCS encompasses a very heterogeneous patient population with presumably different pathomechanisms, our focus was solely on adult PCS patients meeting the ME/CFS criteria. Therefore, our findings may not be applicable to all PCS patients. Particularly our findings regarding the feasibility and safety of this procedure cannot directly be applied to the most severe, home bound patient group, since they were excluded from the study. In conclusion, our study suggests that IA treatment may result in rapid clinical improvement in a subset of patients. Further trials with both more patients and clinical outcomes reported are needed to confirm our findings. Our study serves as proof of concept for the initiation of clinical trials using drugs specifically designed to target autoantibodies. Targeting B cells may offer a promising approach to long-term symptom relief by addressing the underlying mechanisms driving autoantibody generation. | Other | biomedical | en | 0.999995 |
PMC11699804 | The differential diagnosis for pathogens in osteomyelitis varies depending on anatomic location and host risk factors. Vertebral osteomyelitis classically stems through hematogenous spread from a distant primary source . The etiology for vertebral osteomyelitis is typically gram-positive pathogens, regardless of age, gender, or level of spinal involvement. In males younger than 60, Staphylococcus , Streptococcus , and Enterococcus combine to cause 86 % of cases , with Staph. aureus accounting for most of these cases , . Less than 2 % of cases are due to anaerobes . We present a rarely encountered culprit of osteomyelitis. He was known to have a UPJ obstruction since his 20 s, when it was incidentally identified during trauma work-up after he was involved in a car accident. He did not have any intra-abdominal or thoracic injuries related to this accident. Since that time, he had this UPJ obstruction managed with chronic stent exchanges. He did undergo two endoscopic balloon dilation procedures for this, one in the late 1990 's and one in the early 2000's, neither of which were successful. Recently he was evaluated by urology, and it was recommended that the patient undergoes left nephrectomy due to nonfunctional left kidney after a kidney scan was done. Surgery had not yet been performed. He has had several urinary tract infections throughout his adult life, for which he was treated with oral antibiotics. He has not required any antibiotics within the last year. His white blood cell count was 11.2 k/µL (reference range 4.5–11.0) with 60 % neutrophils (normal 41–77 %). CRP was 3.88 mg/dL (reference range <1.00), and ESR was 43 mm/hour (reference range 0–20). Urinalysis was nitrite negative and had 3 + leukocytes (reference negative). There were 2–10 white blood cells/hpf seen on microscopy (reference 0–2), in the setting of 0–2 squamous epithelial cells/hpf (reference 0–5). Urine cultures and blood cultures were obtained. Given overall clinical stability, he was monitored off antimicrobials. CT abdomen/pelvis was obtained and revealed the development of cortical irregularity and osteolysis involving the L4–5 endplates and posterior superior aspect of the L5 vertebral body. There was also paravertebral edema about the L4 and L5 levels with new intermediate density material posterior to the L5 vertebral body, which resulted in moderate central canal narrowing. It was also noted that the left nephro-ureteral stent was mispositioned with the superior cope loop in the mid ureter, with some mild wall thickening of the proximal left ureter. MRI of the Lumbar Spine revealed loss of normal disc height with an abnormal fluid signal and diffuse contrast enhancement of the majority of the L4 and L5 vertebral bodies. There was enhancement in the epidural space, likely contiguous inflammation. Findings were compatible with spondylodiscitis at L4-L5 with contiguous phlegmon into the epidural space. Fig. 1 MRI Lumbar Spine. Fig. 1 Urine culture ultimately resulted with < 100k CFU/mL urogenital/skin microbiota. On day 2 of admission, the patient underwent a L4/5 disc biopsy in interventional radiology. Pathology revealed focal necrosis and acute inflammation (discitis) with adjacent granulation tissue. AFB stains were negative for acid-fast bacilli, and GMS stains were negative for fungal organisms. Biopsy cultures remained negative on day 5 of admission. Two sets of peripheral blood cultures were also negative at 5 days. Transthoracic echocardiogram was obtained and was negative for vegetations. On day 5, he underwent a repeat L4/5-disc biopsy. Results of repeat L4/5 disc biopsy were pending at the time of discharge on hospital day 5. He was started on empiric IV Daptomycin and IV Cefepime. The biopsy culture resulted with Gardnerella vaginalis on both routine and anaerobic cultures 12 days after initial admission. At that time, IV Daptomycin and IV Cefepime were stopped. Alternatively, oral Metronidazole 500 mg three times daily was started. He completed 10 weeks of Metronidazole therapy, with significant improvement in his pain. He ultimately underwent left nephrectomy 3 months following this and had no re-admissions since. To our best knowledge, we present the first case of G. vaginalis vertebral osteomyelitis in a male. G. vaginalis is a rare cause of bone and joint infections. On average, anaerobes account for < 2 % of cases of vertebral osteomyelitis , and < 1 % of osteomyelitis in general . Furthermore, G. vaginalis is even less common, particularly in males . G. vaginalis ’ main association is with bacterial vaginosis . Several case reports to-date of G. vaginalis bone and joint infections have occurred in concurrence with G. vaginalis colonization of the genitourinary tract , . Table 1 outlines all identified cases of G. vaginalis bone and joint infections in literature. To date, there have only been 3 cases of vertebral osteomyelitis reports in females , , , 1 case of native-joint septic arthritis in a female , 3 cases of prosthetic joint infections in females , , , 1 case of prosthetic joint infection in a male , and 1 case of skull osteomyelitis in a male infant whose gestation was complicated by bacterial vaginosis . All cases of joint infections have been in the hip, and all adult cases of vertebral osteomyelitis in adults have been in the lumbar spine, suggesting a possible anatomic association with the GU tract. A variety of mechanisms have been proposed for genitourinary colonization/infection leading to lumbar spondylodiscitis, including hematogenous spread through the posterior venous plexus or Baston’s plexus , . Table 1 Case reports of Gardnerella Vaginalis bone/joint infections in literature. Table 1 Reference Reference # Year * Age Gender Lesion GV colonization Polymicrobial Surgical Management Antibiotic Therapy ** Outcome Nightingale et al. 7 1986 Infant M Parietal OM Yes (in mother) No Debridement Ampicillin Cure Hodge et al. 9 1995 50 F Vertebral OM Unknown No No Ampicillin Cure Graham et al. 10 2009 38 F Vertebral OM No No No Clindamycin Cure Sivadon-Tardy et al. 11 2009 48 F Hip Septic Arthritis No Yes Debridement Amoxicillin Cure Hoarau et al. 6 2012 71 F Hip PJI Unknown Yes 1-stage revision TMP-SMX Cure Thomas et al. 8 2019 68 F Hip PJI Yes No 1-stage revision Amoxicillin Cure Thomas et al. 8 2019 32 F Hip PJI Yes No 1-stage revision Clindamycin Cure Kim et al. 3 2021 61 F Vertebral OM No Yes No Metronidazole Cure Saricaoglu et al. 5 2022 45 M Hip PJI No Yes Debridement/retention Clindamycin Cure Belote et al. present case 2024 55 M Vertebral OM Unknown No No Metronidazole Cure M: Male F: Female OM: Osteomyelitis GV: Gardnerella vaginalis PJI: Prosthetic Joint Infection * Year of publication ** Antibiotic used for main treatment course G. vaginalis is a small, facultative anaerobic gram-variable rod , . It is difficult to identify, difficult to culture, and less virulent , , than more common bacteria associated with native vertebral osteomyelitis. G. vaginalis can form biofilms , , including on contraceptive intravaginal ring, but there is no data on extra-vaginal foreign bodies, or in males . It can colonize the urethra in up to 4.5–11.4 % of males , and has been shown to colonize extravaginal mucosa in women . We suspect that our patient had developed colonization of his left ureteral stent, given the noted wall thickening of the adjacent ureter on CT scan. It also may have been the “urogenital flora” grown on urine culture. Unfortunately, this specimen was not available for further analysis as his biopsy culture resulted 12 days after his original urine culture, and routine urine samples at our institution are discarded in that timeframe. We suspect that colonization of his ureteral stent with G. vaginalis is likely what predisposed him to develop native vertebral osteomyelitis. He improved on culture directed therapy, suggesting that the identified G. vaginalis was truly pathogenic. Our case also highlights the diagnostic importance of withholding antibiotics for osteomyelitis in the setting of clinical stability, as per Infectious Disease Society of America (IDSA) Guidelines . Biopsy results should guide therapy. Image-guided biopsy for lumbar spondylodiscitis has variable sensitivity, with reports as low as 40 % . In the setting of a negative initial biopsy, IDSA guidelines recommend a 2nd biopsy to be sent for culture , as was performed in this case. Were repeat biopsy not pursued in this case, he likely would have developed empiric treatment failure and developed worsening symptoms, and potentially hospital re-admission. Holding antibiotics after initial biopsy in this case proved highly beneficial. | Clinical case | biomedical | en | 0.999996 |
PMC11699842 | Organophosphate (OP) compounds are formed through esterification between phosphoric acid and alcohol. Their uses range from pesticides and herbicides to nerve agents in chemical warfare . An example is diazinon, which was industrialised in 1952 by a Swiss chemical company and became the most widely used pesticide in the United States of America. It was cancelled for residential use in 2000 by the US Environmental Protection Agency to reduce the risk of pesticides at home, especially to children . Such acts have reduced the prevalence of OP poisoning in more developed countries, but it remains prevalent in less developed countries often presenting with non-accidental poisonings . An increased interest in OP poisoning in the United Kingdom (UK) stems from recent poisonings such as the Skripel family in March 2018 in Salisbury . In June 2022, the North Atlantic Treaty Organisation (NATO) identified that the deliberate use of chemical, biological, radiological and nuclear materials had the potential to “sow panic and strain national response capabilities” . OP causes irreversible phosphorylation of the acetylcholinesterase enzymes leading to a spectrum of pathology, depending on the time and dose of the agent. These range from cholinergic crisis and intermediate syndrome to OP-induced delayed neuropathy and chronic OP-induced neuropsychiatric disorder . The method of intoxication is by absorption through the skin, gut or bronchi following inhalation as in this case. The suggested management of OP poisoning involves atropine, pralidoxime (a cholinesterase reactivator), benzodiazepines and supportive care . The evidence and advice regarding these treatments can vary and management of such patients is heterogeneous from country to country. Specifically, OP poisoning raises challenges in muscle relaxant dosing with limited literature to support a specific agent or dose. 08:16: LAS arrived. Examination at the scene found the patient to be tachypnoeic at 40 breaths per minute, tachycardic at 123 beats per minute and with a Glasgow Coma Scale score of 11 (E3, V3, M5). Further findings included a blood sugar of 10.1 millimoles per litre (mmol/L), diaphoresis, pallor, hypothermia (36.1 degrees Celsius), shivering and miosis. Fasciculations were noted but she had no focal neurology. LAS administered 400 µg naloxone intramuscularly with no response and transported via blue light to A&E. 10:20: The patient continued to exhibit widespread fasciculations, confusion, and tachycardia with a heart rate of 120 bpm. Oxygen saturations were 92% on room air, and her blood pressure was 99/65 millimetres of mercury (mmHg). Oxygen was administered via a Mapleson C circuit before induction using fentanyl (1 microgram per kilogram) and 2 mg/kg of ketamine and rocuronium. A grade 1 view on laryngoscopy showed fully relaxed vocal cords. Other cases report non-accidental ingestion of OP with 30% of suicide cases globally due to OP intoxication and most patients received atropine and pralidoxime. A UK-based report treated with atropine and intubated achieved a similar recovery to this patient, without pralidoxime . One paper suggested OP poisoning mortality rates ranged from 3% to 25% worldwide . The UK NPIS report 2023 found, from 579 cases of pesticide poisoning, 81.3% were unintentional acute cases and only 11% of cases were graded severe, which this patient would be categorised into . Ketamine 2 mg/kg due to the patient’s haemodynamic instability and fentanyl 1 mcg/kg were administered. The challenging discussion was the type and dose of muscle relaxant. Suxamethonium is a more potent agonist with greater affinity for the cholinergic receptors due to its structure of two acetylcholine (ACh) molecules joined. Its effects will be exacerbated in the presence of a cholinesterase inhibitor . It is contraindicated due to the risk of prolonged respiratory paralysis and extra junctional receptor side effects following muscle injury suggested by a rising creatine kinase, such as hyperkalemia. Rocuronium at 2 mg/kg achieved suitable vocal cord relaxation within 45 seconds of administration. An increased dose was necessary to overcome increased ACh at the NMJ. In the unlikely event that extubation was required, sugammadex could be considered as a reversal agent. It would be interesting to assess the level and type of muscular blockade prior to and post administration of muscle relaxant using a train-of-four nerve stimulator. This could be tested in a future similar situation if the patient is in a condition where pain from stimulation will not be felt, such as post-induction and pre-muscle relaxant, if starved and a modified rapid sequence induction is justified. The toxicology advice was to administer atropine only if there was high secretion load or severe bradycardia, due to the risk of worsening agitation, and initially, pralidoxime was recommended as per ToxBase guidelines; however, later re-advised to hold due to the risk of hypotension and limited evidence. The mainstay of treatment for this patient was intubation and ventilation followed by supportive level 3 care in the ICU. It is interesting that despite an anti-cholinesterase activity of <1000 units/litre, no antidote was required in this patient. The authors question whether the type of OP may affect the nicotinic and muscarinic receptors differently and therefore the traditional treatment should be tailored to the receptor-type effects, as in this case. Muscarinic effects include SLUDGE symptoms (salivation, lacrimation, urination, defecation, gastric upset and emesis) and bradycardia whilst nicotinic effects include skeletal muscle contraction, weakness, cramps, tachycardia and seizures . The intermediate syndrome has a varying level of severity and often is undiagnosed unless significant respiratory insufficiency. The fatigue symptoms reported in the days following discharge by this patient may have been the effects of mild intermediate syndrome, and further suggestion of nicotinic involvement . This case study demonstrated the importance of proactive supportive management for patients with OP poisoning and underscored the need for early intubation when NMJ involvement is suspected. Unlike other published case reports, no "traditional" antidote was given in this case of severe OP poisoning with an anticholinesterase level <1000 units/litre. The patient's condition improved within two days after which she was discharged home. There were no staff symptoms from contamination following appropriate personal protective equipment (PPE) and decontamination. Intubation was easy and successful with an increased dose of muscle relaxant. Further investigation of the type and density of neuromuscular blockade in the future may aid in further tailoring of muscle relaxant dosing as opposed to a significantly increased dose. The symptoms and signs exhibited by this patient may suggest the varying effects of OP on the ACh receptors, specifically nicotinic rather than solely muscarinic symptoms. | Review | biomedical | en | 0.999995 |
PMC11699872 | Uterine mesothelial cysts are extremely rare. Intra-abdominal mesothelial cysts were first described in 1979 , and approximately 150 cases have been reported in the literature to date. To the best of our knowledge, only five of these cases are associated with the uterus, and only one published in 2019, as in our case, developed within the myometrium . The factors triggering the growth of mesothelial cysts remain unknown, although developmental anomalies are often suspected. A history of abdominal surgery, pelvic inflammation, endometriosis, or mesothelial cysts of the round ligament are considered potential associations . Herein, we present a case of a uterine mesothelial cyst that was surgically treated under the preoperative diagnosis of a degenerated leiomyoma. A 41-year-old female patient presented to the gynecology clinic with complaints of abdominal pain. Her medical history revealed two pregnancies, one ending in cesarean delivery years ago and the other in curettage, with no additional comorbidities or surgical history. Transvaginal ultrasonography demonstrated an enlarged uterus with a hypoechoic intramural cystic mass measuring 7.2 × 3.3 × 3.6 cm in the posterior uterine corpus. The endometrium appeared thin and regular, and the bilateral adnexal regions were unremarkable. Computed tomography further identified the lesion as a hypodense mass in the uterine corpus measuring 7.2 × 3.3 × 3.6 cm . Upon incision, no solid or papillary structures were observed on the cyst wall, although septation was noted in one area. Microscopic evaluation revealed a cystic lesion lined with a single layer of benign cuboidal mesothelial cells located within the myometrium, unrelated to the endometrium or serosa. Immunohistochemical studies showed positive staining of the cyst epithelium with calretinin, D2-40, WT-1, ER, and PR . Since 1985, no cases of mesothelial cysts in the uterine myometrium have been reported in the literature, except for one case in 2019, which, similar to our case, involved a mesothelial cyst developing within the myometrium . Of the 150 cases of mesothelial cysts reported in the literature, only five were uterine-related. One was described as a multicystic lesion attached to the serosa by a pedicle between the left uterine fundus and ovary , another presented as multiple small cysts on the serosal surface of the uterine corpus , one resembled our case within the myometrium , and the remaining two were reported as lesions adhering to the myometrium on the posterior uterine wall . Based on the current literature, our case represents the second instance of a mesothelial cyst developing within the myometrium. Due to their prevalence in women of reproductive age, some studies suggest a potential relationship between sex hormones and mesothelial cysts. However, since mesothelial cysts of the round ligament do not demonstrate immunohistochemical expression of ER or PR, some authors propose that these lesions are independent of sex hormones . In the five uterine mesothelial cyst cases, the patients were aged 27-47 years, but the menopause status of women in their 40s is unknown, and while ER and PR expressions were negative in one of the five articles, immunohistochemical studies related to ER and PR were not reported in the other four articles . The developmental process of these cysts remains controversial due to the limited number of reported cases and the need for further research. Mesothelial cysts share similar morphological and histopathological features with benign cystic mesothelioma . Both can manifest as inclusion cysts within the pelvic cavity. While mesothelial cysts are typically solitary, unilocular, and benign, with epithelia composed of well-differentiated mesothelial cells, benign cystic mesothelioma is often multilocular and considered a reactive lesion or a neoplasm with low malignant potential and a tendency for recurrence . Uterine mesothelial cysts lack specific clinical features. They may be asymptomatic or present with a palpable abdominal mass or pelvic pain in some patients. Radiological diagnosis is challenging due to their similarities with leiomyomas exhibiting cystic degeneration, and they may be misdiagnosed preoperatively as degenerated leiomyomas, as in this case . The characteristic microscopic appearance of a single layer of cuboidal mesothelial cells lining the cyst wall, along with positive staining for specific mesothelial markers such as calretinin, WT-1, mesothelin, and HBME, aids in the diagnosis of pelvic mesothelial cysts . Estrogen and progesterone expression remain variable, and no definitive conclusions have been reached . Although most authors describe mesothelial cysts as benign lesions with low recurrence and malignant potential, cases with recurrence rates of up to 27% in benign cystic mesotheliomas and malignant transformation have been documented in the literature . Treatment options include cyst excision; however, the thin cyst walls often complicate complete excision and result in high recurrence rates. To minimize recurrence, total or partial hysterectomy is considered the optimal approach, especially for older patients or those who do not plan to have children . In conclusion, mesothelial cysts are extremely rare lesions in the uterus, and definitive diagnosis is typically established through pathological examination. Further case reports and advanced studies will help us better understand the development process of this lesion. Awareness of this uncommon lesion and its inclusion in the differential diagnosis of pelvic pain in women of reproductive age can facilitate patient management. Although complete excision of the cyst wall may be challenging, the prognosis is favorable, and hysterectomy, when indicated, significantly reduces the risk of recurrence. | Clinical case | biomedical | en | 0.999996 |
PMC11699873 | The internal medicine clinic provides primary care to a diverse population, serving residents of neighboring nations near the border. Effective record-keeping is critical in this dynamic community to ensure continuity of care and facilitate decision-making during emergencies. Despite the importance of advanced care planning (ACP) and advanced medical directives in supporting informed decision-making, a meta-analysis from 2011 to 2016 found that only 36.7% of patients, and 38.2% of those with chronic illnesses, had documented directives . The number of patients with such documentation at this clinic is unknown, and there is no standardized method to educate patients on ACP. This gap highlights the need for early physician-patient discussions and structured resources to improve patient care and preparedness for future healthcare decisions. This study was conducted at the internal medicine clinic and aimed to address ACP and advanced medical directives with patients to improve overall patient care. The objective was to initiate discussions about ACP and provide advance medical directive instructions and resources to all willing patients. This aimed to enhance awareness, education, and the prevalence of advanced medical directives among clinic patients. The study included all patients aged 18 years and older with appointments at the clinic from February 1 to April 1, 2024. Patients under 17 years, those who refused to participate, those who did not complete the post-intervention survey before discharge, and those who answered “No” to Question 6 of the pre-appointment survey were excluded from the study. This single-group, cross-sectional study was conducted without patient randomization, with participants assigned to the study group based on inclusion and exclusion criteria. Resident physicians conducted the study within the internal medicine residency program, and institutional board review (IRB) exemption was granted on January 31, 2024 , after initial submission on December 6, 2023, and necessary modifications. Data collection took place from February 1 to April 1, 2024. Data collected included the patient's age, comorbidities, advance medical directive completion status, and responses to a post-intervention survey when applicable. A numerical code was assigned to each patient, recorded separately in a Coded Identifier List to link pre-appointment and post-intervention surveys, without containing identifying information. The Excel (Microsoft Corp., Redmond, Washington, DC) file containing the data was saved on a secured, password-protected computer in the principal investigator's locked office. All personnel received training on proper data handling to minimize privacy risks, and data were stored on an encrypted device. Additionally, paper documents were destroyed after being digitized. During the study, all eligible patients who agreed to participate received an anonymous pre-intervention survey in English or Spanish at intake to capture baseline information on ACP. This survey assessed whether the patient had an advance medical directive and whether they wanted to receive education on ACP (Appendix A). Patients had three to four minutes to complete this survey to establish baseline knowledge of ACP and determine if they had completed an advance medical directive. Patients could opt out of the study at any time. For those interested in additional information on ACP, internal medicine residents provided standardized education from the National Institute on Aging website. This educational material explained ACP, the nature and importance of advanced medical directives, and the process for completing them. Patients had five to 10 minutes to review the information during the visit. Of the 52 patients who completed the ACP pre-intervention survey, 23 completed the ACP post-intervention survey. The initial question in the survey asked if patients had ever had discussions about advanced medical directives with a physician prior to the visit. Ten patients (43.5%) reported having such discussions in the past, while 13 (56.5%) stated they had never had advance medical directive discussions before . The performed t-test suggested a statistical significance between the patient's age and the likelihood of having had an advanced medical directives discussion prior to this study (p = 0.013). There was also a significant correlation between the number of comorbidities and having had past discussions (p = 0.025). Publishing these findings is extremely significant for several reasons. Firstly, by standardizing ACP discussions, this study demonstrates a clear pathway to improving patient care through better preparation for end-of-life decisions . Ensuring that patients' wishes are known and respected can reduce stress for both patients and their families during critical times, particularly when patients are unable to communicate their preferences . This aligns with prior research by Silveira et al., which highlighted that patients with advanced directives are more likely to receive care consistent with their values and wishes, reducing the burden on surrogate decision-makers . This intervention also provided valuable education to patients, many of whom may not have been previously aware of or understood advanced directives . Education is a critical component in ACP, as demonstrated by Bravo et al., who found that structured educational interventions significantly increased the completion rates of advanced directives among community-dwelling older adults . The inclusion of culturally and linguistically appropriate materials, as implemented in this study, is crucial for ensuring that diverse patient populations can fully engage in ACP discussions . Sudore et al. emphasized the need for culturally sensitive ACP discussions, particularly in diverse populations similar to those served in this study . Moreover, the project’s methodology and findings can serve as a model for other clinics and healthcare institutions . A systematic approach to ACP discussions promotes the integration of standardized practices into routine care, leading to widespread improvements in how advanced directives are handled across different healthcare settings . Studies by Morrison et al. and Detering et al. have further supported that culturally tailored ACP interventions lead to higher engagement and completion rates among minority groups . This is particularly important in primary care settings, where long-term patient-provider relationships can facilitate ongoing ACP discussions, as suggested by Tung et al. . The standardized approach used in this study is in line with best practices outlined by Lund et al., who found that systematic implementation of ACP in clinical settings can lead to higher rates of advance directive completion and greater patient satisfaction . Similar findings were reported by Jimenez et al., who highlighted the global lessons learned from systematic reviews of ACP . Rietjens et al. also emphasized the importance of continuity of care in successful ACP implementation . The results of this study further highlight its significance to current literature on ACP . During the three months, 52 patients completed the pre-intervention survey, with an average age of 59 years . The survey revealed that hypertension, dyslipidemia, and diabetes were the most prevalent diseases among participants, conditions often associated with higher healthcare utilization and potential complications that necessitate clear advance care plans . Notably, only 1.2% of patients reported having advanced directives prior to the study, underscoring a significant gap in ACP documentation in this population . This finding is consistent with national data reported by Yadav et al., which showed that approximately one-third of U.S. adults have completed some form of advance directive . Furthermore, a study by Rao et al. indicated that chronic conditions like those prevalent in this population often correlate with a higher desire for ACP, yet the completion rates remain low due to systemic barriers . The post-intervention survey results showed a substantial increase in the number of patients who had discussed advanced directives, with 43.5% of participants engaging in these conversations . A significant number of patients found the discussions helpful and recognized the importance of having advanced directives, which is supported by evidence from Jimenez et al., who demonstrated that ACP discussions improve patient and family satisfaction with end-of-life care . These results align with findings by Wright et al., who demonstrated that early ACP discussions significantly reduced anxiety and depression among patients with serious illnesses . Similarly, a randomized controlled trial by Houben et al. showed that ACP discussions are associated with better alignment of care with patients' wishes and fewer unwanted hospitalizations near the end of life . Furthermore, the study addresses significant gaps in the literature regarding ACP in specific demographics, particularly older adults with multiple comorbidities . Research by Tung et al. suggests that older adults with multiple comorbidities are more likely to engage in ACP discussions, a finding that was corroborated by this study's data, which highlighted the association between comorbidities and prior ACP discussions . Additionally, studies such as those by Song et al. have shown that tailored interventions, like the ones implemented in this project, can significantly increase the completion rates of advanced directives among patients, particularly in populations with chronic illnesses . Zhang et al. also noted that patients with multiple chronic conditions benefit most from ACP, as it helps align their complex care needs with their values . The implications of these findings are broad, extending beyond individual patient care to inform policy and training programs for healthcare providers . Integrating ACP discussions early in the patient care process, particularly in primary care settings, is critical for ensuring that patients' preferences are documented and respected throughout their healthcare journey . Kavalieratos et al. found that early integration of palliative care, including ACP, is associated with improved patient and caregiver outcomes, including reduced hospitalizations and enhanced quality of life . This is particularly pertinent in a clinic setting where continuity of care and clear communication are crucial for effective patient management. The importance of healthcare provider training in ACP was emphasized by Lum et al., who found that well-trained providers are more likely to initiate and complete ACP discussions . The importance of addressing ACP during public health crises, such as the COVID-19 pandemic, has also been highlighted in recent literature. It was noted that the pandemic has brought ACP to the forefront, emphasizing the need for healthcare providers to engage in these discussions proactively . This aligns with the findings from this study, which underscore the necessity of standardized ACP practices to prepare for unforeseen circumstances and ensure that patient's wishes are respected even during crises . A study by Curtis et al. during the pandemic also highlighted the critical role of ACP in reducing the burden on healthcare systems by ensuring that patient care aligns with their preferences, particularly when resources are limited . In addition to primary care and public health settings, ACP has been shown to have significant implications in specialty care. For instance, Mack et al. demonstrated that end-of-life discussions in oncology settings are associated with better patient outcomes and reduced distress . Similarly, Mullick et al. highlighted the importance of integrating ACP into routine practice across various specialties to ensure that patients' wishes are consistently respected . Studies in cardiology and nephrology settings, such as those by Schell et al., have further validated the positive impact of ACP on patient satisfaction and care quality . This project on improving ACP discussions demonstrates a significant advancement in patient care practices . The standardized approach not only educates and empowers patients but also provides a replicable model for other healthcare institutions . The findings, supported by recent literature, underscore the importance of targeted ACP interventions and their positive impact on patient outcomes and healthcare practices . Future research should explore the long-term effects of such interventions on healthcare utilization and patient satisfaction, particularly in diverse and underserved populations. Moreover, the ongoing development of ACP tools and resources, as discussed by Bristowe et al., will be essential in supporting both patients and healthcare providers in the future . This study has several limitations that should be acknowledged. First, the sample size was relatively small, with only 52 patients completing the pre-intervention survey and 23 patients completing the post-intervention survey. This small sample size may limit the generalizability of the findings to the broader population served by the internal medicine clinic. Larger studies with more diverse patient populations are needed to confirm these results and determine whether the observed improvements in ACP discussions can be replicated in different settings. Second, the study was conducted over a short duration of only three months. This limited timeframe may not have been sufficient to capture the long-term effects of the intervention on patient behavior and outcomes. For instance, while the post-intervention survey indicated an increased likelihood of patients completing advanced directives, it remains unclear whether these intentions translated into actual behavior change over time. A longer follow-up period would be necessary to assess the sustainability of the intervention's impact. Finally, the study relied on self-reported data from patients, which may be subject to response bias. Patients may have provided socially desirable answers, particularly regarding their comfort with end-of-life discussions and their intentions to complete advanced directives. Additionally, the study did not include a control group, making it difficult to determine whether the observed changes were solely attributable to the intervention or if other external factors may have influenced the results. Future studies should consider including a control group and using objective measures, such as the actual completion rates of advanced directives, to strengthen the validity of the findings. | Other | biomedical | en | 0.999996 |
PMC11699881 | Carotid cavernous fistulas are a serious condition that involves spontaneous or trauma-induced disruption in the carotid arteries within the cavernous sinus. This disruption causes blood to leak into the sinus, which impedes the function of cranial nerves III, IV, V1, V2, and VI . Early signs of CCF often include proptosis, glaucoma, severe headaches, partial blindness, and conjunctival hyperemia, especially in patients with preexisting conditions such as atherosclerosis, hypertension, and diabetes. If left untreated, patients can develop periorbital disfigurement and permanent vision loss . Despite effective methods of diagnosis using angiography and blood vessel repair, this condition is often misdiagnosed due to it making up only 1.5% of endovascular-treated ruptured aneurysms and its similarity in presentation to cerebrospinal fluid (CSF) leaks . Therefore, CCFs have been under-investigated, and additional research is required to delineate the cause, unique features, and proper treatment of this phenomenon. Often misdiagnosed due to its ambiguous presentation, we present a successful clinical workup and treatment of a 78-year-old female patient who developed a CCF in the setting of spinal fusion. We suggest a potential relationship between prolonged prone positioning during surgical intervention and the occurrence of CCF. A 78-year-old female patient presented for bilateral lateral radicular leg discomfort and back pain refractory to conservative management including physical therapy and anti-inflammatory medications. Magnetic resonance imaging (MRI) of the lumbar spine revealed significant degenerative spondylosis of L4-S1 levels including severe lateral recess stenosis at L4-L5 and bilateral pars defects at L5-S1 . She was neurologically intact with no remarkable findings on physical exam. Operative management was pursued due to her symptoms and imaging findings. She underwent a successful L4-S1 posterior instrumented fusion with partial laminectomies at L4 and L5 along with an L4-L5 bilateral facetectomy. There was no significant intraoperative fluid leak. Estimated blood loss was minimal, and the procedure lasted approximately 228 minutes with the patient prone on a Jackson spine table. Six weeks following the surgery, the patient presented for a follow-up with complaints of bifrontal headaches that were worse in the morning for the past two weeks. She also reported neck discomfort and sharp discomfort whenever she turned her head to one side or extended her neck. The headaches were not positional nor particularly severe. There was no evidence of a spinal fluid leak from the incision nor palpable fluid collection appreciated on the physical exam. An x-ray of the cervical spine demonstrated degenerative changes from C4 through C7 with complete loss in disc space height at each level from C4 inferiorly . The patient presented to the Emergency Department on two separate occasions in the following two weeks; first, she had a pounding headache in bilateral temples with swelling below her left eye and an episode of emesis the evening prior. She was also noted to have conjunctivitis in both eyes and was given antibiotic eye drops. She presented the second time with complaints of a constant headache in the middle of the night, nausea, and new onset blurry vision in her left eye. The patient was also hypertensive with 170 systolic blood pressure. A brain CT scan showed no evidence of an acute intracranial process. Basic lab workup was unremarkable on both visits. The patient was discharged the second time with prescriptions for Percocet and Zofran and was recommended to follow up with her neurologist. Eight weeks following her fusion, the patient was struggling with worsening headaches and nausea. A lumbar spine MRI showed a fluid collection adjacent to the dura with a possible dural tear . Her worsening symptoms prompted a return to the operating room for wound exploration and repair of a suspected CSF leak. There was no apparent CSF leak. Valsalva was performed with no obvious egress of spinal fluid. Given that there may have been an occult non-identifiable leak the dura was covered with two layers of Surgicel (Ethicon, Johnson & Johnson, USA) followed by Adherus (HyperBranch Medical Technology, Durham, NC, USA) tissue sealant. A lumbar drain was placed through the next most rostral intact interspinous space brought out through a separate stab incision. A 10-round Jackson-Pratt drain was tunneled into the sub-fascial space and brought out through an additional stab incision. Following the surgery, she had two episodes of emesis with continued headaches and nausea. However, she experienced significant improvement in her headaches and nausea over the subsequent days. She was discharged one week later following drain removal and complete resolution of her symptoms. Two weeks following discharge, she presented to the emergency department again for continued headaches, left conjunctival hemorrhage, mild proptosis, diplopia, and a cranial nerve (CN) III palsy. Given the unclear etiology for her headaches and new exam findings, there was a concern for Tolosa-Hunt syndrome or a carotid-cavernous fistula which prompted a brain MRI. Imaging demonstrated prominent superior ophthalmic veins, with the right being greater than the left . A left carotid-cavernous fistula was confirmed using diagnostic angiography. The patient underwent transvenous embolization for carotid-cavernous fistula in the intercavernous and left cavernous sinus. The fistula was nearly completely occluded with a significant reduction of flow into the fistula and gradual occlusion noted during angiography from the arterial supply . She had a successful coil embolization of a Barrow Type C carotid-cavernous fistula. Following the procedure, the patient had a significant improvement in her symptoms and was discharged three days later. Her postoperative course was significant for an improvement in her headaches and a lingering CN III and VI palsy predominantly of the left eye. Carotid-cavernous fistulas (CCF) are pathological shunts between the cavernous sinus and the carotid artery or its meningeal branches. The Barrow classification system anatomically describes four types of CCFs. Type A CCFs are direct, high-flow communications between the cavernous sinus and the internal carotid artery (ICA) as it traverses the sinus. Low-flow fistulas indirectly connect the carotid artery to the cavernous sinus via the ICA meningeal branch (Type B), external carotid artery meningeal branch (Type C), or both branches (Type D) . Several etiologies regarding the pathogenesis of CCF have been described. Type A CCFs are often associated with trauma, ruptured ICA aneurysms, connective tissue diseases, or iatrogenic injury . Type A CCF has been reported after various procedures, particularly those involving direct carotid manipulation or involvement of adjacent structures; procedures reported in association with Type A CCF include carotid endarterectomy, mechanical thrombectomy, transsphenoidal exploration, craniotomy and ocular surgery . Traumatic CCFs may occur as a result of bony fragment damage to the vessel wall secondary to basilar skull fracture or shear forces directly damaging the vessel wall . Indirect CCFs most commonly affect elderly women and are classically associated with ICA dissection, hypertension, and connective tissue diseases, including atherosclerosis, fibromuscular dysplasia, and Ehlers-Danlos syndrome . The presenting signs and symptoms of CCF are variable, depending on the characteristics of the CCF. Direct CCF often presents with conjunctival injection, blurred vision, headache, pulsating proptosis, and strabismus, most often affecting the sixth cranial nerve (the only cranial nerve traversing the interior of the cavernous sinus) . Low-flow CCFs often follow an insidious course, and conjunctival injection is typically the predominant physical complaint . Notably, low-flow CCFs are especially subject to misdiagnosis; our case represents a delayed diagnosis due to the patient’s recent spinal surgery and symptomatology mimicking a dural tear. The diagnosis of CCF is dependent upon imaging. Computed tomography (CT) and magnetic resonance (MR) imaging with or without angiography are often the first modalities utilized in assessing possible CCF and may be useful in diagnosing CCF; however, digital subtraction angiography (DSA) is currently considered the “gold standard” imaging modality for CCF because it demonstrates the cavernous sinus filling and drainage patterns . Strong clinical suspicion or CT/MRI evidence of possible CCF should prompt DSA . The exact treatment strategy is dependent upon the characteristics of the CCF. Direct CCFs are most often treated through endovascular approaches, with embolic materials, such as coils, acrylic glue, or ethylene vinyl alcohol copolymer, injected into the cavernous sinus to close fistula patency . Endoluminal stenting may be utilized to prevent subsequent embolism and promote endothelial proliferation . Twenty to sixty percent of indirect CCFs spontaneously resolve; thus, conservative management is usually attempted before intervention . External manual carotid compression with the contralateral hand several times per day has been shown to promote fistula closure . If surgical intervention is indicated, the same endovascular techniques used to repair direct CCFs are the first-line treatment modalities . However, because indirect CCFs involve smaller, tortuous carotid branches, endovascular therapy is not as effective compared to direct CCFs . Overall, endovascular treatment has a greater than 80% cure rate for CCF . If first-line treatments fail, open surgical ICA ligation can be attempted . Our patient was successfully treated with CCF coil embolization. CCFs are easily misdiagnosed, as CCF presentations can be highly variable and may be similar to several different diseases . Specific clinical histories may further confound the diagnosis of CCF. For example, Jain et al. describe a CCF that was initially misdiagnosed as hemorrhagic choroidal detachment following ocular surgery . Similarly, our patient's history of recent spinal surgery made a CSF leak a more plausible diagnosis than CCF. CSF leaks can present very similarly to CCF, with headaches, nausea, neck or back tightness, and cranial nerve palsies, being commonly shared symptoms . The diagnosis of CCF in our case was delayed because the patient’s recent surgical history made a dural tear more plausible. However, surgical exploration and drainage did not improve the patient’s symptoms, suggesting a dural tear was not present. After ruling out a dural tear, subsequent workup led to the diagnosis and treatment of CCF. To our knowledge, there is no association between CCF and spinal surgery or prolonged periods of prone positioning. There have been no reported cases of CCF following surgery not involving the carotid artery or related structures; all reported iatrogenic causes of CCF involve direct carotid involvement or surgery to adjacent structures . The patient has no known vascular diseases and suffered no recent trauma. Indirect CCFs (such as the Type C CCF seen in this patient) disproportionately affect elderly women . We hypothesize that perhaps a prolonged period spent in the prone position during her spinal surgery is responsible for the CCF in a patient whose demographics predisposed her to CCF. Our case represents the first reported CCF occurring in conjunction with spine surgery. Although unsubstantiated, we believe there may be an association between a prolonged period in the prone position and the development of CCF. Furthermore, we would like to document our clinical workup; in which the clinical history and symptomatology suggested a dural tear, although subsequent workup led to the diagnosis of CCF. This case demonstrates a difficult diagnosis of CCF and its potential relationship to spinal surgery. | Clinical case | clinical | en | 0.999997 |
PMC11699977 | The increased incidence of second malignant neoplasms (SMNs) after Hodgkin lymphoma (HL) has been well-documented in the literature . In the head and neck region, surgeons encountering simultaneous primary malignancies like HL and squamous cell carcinoma (SCC) of the tongue are exceedingly rare, presenting unique diagnostic and treatment challenges . This case, a notable addition to the limited literature, underscores the intricacy of managing such dual malignancies in the head and neck region, emphasizing the importance of a comprehensive, multidisciplinary approach to these complex clinical scenarios. Physical examination revealed a left lateral posterior tongue lesion and an enlarged, tender left level 2 lymph node. Further examination with a flexible scope showed a nasopharyngeal mass and the vocal cords were bilaterally movable with no masses. Otoscopy demonstrated a clear and intact tympanic membrane. Biopsies showed the existence of invasive, moderately differentiated SCC in the left lateral posterior tongue lesion, nonetheless, there was no evidence of malignancy infiltration in the nasopharynx. The imaging studies provided a comprehensive overview of the patient's condition. The PET-CT scan displayed prominent hypermetabolic activity in the oropharynx, specifically in the pre-epiglottic region and bilateral palatine tonsils, with significant metabolic activity in the left upper cervical lymph node, raising concerns for metastasis . These imaging findings, collectively, indicated a complex scenario with both progressive and regressive elements in different areas. The notable hypermetabolic activity and lymph node involvement initially aimed towards metastasis from the SCC of the tongue. A left partial glossectomy, bilateral tonsillectomy, and left radical neck dissection with nasopharyngeal biopsy were determined following a discussion of the case on the tumor board. However, the intraoperative pathology revealed a dual pathology. Contrary to the metastatic expectations from the tongue SCC , the lymph nodes were predominantly affected by HL . HL predominantly manifests in lymphatic tissues but can occasionally present in extra-nodal sites, including the head and neck region . HL is commonly seen in late adulthood, the occurrence alongside SCC of the tongue, as presented in this case report, highlights a rare and intricate clinical scenario . The coexistence of these two distinct malignancies is not frequently documented, aligning with the literature that states multiple malignancies account for a small percentage (2-11%) of all head and neck malignancies . The increased occurrence of subsequent malignancies in HL survivors, as compared to the general populace, is partially a consequence of the long-term carcinogenic effects of chemotherapy and radiotherapy treatments. Furthermore, individuals who have overcome HL may possess a heightened predisposition towards the emergence of SMNs relative to survivors of other malignancies, a susceptibility that could be attributed to genetic factors inherent to HL . SCC of the tongue is a highly prevalent malignancy that is more common in the head and neck regions. However, the simultaneous development of HL and tongue carcinoma, which is similar to the infrequently documented cases of laryngeal cancer and lymphoma occurring simultaneously, adds complexity to the diagnostic, therapeutic, and prognostication processes . Similar to the case reported by Nigri and Khasgiwala where a patient with laryngeal SCC was later found to have HL, a synchronous lymphoma with laryngeal carcinoma in situ and a mucosa-associated lymphoid tissue (MALT)-type lymphoma with SCC of the larynx were reported as well . Our case underscores the importance of a thorough histopathological examination in cases of suspected or known malignancies in the head and neck region. In the context of extra-laryngeal carcinoma coexisting with lymphoma, our case parallels the complexity seen in other reported cases where patients presented with multiple primary malignancies involving different regions of the head and neck. The development of a second malignancy in the presence of HL, while not as common as with well-differentiated lymphocytic lymphomas, remains a significant clinical consideration, especially in the head and neck region where lymphatic and mucosal tissues interact closely . While non-Hodgkin lymphoma (NHL) has a noted association with human immunodeficiency virus (HIV)-infected patients, this association is less clear with HL . Our patient's clinical presentation, without HIV infection, aligns with the typical demographic profile for HL. The decision-making process in such cases is nuanced and requires a balance between aggressive treatment for local control and the careful management of potential systemic disease spread . Predisposing factors for the development of SCC, including chronic illness, smoking, and prior chemotherapy or radiotherapy, were considered in the management of our patient . However, our case featured the synchronous development of both tumors without a history of the common predisposing factors, presenting a unique clinical picture. The diagnosis and management of our patient highlighted the necessity for comprehensive and thorough pre-operative examinations to ensure all potential primary sites and regions of tumor involvement are adequately assessed. This case contributes to the growing body of literature on the rare but clinically significant occurrence of concurrent HL and SCC of the tongue, emphasizing the need for meticulous diagnostic workup and individualized, multidisciplinary treatment approaches in managing such complex clinical scenarios. We present an extremely rare case of a patient with classical HL who subsequently developed invasive SCC of the tongue. This case highlights the importance of ongoing surveillance of cancer survivors and the potential for the development of secondary malignancies. A multidisciplinary approach is crucial for the diagnosis, treatment, and follow-up of patients with coexisting primary diseases. | Clinical case | biomedical | en | 0.999999 |
PMC11699978 | Penetrating thoracic injuries, especially those affecting cardiac structures, pose significant clinical challenges due to high mortality rates . Cardiac injuries involving the right atrium have a dire prognosis due to risks of rapid bleeding, cardiac tamponade, and hemodynamic instability. Timely identification and surgical intervention are crucial for improving survival rates, as even slight treatment delays can have severe outcomes . Although penetrating trauma is less common than blunt trauma as it accounts for only 0.1% of most trauma admissions, it results in higher fatality rates due to proximity to critical cardiac and vascular structures . Penetrating cardiac injuries, often from stabbings, are typically linked to severe assaults and accidents . The anatomical vulnerability of the right atrium may result in life-threatening bleeding. However, timely intervention remains problematic in resource-limited settings, with positive outcomes dependent on early diagnosis and skilled surgical personnel . Penetrating injuries to the anterior chest wall can result in fatal damage to various thoracic structures, yet reports on these injuries and their management are scarce . Furthermore, documenting such cases is essential for enhancing surgical techniques, particularly in low-resource environments . This report discusses a case of penetrating chest trauma with a right atrium injury from a stab wound, effectively managed through rapid resuscitation and decisive surgical intervention. A 24-year-old male patient presented to the emergency department with a documented history of stab wounds located in the anterior precordial region, which penetrated the chest on the right side, approximately three hours post injury. Upon examination, the patient demonstrated a penetrating injury measuring 1.5x3 cm situated in the right fourth intercostal space within the parasternal region with an additional penetrating wound measuring 1x3 cm located at the fifth intercostal space along the midclavicular line . The patient was assessed as semi-conscious and reported experiencing intense pain on the right side of the chest and respiratory distress while exhibiting signs of hemodynamic instability and an absence of air entry in the right thoracic cavity. The initial evaluation indicated a blood pressure of approximately 80/50 mmHg, a pulse rate of 150 beats per minute, a respiratory rate of 30 breaths per minute, jugular vein distention, and muffled heart sounds. Oxygen saturation was recorded at 90% while the patient received 2 liters of supplemental oxygen. After initial resuscitation efforts, the blood pressure improved to 90/50 mmHg, although the patient continued to experience respiratory difficulties and persistent hemodynamic instability. Immediately, a chest tube was inserted to address the right-sided hemothorax, evacuating 500 ml of blood upon insertion and continued to increase. Due to the patient's unstable condition, no radiologic investigations were undertaken. Within 15 minutes of hospital arrival, the patient was promptly transferred to the operating room after initial resuscitation with fluids and blood transfusion. A central venous catheter was inserted, and a broad-spectrum antibiotic was administered following induction. Firstly, a diagnostic laparoscopy was made to investigate the abdomen. The diaphragm, liver, spleen, and other abdominal organs were all examined and found intact. To treat the probable hemothorax and cardiac tamponade, the patient was positioned supine with the right side of the chest slightly elevated. Upon examination of the penetrating chest wounds, active bleeding from multiple intercostal vessels was identified and promptly addressed through suturing. The thoracic cavity was explored to palpate the diaphragm and assess for potential injuries. A defect in the pericardium was detected, necessitating lateral extension of the wound to evaluate the status of the underlying lungs and pericardium. During the surgical intervention, a pericardial tear measuring approximately 1.5×1.5 cm was observed, accompanied by clot formation and hemopericardium . Following the evacuation of the clot and blood from the pericardial space, a laceration and rupture of the right atrial wall were observed and briefly stabilized using the index finger during the thoracotomy . A tangential Satinsky clamp was applied to the lacerated area of the right atrial wall . The heart rate decreased from 160 to 115 beats per minute, and blood pressure improved to 90/60 mmHg. The clamped right atrial wall was repaired utilizing a 5-0 double-arm polypropylene suture in a double-layer technique until hemostasis was successfully achieved . The thoracic cavity was subsequently re-examined, the lung injury was addressed, and comprehensive hemostasis was ensured. The thoracic cavity was then rinsed with normal saline and closed in layers following the insertion of two chest drains-one positioned in the pericardium and the other within the chest cavity. The total blood transfusion during the operation was three packed cells, the total operative time was 120 minutes, and the total bleeding was 2300 cc. Penetrating thoracic trauma resulting in right atrial rupture constitutes a rare yet critical injury that demands immediate assessment and surgical intervention . Due to its specific anatomical positioning, the heart is particularly vulnerable to penetrating injuries, with right atrial rupture posing a considerable mortality risk if not addressed expeditiously . The case reported by Valdés-Dupeyrón et al. exemplifies the imperative need for rapid resuscitation and suitable surgical management to secure patient survival, as was seen in our case . In the presented case, the patient sustained a penetrating stab wound to the right thoracic region, leading to hemopericardium and rupture of the right atrial wall. The successful application of the vascular clamp was followed by meticulous suturing of the atrial rupture, resulting in effective hemostatic control and stabilizing the patient's clinical status. Deploying double-arm polypropylene sutures in a layered fashion was instrumental in achieving hemostasis, a technique well-established in cardiac surgical practice for managing such catastrophic injuries . Historically, cardiac injuries have been associated with dire prognoses and were deemed intractable; presently, approximately 90% of individuals succumb before arrival at the emergency department . Numerous researchers have identified correlations between mortality and the hemodynamic status of the patient upon admission, the type of weapon utilized, surgical findings, and the intricacy of the repair process . In our clinical scenario, the medical emergency team's intervention was indispensable for preserving these patients' lives. Cardiac injury should be anticipated in any patient presenting with penetrating injuries to the thoracic region, particularly on the anterior aspect of the thorax, predominantly on the left side, as well as in the upper abdomen and neck . Numerous researchers acknowledge that the survival rate following a penetrating cardiac injury is likely contingent upon the timeliness of medical intervention . Stranch et al. discovered a statistically significant correlation between the delay in hospital arrival, the clinical status upon admission, the mechanism of injury, and the implementation of aggressive surgical treatment with patient survival rates . The time between injury and operation for penetrating heart damage must be brief. Isaza et al. observed that the average was 60 minutes . Survival after a penetrating cardiac injury appears to be less likely in a patient five hours after the trauma. However, penetrating wounds in the anatomical area known as the "cardiac box" should raise the most concerns for penetrating heart damage . Focused and coordinated surveys, as well as resuscitation, are beneficial to any patient with penetrating thoracic trauma. In the present case, the time to surgical intervention was about three hours. In another report, Gucho et al. reported a delayed presentation of penetrating cardiac injury for five hours in a 21-year-old patient with a stab wound on his right chest's fourth intercostal space . Penetrating traumas to any of the cardiac chambers may precipitate acute cardiac tamponade and subsequent mortality rapidly. Hemorrhagic flow resulting from a lacerated pericardial injury will disseminate into the hemithorax, ultimately culminating in a fatality. Consequently, the pericardium serves a critical role in averting lethal exsanguination and enables patients to endure sufficiently long to attain medical intervention at a trauma center. Patients may exhibit various degrees of hemodynamic instability as a consequence of pericardial tamponade . Notably, the classical clinical manifestation of Beck’s triad (characterized by muffled heart sounds, jugular venous distension, and hypotension) or Kussmaul’s sign (indicative of jugular venous distension upon inspiration) is observed in merely 10% of patients presenting with pericardial tamponade . Penetrating trauma can result in a pneumothorax or haemothorax with significant blood loss. Some individuals see fast deterioration following chest injuries. However, with correct care, they can quickly improve. In patients with penetrating injuries, surgical operations are typically suggested, although diagnostic examinations are less necessary than in blunt trauma . Various surgical strategies exist for addressing penetrating cardiac trauma, including left anterior thoracotomy, right anterior thoracotomy, pericardial window, and median sternotomy. The latter approach is prevalent owing to its extensive exposure, albeit it is not as expeditious as other techniques . In our case, we opted for an abdominal laparoscopic diagnostic procedure without prior radiological assessment due to the patient's declining condition. Consequently, we prioritized abdominal examination over thoracic assessment, believing the abdominal and hepatic injuries were of greater severity. Upon negative findings, the surgical strategy shifted to thoracotomy and pericardial exploration, resulting in the identification and repair of the atrial injury; a comparable case with certain deviations was documented by Alfraidy et al. . In a separate report by Valdés-Dupeyrón et al., the authors conducted a left thoracotomy combined with a transverse sternotomy in one patient, where a conventional sternotomy would have delayed vascular control . Penetrating thoracic trauma that affects the right atrium represents a rare yet potentially life-threatening condition that requires immediate assessment and intervention. This case exemplifies the critical nature of rapid diagnosis, proficient resuscitation, and thorough surgical repair in managing such injuries. The effective utilization of a tangential Satinsky clamp coupled with double-layer polypropylene sutures to control bleeding demonstrates that survival can be achieved even in severe cases of cardiac injury. Furthermore, this case further emphasizes the need to follow standard principles of resuscitation and guidelines for the management of thoracic trauma in places with a high incidence of penetrating chest trauma. Applying clinical diligence and the use of imaging like CT trauma series after stabilizing these patients could avoid potentially morbid negative explorations. Even in resource-poor settings, the use of emergency room/bedside ultrasound or diagnostic peritoneal lavage for abdominal injuries could avoid such aggressive exploratory attempts. | Other | biomedical | en | 0.999997 |
PMC11699980 | Head and neck cancer (HNC) refers to a group of heterogeneous tumors that originate, except for skin tumors, in the mucosa of the upper gastrointestinal tract, especially in the oral cavity, pharynx, and larynx . It ranks seventh in incidence and eighth in mortality estimates among the most prevalent cancers worldwide, excluding nonmelanoma skin cancer . HNC is a multifactorial disease whose risk factors arise from the interaction between environmental influences and genetic inheritance . Among environmental factors, smoking has emerged as the greatest risk factor, followed by alcohol consumption. When consumed simultaneously, risk increases from 10 to 100 times [ 4 – 6 ]. As HNC affects regions directly related to the digestive tract (reducing food intake) and induces a hypercatabolic state, carriers show significant weight loss from the time of diagnosis . Such nutritional decline is consistently associated with nutritional impact symptoms (NISs). NISs are physiological alterations caused by cancer that can compromise oral intake, including pain, dry mouth, difficulty swallowing, reduced appetite, and vomiting, among others . Approximately 96% of the population with HNC show one or more NISs before starting treatment . Individuals with HNC and multiple NISs are more likely to experience reduced food intake, weight loss, functional capacity dysfunction, and decreased survival [ 12 – 14 ]. Systematic assessment and management of these symptoms before the start of treatment are crucial to prevent serious nutritional complications that often develop during antineoplastic therapy . An early approach allows for the implementation of appropriate nutritional and therapeutic strategies that can not only mitigate the progression of symptoms but also significantly improve the patients' quality of life. Furthermore, early treatment of NIS can prevent patients from reaching a state of severe malnutrition, which would further compromise the effectiveness of the treatment and survival . Therefore, early recognition (pretreatment) of patients with a higher burden of NIS is crucial and provides insights that can assist in appropriate therapy before the development of malnutrition and associated factors. This study aimed to analyze the presence and severity of NISs and their associated factors in individuals with HNC before treatment. This is an epidemiological, analytical, and cross-sectional investigation of individuals with HNC who were treated at a reference oncology hospital in Greater Vitória, Espírito Santo, Brazil. The data represent the baseline of a longitudinal study titled “Nutritional Indicators, Mortality, and Associated Factors: A Hospital-Based Study in Individuals with HNC.” The target population consisted of patients diagnosed with squamous cell carcinoma (mouth, larynx, oropharynx, and hypopharynx) of all sexes who were aged 18 years and above. To calculate the sample size, a sample universe of 200 patients was considered, corresponding to the average annual number of first consultation visits at the HNC outpatient clinic, a prevalence of 50% (to maximize the sample), a sampling error of 5%, and a confidence interval of 95%. The resulting minimum sample size totaled 132 individuals. The calculation was performed using Epi Info 7.2 software. Participants of all sexes who were aged 18 years or above, diagnosed with squamous cell carcinoma of the oral cavity, larynx, oropharynx, and hypopharynx, and without previous treatment, were included. Cases were confirmed by histology and classified according to the ICD-10-03 into the following topographies: base of tongue (C01), other and unspecified parts of tongue (C02), gum (C03), floor of mouth (C04), palate (C05), other and unspecified parts of mouth (C06), tonsil (C09), oropharynx (C10), pyriform sinus (C12), hypopharynx (C13), and larynx (C32). Subsequently, the ICD codes were grouped into four topographic categories: oral cavity (C01, C02, C03, C04, and C05), oropharynx (C05.1, C09, and C10), larynx (C32), and hypopharynx (C12 and C13). Patients were excluded from the study if they had a diagnosis of recurrent squamous cell carcinoma, had previously received cancer treatment, presented with multiple tumors, or lacked the clinical and mental capacity to respond to the administered questionnaire. A questionnaire was administered to evaluate patients' sociodemographic (sex, age, race/ethnicity, education, and income), lifestyle (smoking and alcohol consumption), and clinical data (tumor site and staging). It is worth noting that sociodemographic variables and lifestyle habits were self-reported. The Head and Neck Symptom Checklist (HNSC), a validated tool in individuals with HNC , was used to assess NIS. It consists of 17 symptoms assessed on a five-point Likert scale ranging from “1, not at all” to “5, very much.” For analysis, all 17 symptom scores were added, resulting in a total score that can range from 17 (no symptoms) to 85 (highest score for each symptom on the list) . The Nutritional Risk Screening tool was used to assess nutritional risk. It is a validated instrument and is based on data on dietary intake in the previous week, body mass index (BMI), weight loss in the last three months, age, and disease severity. Patients are scored according to malnutrition and the severity of the underlying disease, classified for each variable as absent (0), mild (1), or severe (2). Patients with a score of three or more are classified as at nutritional risk, while those with a score below three should be reassessed . In addition, anthropometric measurements (weight and height) were collected by the researchers, and body composition measurements (appendicular skeletal muscle mass) were obtained using a portable InBody 120 bioimpedance device. Data were evaluated on Research Electronic Data Capture , uploaded to the cloud of the Federal University of Espírito Santo, and organized and analyzed on R (4.3.1) for Windows. Absolute and relative frequencies were used to describe the categorical variables. Measures of central tendency (means and medians) and dispersion (standard deviations and interquartile ranges) were used to describe continuous variables. NIS scores (17–85 points) were used as the dependent variable. A multiple linear regression was performed to quantify the participation of independent variables (sex, age, staging, education, income, topography, race/color, smoking, alcohol consumption, nutritional risk, BMI, and ASMM) in the outcome. The significance level for all tests was set at 5%. The sample consisted of 132 participants, with a higher proportion of older adults (61%)—with a mean age of 61 years, men (77%), mixed-race people (54%), with less than eight years of education (71%), and a family income of less than or equal to two minimum wages (76%). In terms of lifestyle habits, most participants reported being former smokers (52%) and former drinkers (65%). However, even after diagnosis, 19% continued to consume alcohol. The most prevalent topography was in the oral cavity (45%), with stage IV tumors (53%). Although 43% of participants were overweight (showing overweight and obesity), 45% of the population were at nutritional risk according to the NRS at the time of diagnosis ( Table 1 ). Regarding NIS, 95% of participants showed one or more NISs ( n = 126), with the most recurrent symptoms being pain (68.93%), difficulty chewing (60.60%), thick saliva (59.84%), anxiety (58.33%), sore mouth (52.27%), and difficulty swallowing (50.0%) ( Table 2 ). Table 3 shows the data on the association between sociodemographic, lifestyle, and clinical variables and the total score of NISs. Topography, smoking, and nutritional risk showed an association. Cancer in the larynx ( p =0.031) had an estimated reduction of 6.67 points in NIS scores in comparison to that in the oral cavity. Regarding smoking, former smokers ( p =0.019) showed an estimated reduction of 5.87 points in NIS scores than smokers. Finally, participants with nutritional risk ( p =0.009) have an estimated increase of 6.15 points in total NIS scores when compared to those without nutritional risks (see Table 4 ). This study evinced a high severity and quantity of NIS in its population. About 9 out of 10 participants reported one or more NISs before treatment, corroborating the findings by Farhangfar et al. , who observed that 94% of their HNC cohort had one or more NISs before antineoplastic therapy. NISs lead to a series of consequences for individuals, inducing an inflammatory response that limits energy and protein intake and contributes to increased stress and weight loss before and even during treatment . Additionally, NIS presence and severity tend to increase during treatment , which can impact treatment efficacy and sometimes necessitate treatment interruption until the specific symptom is reversed and the individual can resume therapy. Jin et al. showed that NIS worsened during radiotherapy early on, emphasizing the importance of managing these symptoms before starting therapy. The most recurrent symptoms included pain (68.93%), difficulty chewing (60.60%), thick saliva (59.84%), anxiety (58.33%), sore mouth (52.27%), and difficulty swallowing (dysphagia) (50.0%). Results showed symptom clusters similar to previous studies. Kubrak, Olson, and Baracos found that the most common symptoms before treatment were pain (33%) and dysphagia (29%). Farhangfar et al. reported that the most prevalent symptoms were pain (63.6%), anxiety (62.9%), and lack of energy (58.5%). Furthermore, Granstrom et al. showed that the highest NIS scores at baseline occurred for pain (96%), anxiety (96%), and sore mouth (95%). Despite the discrepancies in symptoms across studies, pain is commonly the most reported symptom, typically ranking first in proportions. This study may explain this fact by the predominant location (the oral cavity) and the advanced stage of the disease (stage IV). This study found an association between tumor location and NIS score before treatment. Individuals with HNC in the larynx had NIS lower quantity and/or severity than those with cancer in their oral cavity. These findings resemble those by Granstrom et al. , who found a higher NIS score in individuals with HNC in the oral cavity and oropharynx than in those with larynx cancer during and after treatment. The main signs and symptoms in laryngeal cancer include hoarseness, pain during swallowing, changes in voice quality, sensation of lumps in the throat, difficulty breathing, or even shortness of breath . Of these reported symptoms, the HNSC—the tool chosen for the study—includes only one: odynophagia (painful swallowing), with only 12% of the population reporting this type of NIS. Therefore, a partial explanation for a lower presence and/or severity of symptoms in laryngeal cancer may be due to the chosen tool ignoring the other characteristic symptoms of this type of cancer. Individuals who quit smoking had lower NIS presence and/or severity than those who still smoked. Smoking is a well-established direct risk factor in the literature, considered the primary risk factor in HNC development . Tobacco acts as a factor for tumor growth, and cigarette smoke acts as a mutagenic and DNA-damaging agent that drives tumor initiation in normal epithelial cells . Thus, individuals who continue smoking after diagnosis contribute to this entire cascade driving the tumor, which is consistently related to a higher burden or severity of NIS. The presence of nutritional risk in individuals with HNC is relatively high from the time of diagnosis. Overall, 45% of the population in this study was at nutritional risk, agreeing with the findings by Wang et al. and by Bossi et al. , who found around 30.3% of the HNC population showing nutritional risk at the time of diagnosis. Furthermore, the presence of nutritional risk and total NIS scores showed an association. Individuals at nutritional risk had a higher presence or severity of NIS than those without nutritional risk. According to Wang et al. , NISs are positively correlated with nutritional risk in HNC patients in the perioperative period, resulting in higher scores according to the NRS-2002. This study has limitations. Its cross-sectional design implies an analysis of NIS at a single point in time, which affects causality. Additionally, a multicenter evaluation with a larger number of patients could provide more accurate assumptions. However, the findings in this study complement the literature and show that health education and nutritional monitoring should be provided to patients from the moment of diagnosis to help them understand the importance of nutritional treatment, ensuring adequate nutritional intake and improving their nutritional status. | Other | biomedical | en | 0.999997 |
PMC11699984 | From parasitic health factors of equine, strongyle nematode parasites embrace the first place. The strongyles are nematode parasites found in the large intestine, specifically in the caecum and colon of equines. Strongyles of equines are categorized as large strangles and small strongyles (cytostome). The large strongyles comprise the three most important species found in equines Strongylus vulgaris ( S. vulgaris ), Strongylus edentatus ( S. edentatus ), and Strongylus equinus ( S. equinus ). S. vulgaris , S. edentates , and S. equinus are commonly known as the double-tooth strongyle, the toothless strongyle, and the triple-toothed strongyle, respectively. S. vulgaris is smaller than the other two large strongyle species . Among the gastrointestinal nematodes of horses, large strongyle infections, particularly S. vulgaris , have long been considered one of the most common and pathogenic parasites affecting horses . Strongylosis poses a serious problem in young horses raised on permanent horse pastures, although cases of severe disease may also occur in adult animals kept in suburban paddocks and subjected to overcrowding and poor management . Strongylosis has been reported worldwide and affects nearly 90% of the horse population . In Ethiopia, equines make significant contributions to the national economy, yet parasitic helminths are among the most common factors hindering their maximum utilization. These parasites cause varying degrees of damage depending on the species, nutritional status, and immune system of the equines, resulting in decreased performance and productivity, as well as increased morbidity and mortality . The prevalence and types of internal parasites affecting equids are widespread, with equines being continually exposed throughout their lives . S. vulgaris and S. edentatus are among the most common equine health problems caused by strongyle species in Ethiopia, with S. equinus being less common . Although strongyle infections pose significant concerns for both the economy and equine health in Ethiopia, there is limited availability of recent data that shows the current status of strongyles, particularly in and around Bishoftu, Oromia, Ethiopia. Therefore, the objectives of this study were to determine the prevalence of strongyle parasites, to evaluate associated risk factors, and to identify the important genera of strongyles in horses and donkeys. The studied animals were 364 equines (298 horses and 66 donkeys) in and around Bishoftu town managed under a traditional extensive management system that originated from five localities (Kality, Dambi, Dalota, Bambogaya, and G/Gorba). The study encompassed both clinically suspected and healthy groups of donkeys and horses. The study animals were primarily used for traction, transport, and cart pulling, from which samples were obtained. During sampling, the animal species, sex, age, and body condition were recorded. The ages of the equines were determined from birth records of owner history and dentition characteristics. The body condition scoring was based on the criteria of donkey sanctuary and was classified into poor, moderate, and good. Faecal samples were collected exclusively from animals that had not been treated or dewormed in the 3 months preceding the study to ensure unbiased results. A cross-sectional study was conducted from February 2022 to July 2022 to determine the prevalence of strongyle parasites in horses and donkeys, as well as to evaluate associated risk factors in and around Bishoftu. The study animal was conducted by using a simple random sampling method to examine the prevalence of equine (horses and donkeys) strongyles parasites. During the study, 364 equines (298 horses and 66 donkeys) were randomly selected based proportionality of their total numbers that were presented in and around Bishoftu town (Kality, Dambi, Dalota, Bambogaya, and G/Gorba). Five kebeles (peasant associations) were randomly selected using a sampling frame created from a complete list of kebeles in Bishoftu town. Similarly, the total population of each horses and donkeys in each kebele was determined, and the number of animals to be sampled was proportionally allocated based on their population in each kebele. Individual study animals were then randomly selected using a lottery system. The consent of their owners was obtained and conducted a brief interview. Faecal samples were collected directly from the rectum of the selected animals by the researchers to ensure sample quality and minimize bias. The required sample size of the study was determined by using the formula given by , with a 75.8% expected prevalence that was previously done, a 95% confidence level, and 5% desired absolute precision. (1) n = 1.96 2 Pexp 1 − Pexp d 2 , where n is the sample size, Z (1.96) is the statistic corresponding to the level of confidence 95%, Pexp is the expected prevalence, and d is the desired absolute precision 5%. Therefore, based on the above formula, the desired sample size was 282 equines. However, the sample size was increased to 364, to increase the precision of the research and to obtain enough fecal egg outputs. With gloved hands, approximately 10 g of fecal samples were directly collected from the rectum of restrained donkeys or horses in the clean sampling plastic bottle. Following collection, each sample was uniquely labeled with the animal's identification parameters, including species, age, sex, origin/site, and body condition score, and kept in the ice box. The collected samples were then packed and transported to the Addis Ababa University Veterinary Parasitology Laboratory for immediate examination of strongyle parasite eggs. If immediate processing is not possible, the sample was stored at +4°C in the refrigerator. The fecal samples were subjected to coprological examination for the fecal flotation technique. Approximately 3 g of feces were measured and placed into a glass beaker, followed by the addition of 40 mL of flotation fluid (magnesium sulfate). The mixture was continuously stirred using a glass rod, and the dissolved suspension was strained through a tea strainer into another beaker. The suspension was then transferred to a test tube until a meniscus formed at the top of the tube, and a cover slip was gently placed over the meniscus, allowing it to stand for 20 min . The coverslip was removed and placed gently on a clean glass slide and examined under the microscope at low power magnification (10x) . It is important to recognize that the eggs of all equine strongyles are nearly identical and cannot be distinguished based on egg morphology alone, whether at the subfamily, genus, or species level. To achieve differentiation, the eggs can be cultured to hatch and develop to the L3 stage. This process involves a coproculture (fecal culture), followed by the Baermann technique and microscopic larval identification . The modified Baermann technique was employed to isolate the third larval stage of nematode parasites from fecal cultures. First, a pooled fecal sample was collected from naturally infected horses that tested positive. The fecal culture of eggs to third-stage larvae was then carried out to differentiate the genus of equine strongyles. Ten g of fecal samples containing strongyle-type eggs were pooled, finely ground using a mortar and pestle, and cultured in a Petri dish with a small amount of water to moisten the sample. The samples were incubated at 27°C for 7 days and mixed periodically. The larvae were then recovered using the modified Baermann technique . After collection, the third-stage larvae were mounted on slides, killed with Lugol's iodine, and identified under a microscope to the genus level based on morphological characteristics as described by . The data collected from the study were coded and entered in a Microsoft Excel spreadsheet 2007, and the statistical analysis was performed using SPSS Version 16 software packages. The chi-square test was used to assess the difference in the prevalence of strongyles nematode parasites among different variables such as equine species, sex, age, body condition, and origin. The percentage was used to calculate the prevalence rate of strongyles parasites. In all cases, 95% confidence interval (CI) and p < 0.05 were considered for a statistically significant difference. In the current study, the data distribution was a non-normal distribution. Based on the coprological analysis (egg morphology) (Supporting Information S1 ), the overall prevalence of strongyles nematode parasites of equine in the study area was found to be 56.6% (206/364) with a prevalence of 54.7% and 65.2% in horses and donkeys, respectively ( Table 1 ). The analysis highlights significant associations between risk factors such as sex ( χ 2 = 16.10 and p ≤ 0.001), age ( χ 2 = 14.78 and p ≤ 0.001), and body condition ( χ 2 = 8.60 and p = 0.013) with the prevalence of stronglyles infection in horse. However, prevalence, while origin shows no significant variation ( χ 2 = 3.32 and p = 0.506) in ( Table 2 ). The chi-square analysis revealed that there was no significant difference in the occurrence of strongyles parasitic infection among the equine species between horse and donkey ( χ 2 = 2.404 and p = 0.121) ( Table 3 ). Regarding body condition of the equines, the highest relative prevalence was observed in poor (64%) than those in medium (54%) and good (50%). Prevalence in female (70.7%) animals was higher than in male (52%). The prevalence of strangles nematode parasites according to age group was 59.3% and 42.1% in adult and young equines, respectively. The prevalence of strongyles parasite was insignificantly varied between sexes ( χ 2 = 2.644 and p = 0.266) and age of the equines ( χ 2 = 0.609 and p = 0.435) but it was significantly varied among body conditions ( χ 2 = 76.536 and p ≤ 0.001) ( Table 3 ). Among the five origins or sites study areas, the highest prevalence in horses and donkeys was revealed from Kality followed by Dalota, Bambogaya, Dambi, and G/Gorba, with the prevalence of 22%, 19.8%, 19.8%, 19.2%, and 19.2%, respectively. The study showed no statistically significant difference ( χ 2 = 3.205 and p = 0.524) between the study area and the prevalence of gastrointestinal parasites in both horses and mules . Larvascopy examination analysis revealed that the two genera of strongyles were identified by the morphological characteristics of third-stage larvae stages. These two important identified genera were Cyathostomum and Strongylus . Cyathostomum spp. and were characterized by short, small, and thin larvae with a thin and long tail, and Strongylus was long, large, and broad larvae with a short esophagus and well-defined gut cells with a long tail as shown in Supporting Information S2 . The coprological examination done for the current study using floatation techniques revealed an overall prevalence of strongyle nematode parasites in and around Bishoftu town to be 56.6%. This finding was closely aligned with previous reports by , who reported a prevalence of 47.4% in horses and donkeys in and around Kombolcha town, Ethiopia. The current study finding's prevalence was lower than the reports of , who documented prevalence rates of 64.61%, 100%, 99%, and 100% for Menz keya gerbil district, East Shewa, Adaa, and Akaki of East Shewa, respectively. The variation in the prevalence reported might be due to variations in sample size and sampling time as seasonality affects the occurrence of the parasites, feeding practice, and deworming habits . In the current finding, the prevalence of 65.2% and 54.7% were recorded in donkeys and horses, respectively, the difference being statically nonsignificant ( p > 0.05). The present study result is relatively in line with the works of , who reported a prevalence of 70.8% in donkeys and 58.5% in horses from South Wollo. This finding was higher than the reports of , who reported a prevalence of 44.55% in donkeys and 48.2% in horses. However, the current study's results were lower than the reports of , which reported a prevalence of 87.8% in donkeys and 66.7% in horses from Gondar, the reports of , which revealed 100% and 99% prevalence in donkeys and horses, respectively, from East Shewa-Adaa, and another study that reported a higher prevalence of 99.15% in Sudan . The higher prevalence in donkeys might be attributed to differences in feeding and deworming activities, while the lower prevalence in horses could be due to their predominantly being cart horses in the study area, which are less exposed and sometimes completely restricted from pasture and grazing. Furthermore, differences in prevalence in different areas might be attributed to variations in sampling areas, feeding systems, and accessibility to deworming and health services . Regarding the body condition score of equines, the prevalence of strongyle parasites was 64%, 54%, and 50% in poor, medium, and good body-conditioned equines, respectively, and there was a statistically significant association ( p < 0.05) between the existence of strongyle infection and the body condition score of equines. This finding is in agreement with previous reports by , who reported a statistically significant association ( p < 0.05) between the occurrence of strongyle infection and the body condition score of equines. However, there was no significant difference in the prevalence of strongyle parasites in the equines from different origins of the study area. This is similar to the findings of . This may be due to the similarity in the agroecology of the study areas, the epidemiology of the parasites, and the management systems used for the equine species. In this study, there was no significant difference ( p > 0.05) observed in the prevalence of the parasites between young and adult equines. The highest prevalence of strongyle parasite infestation was observed in adults (53%) compared with young equines (3.6%). It may be attributed to the declining body condition and immunity of adult equines compared with young ones, as older equines are more frequently exposed to strongyle parasites due to extensive work overload and undernourished conditions . In the present study, the equine strongyle parasites genera Srongylus and Cyathostomum were identified. These findings were conducted based on characteristics of strongyle nematode larva morphology similar to other studies conducted worldwide as well as in Ethiopia. This finding supports previous reports of in Cambridge , in Brazil , in the United States , and in Ethiopia. The current study revealed that equine strongyle infections are highly prevalent in the study area, indicating a significant health concern for equines. This highlights a critical gap in the control and prevention of parasitic diseases, suggesting that they are often overlooked or inadequately addressed. To reduce the prevalence of strongyle infections and improve the health and productivity of equines, it is essential to implement a strategic, programmatic approach to deworming. Moreover, this study has certain limitations, including the lack of an in-depth assessment of deworming practices, determination of infection levels, and evaluation of the efficacy of anthelmintic treatments. As such, it is recommended that future research builds upon this study as a baseline, conducting more comprehensive investigations to provide a more thorough, significant, and valuable contribution to equine owners, veterinarians, policymakers, and government agencies. These efforts will be crucial for developing effective interventions to control strongyle infections and improve overall equine health. The current research underscores the vital role of equines as a means of transportation in rural and semiurban areas. However, strongyle parasites pose a significant threat to equine species and have a considerable economic impact. Equines often graze freely in pastures, exposing them to a high risk of parasitic diseases. Parasitized equines exhibit poor immunity and health status, leading to decreased working performance. Therefore, as a recommendation to control the burden of parasites, regular and strategic deworming programs using effective anthelmintics should be implemented consistently. Moreover, to obtain a clearer epidemiological picture, molecular characterization of parasites and assessment of anthelmintic resistance profiles should be conducted. | Study | biomedical | en | 0.999996 |
PMC11699985 | Sudden cardiac death (SCD) is an unexpected lethal cessation of cardiac function that accounts for approximately half of the 17 million cardiovascular-related annual deceases that are estimated worldwide . One of the significant causes of SCD is dilated cardiomyopathy (DCM), which is the leading global indication for heart transplantation (HT) . DCM is defined as a condition in which the ventricles of the heart enlarge and the myocardium walls become thinner, compromising cardiac output and making it difficult to oxygenate the brain and body cells . The pathological manifestations of DCM can vary from mild symptoms of heart failure (HF) in adults to the onset of SCD at an early age . The prevalence of DCM in HF has been reported of approximately 1:250–400 and 5–7 new cases per 100,000 persons per year , although an accurate update estimation is difficult to assess. Around 40% of familial DCM cases can be clarified by genetic variants, and most of them were inherited as an autosomal-dominant disease in families . Diagnosis of SCD syndromes may be the most pressing challenge in cardiology due to its complex physiopathology, unknown molecular mechanisms and inadequacy of current genomic tools. Moreover, this challenge is compounded by difficulty in managing cardiac tissues. However, researchers continue to find new gene variants in many malignant cardiac cases such as DCM [ 6 – 8 ], that could be the cause of its etiology, which highlight the importance of genetic testing in certain individuals to implement an adequate preventive and personalized treatment plan if needed. The current study describes the clinical features of a severe DCM in a 1-year-old infant and links this pathogenesis with the presence of two rare and presumptive deleterious genetic variants that change well evolutionary conserved regions in the ACTC1 and TTN structural genes. Additionally, the ACTC1 mutation appears to have a de novo origin, which highly supports its probable pathogenicity. Overall, the identification of these mutations broadens the base of pathogenic variants linked to SCD. Genomic DNA was extracted from peripheral blood lymphocytes using a commercial kit (Gentra System, Puregene, Valencia, California, the United States of America). The Supporting Tables S2 and S3 describe the library of 48 structure-related and 84 electrical-related genes, respectively, that were amplified using the Ion AmpliSeq Library Preparation protocol (Thermo Fisher Scientific, Waltham, Massachusetts, the United States of America). Clonal fragments were loaded into an Ion 318 Chip Kit v2 BC with the Ion Chef system and analyzed by NGS using the Ion Torrent Personal Genome Machine. After variant calling from the Ion Reporter software, genetic variants were further selected for identification of candidate deleterious mutations, selecting only those with MAF under 0.03, by comparison to the reference genomic DNA sequences from the 1000 genomes' project , as well as other previously described criteria , which include the score evaluation of each mutation according to the in silico prediction tests PolyPhen-2, PROVEAN, and SIFT. Genomic DNA (80–120 ng) was purified postamplification using the ExoSAP-IT kit (Applied Biosystems, Foster City, California, the United States of America), and sanger sequencing was performed with dideoxynucleotides using 0.8 μL of BigDye terminator v3.1 (Thermo Fisher Scientific, Waltham, Massachusetts, the United States of America) in the appropriate buffer, 0.5 μ M of each primer in separated reactions, 1 μL of PCR product, and 4 μL of HPLC grade water. Final PCR products were purified using Sephadex columns G-50 (Sigma-Aldrich, St. Louis, Missouri, the United States of America), and sequencing reactions were performed in an ABI PRISM 3100-Avant Genetic Analyzer (Applied Biosystems, Foster City, California, the United States of America). Electropherogram analysis of both DNA strands was achieved using sequencing analysis v.5.3 software (Applied Biosystems, Foster City, California, the United States of America). Protein structural homology of wild-type cardiac actin and its found variant were performed using the online workspace of SWISS-MODEL . To generate the 3D protein structure, files from the protein data bank (PDB) were visualized in PyMOL 2.4.0 software (DeLano Scientific) , and the corresponding analysis of protein features was conducted. In parallel, the structure of wild-type cardiac titin and its variant were analyzed following the method of Cukier . We used the probability of occupancy for each pair of amino acids in dihedral angle space to generate an ensemble of conformations of the peptide encompassing five residues flanking each side of the mutant. Each eleven-residue peptide had 1300 allowed conformations generated after removing the conformations with high-energy overlap of atoms that violate the Ramachandran map. The range of conformations and the end-to-end distance for each peptide was analyzed. To assess the evolutionary importance of the amino acid change provoked by confirmed mutations, a comparison across different species was used to determine the amino acidic grade of conservation, wielding multiple protein alignment tools with the interest protein sequence from different species. After performing Sanger sequencing of the available proband's relatives for the confirmed variants, a pedigree tree was constructed to elucidate the heritability pattern. A one-year-old Mexican boy who presented severe HF and DCM was properly diagnosed based on the current classification . His parents and brother were apparently healthy individuals with no reported clinical history of SCD syndromes nor cardiac-related deaths within their family. The parents reported that the symptoms in the patient began during the first months of life but had rapidly intensified in the following weeks. The patient was diaphoretic, tachycardic, tachypneic, and hypotensive due to inadequate cardiac output. Palpation of the precordium demonstrated an apical impulse displaced downward and laterally a systolic murmur Grade III/VI and an S3-S4 gallop rhythm were audible in all the cardiac auscultatory focus. The twelve-lead electrocardiogram showed sinus tachycardia of 159 bpm, left increased precordial lead voltages secondary to ventricular dilation, an incomplete bundle branch left ventricle (BBLV) was apparent and abnormalities in the repolarization were noticed with inverted T waves in V3-V6 leads. Chest roentgenography demonstrated marked cardiomegaly. The cardiothymic silhouette was enlarged mainly because of left atrial and ventricular dilation. Pulmonary venous congestion and pulmonary edema were evident as well. Echocardiography confirmed the diagnosis of DCM with the left auricle and LV severely enlarged. End-diastolic and end-systolic LV diameters were increased (diameter at the end of LV diastole of 34 mm; z score +5.5, LV diameter at the end of systole of 31 mm; z score +7.1). The interventricular septum was bulging into the right ventricle, and the interventricular septum and free wall of the LV appeared diminished. Systolic function was decreased, and LV ejection fraction (LVEF) was calculated at 7%. The origin of both coronary arteries presented normally. The initial treatment of the patient was based on intravenous diuretics with combined inotropic and vasodilator support (levosimendan and milrinone) with incremental titration as the clinical situation dictated. Once the clinical situation improved and the patient was able to eat, the use of oral beta-blocking agent with bisoprolol was also initiated with incremental titration. In addition to optimal pharmacological management for the HF, with the function of the right ventricle still preserved, the patient underwent surgery for a pulmonary artery band as a bridge therapy for HT based on a recent worldwide experience report . Temporary improvement was obtained, and the patient was sent home with ongoing consultations; however, the child died suddenly at home 6 months later waiting for HT. The NGS analysis in the patient suffering DCM yielded 243 Mb of read bases from which 95.61% obtained at least a Q20 score. 99.1% of the reads were on target, and 97.99% of them read at least 20x more with mean sequencing depth of 593 reads per amplicon. A summary of the coverage analysis data of the NGS is described in the Supporting Table S1 . Alignment and variant calling showed a total of 343 variants, derived only from the 48 structure-related gene panel. After the applied internal filtering criteria, two variant genes were discovered with a heterozygous single-nucleotide variation (SNV) each, whose minor allele frequency (MAF) was cero in the control cohort. Thus, we confirmed the coexpression of a TTN and ACTC1 mutants (Figures 4(a) and 4(b) ). In silico predictors suggested the mutations to be deleterious to a certain extent ( Table 1 ). All of them classified the ACTC1 variant as disease-causing or damaging, while the TTN mutation was denoted by PolyPhen-2 as possibly damaging. Additionally, the conservative analysis revealed that both mutated amino-acid residues were situated in well-conserved domains among many orthologous proteins (Figures 4(c) and 4(d) ), suggesting the significance of the affected protein sites throughout evolution. Interestingly, no mutation in the 84 electrical-gene panel related to cardiac anomalies was found. Sanger test validation of the variants and further genealogy analysis conveyed that the TTN variant was transmitted from the father, which was also acquired by the proband's sibling. However, to our surprise, the ACTC1 variant was not present in the parents nor in the brother ( Figure 4(e) ). As the frequency of spontaneous mutations in human DNA is relatively low due to the DNA polymerase exonuclease activity, we decided to avoid false-positive results by performing Sanger sequencing of the same loci again in the four individuals, which resulted in no sequence changes, validating the presence of both mutations. Quality parameters declared a reliable template for protein 3D modeling (GMQE = 0.99; identity = 98.67%), and the structure of the monomeric actin was successfully constructed. Among several other observed structural shifts, distance changes between Ala222Thr neighboring residues in subdomain four were observed, as well as hydrogen bond rearrangements including a new steric contact between the mutant substitute 222Thr and 224D. The most relevant changes are depicted and described in Figure 5 . In the modeling of the region around the amino acid change in titin, this E11084K mutation appears to hinder the formation of compact states, leading to a larger average end-to-end distance for the peptide , which may impact the spatial distribution in the sarcomere. The presence of genetic deleterious variants expressed in cardiac tissues can lead to structural heart defects. Congenital heart defects compel people susceptible to an unexpected stoppage of the heart by triggering arrhythmias, the principal cause of SCD . These SCD syndromes are mainly explained by the alteration of two groups of cardiac proteins: (1) those that principally constitute the scaffolding of cardiomyocytes and give them support and structure and (2) those related to the action potential and electrical transduction. In a previous study carried out at the cardiomyopathies and arrhythmias research laboratory of The Children's Hospital of Mexico Federico Gómez, DCM was the most frequent variety of cardiomyopathy (62.5%) and the most common disease of all the hereditary SCD syndromes (including cardiomyopathies and hereditary electrical diseases; 42.37%) . The infant presented in this study corresponds to the case with the most malignant presentation (early diagnosis and death) within this cohort of Mexican children from 0 to 18 years with hereditary SCD syndromes, which give support to the clinical significance of the findings described here. To date, variants in over 50 genes have been linked to familial DCM , unfortunately, many of these cases remain without a clear molecular etiology. Next generation sequencing studies performed by our research team showed that the infant case described here did not possess any genetic variations within a selected group of 87 ion channel genes related to cardiac anomalies (unpublished data). Nonetheless, we found two interesting germline variants within the structural cardiac genes ACTC1 and TTN , which aid in facilitating the identification of possible DCM-causing genes. The quality of the data generated though NGS is an important parameter to be considered when used to correlate with clinical diagnosis because it is a mark of the experiment's reliability, also giving an insight about the quality of the technical performance of the experiment. Our results pertain to the recommended sequencing depth (120x) for the diagnosis of genetic variants with clinical significance and the detection of heterozygous SNV (35x) . Moreover, the obtained on-target reads percentage and mapped reads surpassed the threshold of a good-quality library (80% and 100,000, respectively) . Other studies searching for new genetic variants linked to cardiomyopathies by NGS showed similar or fewer quality data points in terms of the abovementioned parameters [ 8 , 9 , 22 – 24 ]. The first genetic mutation related to DCM was identified in ACTC1 (gene encoding for cardiac alpha actin protein) which was segregated in an autosomal-dominant pattern . Since then, several ACTC1 mutations have been identified from which up to 20% can cause DCM , some of them even overlap with different types of cardiac hereditary diseases [ 26 – 30 ]. Cardiac actin is a fundamental protein located in the sarcomere of cardiomyocytes, and it is crucial for myocardium contraction, force generation, and blood pumping. For this reason, it was very interesting to find that the ACTC1 mutation in the subject of study was not segregated by either of the parents, denoting its de novo origin, a very rare type of “spontaneous” acquisition. To the best of our knowledge, there is no peer-reviewed data regarding a phenotypic association of c.664G > A ACTC1 /p.Ala222Thr variant in the available literature or in databases such as PubMed, and it is absent in control cohorts as 1000 genomes, exome sequencing project, the exome aggregation consortium (ExAC), or the genome aggregation database (gnomAD). Notwithstanding, it is of important notice that there are five previous reports of this variant from different submitters in ClinVar database, two of them being classified as likely pathogenic in DCM cases also with a de novo origin. Based on the American College of Medical Genetics and Genomics (ACMG) guidelines and the features we found, our research team also classified the c.664G > A ACTC1 variant as likely pathogenic in the present case (categories PM2, PM6, PP3, and PP5 are met). As nonrelated cases, these findings could remark the potential pathogenicity of the variant. The origin of spontaneous SNV can happen during embryonic development through different mechanisms, and it is estimated that up to 74 SNV can be found in a single germinal genome . The detection of de novo variations is frequently initiated by genetic research, as there are DNA changes that do not come from family inheritance. These de novo mutations are generally very important in the etiology of diseases because they have been occurring outside of the usual parameters of natural selection, so they are more likely to cause undesirable functional consequences . This strongly suggests that ACTC1 mutation may play a pathological role in the studied DCM case. The amino acid change in cardiac actin occurred in a highly conserved region of actin in a variety of species ( Figure 4(c) ) which constitutes a hallmark that connote its possible pathogenic effect. This actin protein variant has a change from alanine to threonine in the 222nd position, in relation to the human genome reference GRCh37.p7. According to X-ray fiber diffraction studies, this position is located in an intra-subdomain four region ( Figure 4(f) ). Mutations in Subdomains 1 and 4 of actin have been recognized to likely affect protein-protein interactions . Subdomain 4 of actin maintains electrostatic interactions with proteins such as tropomyosin . Tropomyosin is a key protein in cardiomyocyte contraction because it acts together with troponin to sterically hamper the union of myosin heads when Ca ++ levels are low . On the other hand, it is scientifically recognized that hydrophilic amino acids of a given polypeptide chain are facing the surface of the molecule in protein folding, while the hydrophobic amino acids are located interiorly. The p.Ala222Thr actin variant provokes exactly the opposite, which could explain our in silico findings where structure and hydrogen bond arrangements change intra-subdomain four. That perhaps leads to defects of actin polymerization in vivo, hampering the formation of filamentous actin and in turn, and the contraction of cardiomyocytes as it was demonstrated for the p.Gly247Asp actin variant, located also in subdomain four . Either conformational or affinity changes between tropomyosin and actin or defects of actin polymerization would lead to cardiac remodeling such as DCM. However, further specific functional studies are needed to corroborate this hypothesis. The index case described here also had a cosegregation of a heterozygous mutation in the TTN gene that encodes titin protein. For a matter of fact, its crucial role and its nucleotide length make TTN the major affected gene in DCM cases . Titin is located in the sarcomeres, between actin and myosin filaments, and it is the longest protein in humans. Its main purpose is to provide cell elasticity and structural network organization, preventing damage caused by cardiomyocyte resistance to contractions and providing passive tension to striated muscle. This ability is mainly attributed to an elastic structure enriched of proline, glutamate, valine, and lysine (PEVK), which constitutes an entropic biological spring and mimics a shock absorber . The conformations of the PEVK region appear to be sensitive to both pH and calcium concentration and, interestingly, the amino acid change in the titin variant that our research group found was a substitution of acidic and negatively charged glutamic acid by an alkaline and positive charged lysine in Position 11,084, which is also a conserved residue among species ( Figure 4(d) ). Specifically, this amino acid position is within the distal I band according to the spatial distribution of TTN in the sarcomere ( Figure 4(f) ) region where truncating variants have already been associated with DCM . However, despite the efforts of the scientific community to assess the real implications of missense TTN variants, the majority of them are still considered ambiguous because it is hard (and sometimes contradictory) to determine their pathogenicity with current approaches, leaving them without both molecular and a precise clinical significance ; functional studies are also difficult to carry out due in part to the nature of titin, which is considered the largest coded protein in humans. In the present study, the c.33250G > A TTN allele is considered as a variant of uncertain significance (VUS) according to the ACMG guidelines because of the contradictory features we found when analyzing the data in the giving clinical and scientific context. The mechanism by which the E11084K mutant contributes to cardiac disease may plausibly be one or more of several related effects. Charge pairs direct formation of compact configurations of the titin chain upon release of tension , so the insertion of a positive charge farther to the N -terminus of the PEVK region may disrupt the formation and stability of the compact or folded states that normally contribute to avoid mechanical damage. Disruption of the ability of titin to extend and collapse may inhibit its mechanosensory mechanism. On the other hand, it is possible that the poly- E motifs in titin interacts with F -actin , affecting the interactions that mediate the mechanical memory of the sarcomere. Previous reports of this TTN variant are currently found in ClinVar database in the clinical context of DCM and other myopathies, six of them being classified as VUS, and two as benign. On the other hand, some observations explain the pathological implications that some missense TTN variants can represent in some individuals, as the one that demonstrated to cause partial protein unfolding and domain destabilization . Also, an in vitro study revealed that induced pluripotent stem cell cardiomyocytes carrying a missense mutation within the sarcomere Z/I junction portion in titin developed a DCM-like phenotype . Nonetheless, it is of noticeable importance that the missense c.33250G > A variant addressed in the present study was observed to be inherited by the father of the study infant subject, which was also acquired by his brother but who did not present any cardiac symptomatology at the time of sampling, suggesting a minor cardiac pathogenic effect when expressed alone. Due to the suggestive pathogenic elements in all the previous examined data, one hypothesis to be considered in this work regarding the onset of DCM in our patient is an autosomal dominant behavior of ACTC1 mutation. Notwithstanding, due to the high complexity of cardiomyopathies, it is also suggestive that the cause of the malignant condition reported here may be derived from a polygenic synergistic effect of both ACTC1 and TTN variants, where this double hit is necessary to impair the phenotypic manifestation of an early and severe DCM. This type of polygenic effect has been also described and demonstrated in vitro and in vivo in another similar case of DCM , where two separate variants in VLN and TPM1 could not be associated with the DCM phenotype when evaluated separately but together, combined with disease-related stress factors. A limitation in our study is that we may be missing other factors that can prompt DCM onset, such as environmental challenges, variants with epigenetic significance, genes in other networks that either directly or indirectly interact with the sequenced structural genes, or rare somatic mutations. Altogether, as many other VUS, it is necessary to go further with this study and to carry out functional experiments to determine whether these two rare variants can trigger DCM and the possible mechanisms involved. Finally, by comparing our results with the available literature, we can conclude there is a diverse genetic background in DCM patients. Our report of the germline variants in TTN and ACTC1 connote the complexity of the possible molecular causes of SCD syndromes and affirm that there are still pathogenic genetic factors we could be facing for the first time, which challenges the prescription of the more appropriate and beneficial treatment. | Review | biomedical | en | 0.999997 |
PMC11699986 | Oral cancer (OC) ranks as the 16th most prevalent malignancy impacting the lips and oral cavity, encompassing the oropharynx, hypopharynx, and larynx . It ranks as the 15th most prevalent cause of mortality worldwide, with significant heterogeneity based on age, gender, race, ethnicity, and socioeconomic status . Despite the existence of several treatment options, the overall five-year survival rate for OC remains approximately 50% . OC, however rare in the Western world, is prevalent in high-risk nations . In 2017, South Asia recorded the highest incidence, death, and national burden of OC . OC ranks third among all malignancies in India and accounts for about 40% of total cancer-related mortality. Approximately 70,000 new cases and more than 48,000 deaths connected with OC occur each year. OC is quite prevalent in India, ranking as the second most common malignancy among males and the fourth among women. The prevalence escalates with age and is more prevalent in men . Oral squamous cell carcinoma (OSCC) was identified as the sixth most prevalent variety in 2016, accounting for 90% of head and neck malignancies . While OSCC can originate from anatomical sites in the head and neck, it predominantly manifests in the oral cavity . The etiology of cancer is mostly linked to detrimental behaviors, including betel nut consumption, smokeless tobacco use, and alcohol abuse, as well as factors such as hereditary predisposition and excessive sun exposure . The prevalence of OC among the younger demographic has lately increased due to filthy sexual practices associated with specific DNA viruses, including human papillomavirus and herpes simplex virus type I . Furthermore, due to the absence of a compulsion for cancer registration in India, the actual incidence and mortality rates are likely to be underestimated . Recognizing the predisposing factors is crucial for facilitating early diagnosis and preventing cancer progression and subsequent mortality. OC can be discovered early by methods such as screening high-risk populations, opportunistic screening in primary healthcare settings, and the prompt identification and reporting of signs and symptoms by individuals and healthcare professionals to confirm a diagnosis . This strategy is highly beneficial, but a lack of information and social stigma may hinder patients from seeking care at an early stage. Limited awareness correlates with reduced survival rates in patients with OCs . Consequently, heightened knowledge of the disease and improved access to healthcare services will facilitate early diagnosis, mitigate disease development, and enhance prevention efforts. Encouraging regular dental exams is recommended to enable the opportunistic evaluation of potential indications and symptoms. Furthermore, this approach would need dentists and other primary healthcare workers to be well trained to recognize the early signs of OC. This study aimed to assess the knowledge and attitudes of dental and medical students regarding OC and to enhance their understanding of the associated hazards. A cross-sectional study was initiated on obtaining approval from the Institutional Ethics Committee at Manipal College of Dental Sciences, MAHE, Mangalore, bearing reference number 16117, from January 2022 to June 2022. A convenience sampling method was used to enroll the undergraduate students of II-year medical ( n = 144, Kasturba Medical College, Mangalore) and dental ( n = 112, Manipal College of Dental Sciences, Mangalore) fraternities. The rationale for selection of II-year participants was to evaluate their awareness prior to the exposure to theoretical and clinical knowledge and evaluation of cancer and related features. The participants were thoroughly instructed about the study objectives before handing out the printed questionnaire. Permissions to administer the questionnaire were obtained from the Heads of the respective institutions. Before administering the printed questionnaire to individual participants, written consent was documented from each participant to ensure their voluntary and active participation in the study. The participants were well-informed that their personal information would be held strictly confidential. The routine evaluation of the oral mucosa was significantly more frequent among medical graduates, with 50.40% reporting this practice compared to only 30.40% of dental graduates ( p =0.002). Some questions showed borderline significance, such as whether they screen the oral mucosa if patients are in high-risk categories ( χ 2 = 3.107, p =0.078), or the presence of ulcers ( χ 2 = 3.22, p =0.073); these results suggest trends that may warrant further investigation. Fewer dental students (44.40%) than medical students (58.80%) reported screening high-risk patients when they do not routinely examine the oral mucosa, and dental students related ulcer to be more related to malignancy than the medical students. Both groups felt adequately informed about the clinical appearance of OC. However, dental graduates demonstrated greater awareness of clinical features such as white patches, proliferative growth, cauliflower-like growths, and pedunculated growths, compared to medical graduates. Medical graduates predominantly identified ulcers as a clinical feature but were less aware of other signs of OC. This study aims to elucidate the behaviors and attitudes of dental and medical students regarding OC, a lethal oral disease, and to enhance knowledge of its related risk factors at an early age. Our study found that medical graduates examined the oral mucosa substantially more than their dentistry and medical student counterparts. This could be attributed to the fact that clinical exposure for medical students start from the II year itself. Approximately 49.6% of medical graduates and 69.6% of dentistry graduates do not assess the oral mucosa. Dental graduates must enhance their thoroughness in examining the oral mucosa and teeth. Early recognition of the significance of teeth and their related structures will enhance their comprehension of deviations from normalcy. Our study noted that, within high-risk categories, oral cavity screening rose to 58.8% in medical groups and 44.4% in dental groups, compared to 50.4% and 30.4% for routine examinations conducted by both groups, respectively. The data indicate that medical students engage in a clinical program starting in their second year, but dental students are introduced to a clinical environment only in their third year. This may explain the results for parameters 1 and 2, wherein medical students might have assessed patients' oral cavities, a scenario that was improbable for dentistry undergraduates. Our findings are consistent with previously published literature . Dental students should be encouraged to periodically visit a clinical setting to enhance their understanding of normal and pathological conditions of the oral cavity. Furthermore, this exposure can be expanded for spontaneous screening of high-risk patients during routine oral examinations, even at the community level. When comparing information about the etiology of OC, both medical and dental students demonstrated sufficient understanding of risk factors such as tobacco use and smoking. Medical graduates predominantly screened for chewing tobacco as the most prevalent high-risk category, whereas dental graduates focused more on smoking. Poor oral hygiene prompted more dentists to assess the oral mucosa than medical graduates. Nonetheless, the students had a limited understanding of additional risk factors related to alcohol usage and viral infections . Dental students had a greater enthusiasm for informing their patients about the risk factors associated with OC than their medical counterparts. This is likely due to the fact that a dental graduate's area of specialization is the oral cavity, in contrast to that of medical students. A study indicates that medical students exhibit a deficient attitude toward OC, attributed to little exposure during their educational experience . To foster awareness, broader comprehension must be imparted to the undergraduate students. The majority of dental students had negative responses regarding the opportunity to evaluate patients with oral lesions, in contrast to medical students. This disparity may be attributed to the variations in curricula among the fraternities. The dentistry group had much greater awareness of the clinical signs and symptoms of OC compared to the medical students. Nonetheless, both medical and dental graduates perceived themselves as sufficiently versed about their clinical presentation. Both student groups concurred that the ulcerative lesions are symptomatic of cancer. Nevertheless, dental graduates exhibited superior awareness of the malignancy of white patches (74.7%), proliferative growth (74.7%), verrucous development (35.6%), and pedunculated growth (27.6%). Nonetheless, literature reports exhibit inconsistencies contingent upon geographical location and several study characteristics . This may indicate enhanced awareness among dental students. The importance of early clinical indicators should be underscored to facilitate prompt diagnosis. In our study, medical graduates predominantly recommend referring patients to a physician when oral lesions are identified, in contrast to dentistry graduates. Enhancing understanding of the dentist's function in OC detection and prevention within the medical curriculum may elevate knowledge of streamlined referral mechanisms. Our findings indicated that, upon graduation, medical and dental students would preferentially refer patients to specialists in oral and maxillofacial surgery or oral medicine. Nonetheless, 12.2% of the medical graduates will refer patients to ENT specialists. Medical graduates possess greater awareness of ENT, a subject that is undervalued in dentistry, potentially explaining the unfamiliarity observed among dental students. Both groups concurred that the optimal recommendation for an OC patient is to an oral and maxillofacial surgeon. The outcome concurred with the extant literature . A knowledgeable and proficient healthcare team is essential to reduce the disparities between patient screening and referral to a specialist. This knowledge will facilitate an efficient referral system to avert unnecessary diagnostic and preventive delays. Medical and dentistry students similarly lack adequate understanding concerning the prevention and detection of mouth cancer and require additional information on the subject. The dentistry graduates would desire substantially more information about OC compared to the medical graduates. The information pack was the predominant request among medical graduates in contrast to seminars requested by dental graduates. Numerous findings have been documented by numerous authors within the pertinent literature. Soares et al. concluded that students had a good knowledge of the etiology of OC and were alert in their examinations to the possibility of detecting malignant lesions. However, they noted that the clinical features of the lesion were not sufficiently clear to the students. They suggested that knowledge of OC, in particular its clinical presentation, needed to be reinforced throughout the undergraduate dental course to enable raising suspicions and making an early diagnosis of lesions . Anushya, Dhanraj, and Keerthi also opined the necessity for an improvement of the teaching program regarding oral examination . Rai et al. and Chan et al. reported general lack of awareness about OC and its associated risk factors among student cohorts (dental and medical) of the Singaporean population and Malaysian population, respectively . They too emphasized upon the need for targeted education and to provide training to improve diagnostic skills for every dental undergraduate. Similar sentiments were echoed in studies conducted by Fotedar et al. , Kumar and Ak, Keser and Pekiner, and Saleem, Mahmoud and Joseph. Given considerable morbidity and effect on quality of life, initiatives to prevent the condition and enhance early detection are essential [ 23 – 26 ]. Nazar, Ariga, and Shyama studied knowledge, attitudes, and practices regarding OC among recently graduated dentists in Kuwait and found that majority of dentists were knowledgeable and aware of the numerous aspects of OC. They also concluded that the training programs in OC education must be prioritized and reinforced, with a particular emphasis on early detection and prevention. They strongly advised that dentists participate in continuing education programs and seminars to increase their understanding of the risk factors and diagnosis of OC . The findings of the present study indicated a disparity in knowledge and awareness of OC between the two study groups. The current curricular design does not allow dental students to see patients during their second year of training, in contrast to medical students, who begin patient interactions at the start of their second year. Engaging second-year dental students as observers in dental screening camps may address this gap. Medeiros et al. assessed knowledge about OC among dental students and Primary Health Care Dentists in Brazil and concluded the need for proper training to examine and identify signs of OC . Algudaibi et al. studied the difference of knowledge of OC between dental and medical practitioners and found that dental practitioners were found to be more knowledgeable about the high OC risk sites and predisposing factors than medical practitioners . As the adage goes “nipping the evil in the bud,” understanding risk factors along with diagnostic instruments to identify early symptoms can help alleviate the burden of OC. There were certain limitations in the study. A convenience sampling was performed; hence, the entire student community of both the institutes was not subjected to the questionnaire. A larger community was not provided with the questionnaire due to the higher risk of dropouts. Only the Mangalore campus of Manipal Academy of Higher Education was enrolled in the study for strategic access to the geographical location. Within the study limitations, it was concluded that the medical students had more knowledge about OC in terms of causes. In contrast, the dental students had more awareness of the signs and symptoms related to OC. It was also seen that dental students sought more information related to OC than medical students. Through this study, an attempt to elicit knowledge and awareness was made, and an introduction of an important step was attempted. The information gained through this questionnaire helps the students change perspectives regarding OC. An educational intervention or early exposure to dental/oral health screening camps should be implemented promptly to sensitize the students and familiarize them with the initial signs and symptoms of OC. The study's extended scope involves conducting further analyses involving multiple institutions and larger sample size to investigate factors affecting OC awareness among medical and dental students, including demographic variables, curriculum content, and clinical exposure, to gain a deeper understanding of the underlying reasons for the observed disparities in knowledge and practices between these groups. | Review | biomedical | en | 0.999996 |
PMC11699989 | Inflammatory bowel disease (IBD) is a chronic nonspecific disease that includes Crohn's disease (CD) and ulcerative colitis (UC). Common clinical symptoms include recurrent abdominal pain, diarrhea, and posterior tenesmus . A primary aim in treating IBD is to enhance the quality of life, given its chronic nature involving alternating between the active and remission phases, requiring lifelong management. The incidence rate of IBD is high in developed countries such as North America and Western Europe. The annual incidence rate can reach 29.3/100,000 people . However, in the past 20 years, the incidence of IBD in Western countries has remained relatively stable or even decreased, while the incidence in Eastern Europe and Asia has shown a continuous increasing trend . More specifically, the incidence of IBD has increased drastically in East Asian countries such as China , South Korea , and Japan . The course of IBD is characterized by alternating active and remission phases, protracted recovery, and the need for lifelong treatment, all of which seriously affect the patient's quality of life. The etiology and pathogenesis of this disease remain unclear but are generally believed to be closely related to genetics, the environment, etc. Currently, drug therapy is the most crucial treatment approach; however, it encounters numerous challenges, such as low response rates and potential side effects. Increasing research attention is directed toward investigating the potential advantages of nonpharmacological interventions, such as physical activity (PA), for patients with IBD. Some evidence based on the population shows that the implementation of an exercise intervention can reduce the risk of various chronic diseases, including cancer and cardiovascular disease . Furthermore, exercise interventions can improve patient outcomes with depression . The impact of exercise and PA on the gastrointestinal tract is an emerging research field. Several studies have shown an inverse relationship between PA and the risk of gastrointestinal related diseases such as colon cancer , diverticular disease , gallstones , or constipation . However, some studies have also shown the harm of exercise to gastrointestinal symptoms such as nausea, heartburn, diarrhea, and gastrointestinal bleeding. In recent years, studies have found that exercise may have multiple health benefits for IBD [ 16 – 18 ]. From the perspective of pathological and physiological mechanisms, regular exercise can reduce visceral fat content, inhibit the release of inflammatory factors, and induce an anti-inflammatory microenvironment, providing theoretical support for exercise assisted treatment of IBD . Recent studies have confirmed the existence of the “gut-muscle” axis , indicating that exercise has a regulatory effect on gut microbiota, which is crucial in the development of IBD . According to the WHO, engaging in PA can lower the risk of disease progression or premature death while also enhancing physical function and overall quality of life in adults with chronic diseases . Nevertheless, research on PA in patients with IBD is still in its preliminary stages . Given the crucial role of PA, this study reviews the current status of PA in patients with IBD with the aim of providing a reference for PA practice. PA involves increased energy expenditure of the body resulting from skeletal muscle contraction, including all types of sports or exercise, such as leisure activities and commuting. PA can be categorized into various types, such as static exercise and dynamic exercise, based on the form of muscle contraction. In addition, PA can be classified into anaerobic exercise and aerobic exercise according to the metabolic law of energy supply during exercise. Based on the background of daily life sources, PA can be categorized into occupational, family (household chores), transportation (such as walking and cycling), and leisure (various sports) . The intensity of these activities directly affects the effectiveness and safety of PA for patients with IBD. Key indicators of activity intensity include reserve oxygen uptake, reserve heart rate, oxygen uptake, heart rate, and metabolic equivalent (MET) . In particular, MET is a globally recognized and widely used approach for quantifying the energy expenditure of PA. One MET is the resting metabolic rate, indicating the amount of oxygen consumed while resting quietly in a chair, approximately 3.5 mL/kg/min. The resting state is 0–1.5 METs, low-intensity PA (such as walking, laundry, and cooking) is 1.5–2.9 METs, moderate-intensity PA (such as brisk walking, heavy cleaning work, and dancing) is 3–5.9 METs, and high-intensity PA (such as running, doing heavy agricultural work, and kicking) is 6 METs or higher . We summarized the exercise modes corresponding to PA of different intensities ( Table 1 ). Patients with IBD should perform PA according to their condition and appropriate intensity levels. Studies have indicated that walking for more than 1.5 h per day for patients with IBD does not lead to further improvements in quality-of-life scores. Thus, additional research is needed to determine the optimal intensity of PA that maximizes benefits for patients with IBD. Currently, consideration can be given to adhering to the 2020 WHO guidelines for PA and sedentary behavior . The guide recommended that adults and patients with chronic disease engage in 150–300 min of moderate-intensity or 75–150 min of high-intensity aerobic exercise per week or an equivalent combination of the two. PA can be measured using two primary approaches: objective and subjective. Objective approaches involve the use of wearable devices such as pedometers, accelerometers, and heart rate monitors . Subjective approaches primarily assess the type, frequency, and duration of PA through self-reported questionnaires, scales, or logs. Examples include the International Physical Activity Questionnaire (IPAQ) , the Global Physical Activity Questionnaire , the Short Questionnaire to Assess Health Enhancing Physical Activity , and the Gordon Leisure Time Questionnaire . Among them, the IPAQ has high reliability and validity and has been extensively employed in numerous countries . The IPAQ can be divided into two versions: long and short. The long version comprises five sections: occupation, household chores, transportation, leisure PA, and sitting. Regarding the frequency and time of PA inquiries in the PA project, each PA intensity was truncated at 180 min. The short test comprises seven questions that assess the total time individuals spend each week in four types of PA: intense PA, moderate PA, walking, and sitting. The IPAQ recommends computing an individual's weekly PA level (MET minute per week). This is done by multiplying the MET value of a specific intensity of PA by the average daily activity time (minute per day) and the number of days per week the activity is performed. The total weekly PA level was then determined by summing the PA levels at different intensities. Epidemiological data indicate that the incidence rate of IBD is relatively high in Europe (505 per 100,000 people), Canada (248 per 100,000 people), and the United States (214 per 100,000 people), among other developed regions . In addition, the incidence rate of IBD annually increases in developing countries and regions, such as Asia, the Middle East, and South America . The etiology of IBD remains unclear, and increasing research is examining the influence of lifestyle factors on IBD onset. The correlation between PA as a lifestyle and IBD onset remains a topic of debate. First, some studies revealed a correlation between the incidence of IBD and occupational characteristics, possibly because of the varying PA levels among different occupational groups. Sonnenberg analyzed the occupational distribution of 12,014 patients with IBD using German social security statistics and discovered that outdoor work and high PA level work were protective factors for IBD onset. Conversely, occupations involving working in air-conditioned environments or irregular shifts for extended periods were associated with an increased risk of IBD. In a prospective study in Denmark, researchers conducted a 5-year or 10-year follow-up survey involving over 2.2 million people, and the findings indicated that sedentary work may increase the risk of developing IBD . Second, there is a relationship between IBD risk and PA levels. Persson et al. conducted a case–control study examining various risk indicators associated with IBD. In an analysis involving 152 patients with CD, 145 patients with UC, and 305 control populations, regular weekly relative ratio (RR) = 0.6, 95% confidence interval (CI): 0.4–0.9) and daily (RR = 0.5) PAs (95% CI: 0.3–0.9) were found to potentially reduce the risk of developing CD. In a case–control study conducted in Slovakia involving 338 adult patients with IBD (190 with CD and 148 with UC), insufficient PA during childhood was associated with an increased incidence of CD (odds ratio (OR) = 2.7, 95% CI: 1.5–5.0) and UC (OR = 2.0, 95% CI: 1.1–3.5). Insufficient PA level was identified as an independent risk factor for IBD onset . In a national health interview survey conducted in the United States with 60,155 participants, 786 individuals (1.3%) had IBD. The study revealed a positive correlation between IBD incidence and PA deficiency (OR = 1.38, 95% CI: 1.16–1.66) . Klein et al. compared environmental factors between newly diagnosed patients with IBD and healthy controls and found that the control group tended to engage in moderate or severe PA before bedtime, whereas patients with IBD reported less PA before bedtime. Furthermore, one extensive prospective cohort study focusing on American women identified a negative relationship between PA and the incidence of CD, but not UC. However, some studies have reported that the incidence of IBD is not correlated with PA levels. For instance, the European Cancer and Nutrition Prospective Study cohort assessed and monitored 300,724 participants, identifying 177 confirmed cases of UC and 75 cases of CD . No significant correlation was observed between PA and UC ( p = 0.97) or CD ( p = 0.42). Furthermore, Halfvarson et al. examined the potential exposure factors for IBD in a genetically identical Swedish–Danish twin cohort and effectively mitigated the potential influence of genetic factors. Their findings revealed no significant difference in PA between the patients and their twins before the diagnosis of IBD . In summary, research findings on the causal relationship between IBD incidence and PA levels are inconsistent ( Table 2 ). This variability could stem from different confounding factors and retrospective biases. Future large-scale studies are necessary to provide more definitive evidence and confirm this causal relationship. Although many patients with IBD recognize the positive impact of PA on their condition and are willing to engage in it, their PA levels are generally lower due to symptoms such as abdominal discomfort, fatigue, pain, negative emotions, and other related factors. Based on a survey conducted by Guthold et al. across 51 countries, 17.7% of healthy adults exhibit insufficient levels of PA . The proportion of patients with IBD with insufficient PA is significantly higher than that of healthy adults. Moreover, there is no substantial difference in PA levels between active and remission patients with CD . Tew, Jones, and Mikocka-Walus reported that 33.3% of patients with IBD do not meet the recommended PA levels . Conversely, Fagan, Osborne, and Schult found that only 66% of patients with IBD in remission or with mild-to-moderate activity met the WHO recommendation of at least 150 min of moderate-intensity aerobic PA per week . Furthermore, patients with IBD typically engage in low-intensity PA. In a survey involving 859 adult patients with IBD conducted by Tew, Jones, and Mikocka-Walus , 57% reported walking as their most common activity, 34% expressed a preference to avoid running or jogging, and only 17% reported participating in high-intensity PA. Thus, when advising patients with IBD about physical activities, it is crucial to consider their preferences and prioritize low-intensity activities to encourage their full participation and enthusiasm. Various factors limit patients with IBD from participating in PA, and effectively identifying these factors can aid in developing targeted strategies to encourage and support their engagement in activities. According to Tew, Jones, and Mikocka-Walus, as the course of IBD progresses, fatigue, anxiety, and depression tend to worsen, leading to a corresponding decrease in the PA levels of patients . In addition, most participants (677/859) in the study indicated that IBD limited their ability to engage in PA, with influencing factors such as abdominal/joint pain (70%), fatigue/fatigue (69%), disease onset (63%), and increased urgency to use the toilet (61%). Furthermore, the PA levels of patients with CD were independently associated with depression, disease activity, and motor dysfunction. An online survey conducted by the Crohn's Disease and Colitis Association in the United Kingdom was used to assess exercise habits among the 918 participants with IBD . The findings indicated that patients with IBD generally accepted PA, with a majority (72%) reporting positive feelings toward PA. However, 80% of the survey respondents indicated that they had to temporarily or permanently stop exercising at some point due to severe disease symptoms. IBD significantly hindered their ability to participate, enjoy, and derive benefits from PA. A semistructured interview study conducted in the Netherlands examined the role of PA in patients with IBD. The findings indicated that 86% of patients experienced positive effects from PA, such as improvement in overall health, quality of life, and self-image. However, symptoms experienced during the active phase of IBD often lead to postponement or discontinuation of PA . According to population-based evidence, PA can lower the risk of several chronic diseases, such as cancer and cardiovascular disease, and enhance patient prognosis . Nevertheless, the safety and potential health benefits of PA for patients with IBD remain unclear. Before recommending specific PA practices, it is essential to understand their impact on the clinical outcomes of patients with IBD, including disease activity, quality of life, and mortality. There are many benefits of PA for people with IBD . We also summarized the impact of PA on the clinical outcomes of IBD ( Table 3 ). Several studies have investigated the correlation between PA and disease activity, but the findings have been inconsistent. PA level may be associated with disease activity in patients with CD compared with those with UC. A cross-sectional survey conducted in the Netherlands revealed a negative relationship between PA levels and disease activity in patients with CD. Patients with higher PA levels had lower disease activity. In contrast, no correlation was observed between PA levels and disease activity in patients with UC. A prospective cohort study conducted in the United States examined the relationship between PA levels and disease activity over 6 months in patients with IBD . The findings revealed that patients with remission-stage CD with higher PA levels experienced a significant decrease in disease activity after 6 months (OR = 0.72, 95% CI: 0.55–0.94, p = 0.02). However, remission-stage UC patients with higher PA levels did not exhibit a significant decrease in disease activity after 6 months (OR = 0.78, 95% CI: 0.54–1.13, p = 0.18). Watanabe et al. investigated the correlation between four types of PA (sedentary, standing, walking, and vigorous activity) and clinical remission in 327 patients with UC. No significant correlation was observed between mucosal healing and any type of PA and clinical remission in patients with UC. However, high vigorous activity levels and total daily METs may be negatively correlated with mucosal healing but not with clinical remission. In a randomized controlled study conducted by Seeger et al., 45 patients with CD in remission or mild activity were randomly assigned to a control group, endurance group, or muscle training group for 3 months, with PA intervention thrice weekly . The findings indicated no significant change in disease activity between the two groups; however, both intervention groups reported improved quality of life. In summary, PA might help mitigate disease activity in patients with IBD, particularly patients with CD. Although the findings from various studies may vary, no negative effect of PA on disease activity has been found. Cucino and Sonnenberg examined the mortality data of patients with IBD from 1991 to 1996 at the National Center for Health Statistics in the United States. They observed that after adjusting for gender and race, patients with IBD in occupations involving physical labor and agriculture (such as farmers and workers) had lower mortality rates compared with those in sedentary occupations involving indoor work (such as salespeople and secretaries). Lo et al. used the Cox proportional hazards model to examine three large cohort data: Nurse Health Study , Nurse Health Study II , and Health Professional Follow-up Study . They observed that among 828 patients with IBD (363 CD and 465 UC), patients with UC with high PA levels had the lowest risk of all-cause mortality (HR = 0.14–0.41, p = 0.002). However, no significant correlation was observed in patients with CD. Engaging in PA can enhance the quality of life for patients with IBD. A cross-sectional study conducted in South Korea investigated the correlation between PA levels and quality of life in patients with IBD . The study included 158 patients with IBD and evaluated their PA levels using the IPAQ, the European Qol Five-Dimensional Questionnaire, and the European Quality of Life Visual Analog Scale. The findings indicated that higher PA levels were associated with improved quality of life, and improvement in leisure activities and nonsweating exercise was particularly associated with improved quality of life. In an intervention study, Loudon et al. developed a 12-week walking plan for patients with sedentary remission or mild active CD. The exercise plan involved walking thrice weekly, and 12 patients completed it. The study revealed that after the intervention, patients exhibited significant improvements in the IBD stress index, quality of life score, BMI, and other outcome indicators. Similarly, Elsenbruch et al. conducted an intervention for patients with UC in remission using a 10-week, 60-h structured training program. This program incorporated stress management training, moderate exercise, Mediterranean diet, behavioral skills, and self-care strategies. Quality of life was better in the intervention group than in the control group. However, no significant impact on clinical or physiological parameters was observed. Ng et al. implemented a low-intensity walking program for 32 patients with CD in remission or mild activity. This program involved walking three times a week, with each session lasting 30 min for 3 months. Their findings indicated that patients who participated in the exercise program experienced a significant improvement in their quality of life, with no adverse effects observed on disease activity. Van Erp et al. developed a 12-week intensive exercise program for patients with IBD in remission. This program included three sessions per week, each consisting of personalized high-intensity aerobic and progressive resistance training lasting for 1 h. Their study demonstrated that a personalized high-intensity exercise program can substantially improve fatigue, quality of life, and cardiopulmonary function in patients with IBD in remission. Recent studies have emphasized the bidirectional relationship between PA and IBD, advocating for the inclusion of PA in IBD management to improve patients' quality of life and reduce IBD-related complications. Our mixed study also showed that recreational PA was associated with improved patient-reported outcomes . In summary, PA can potentially improve the quality of life of patients with IBD, although the PA schemes implemented in current studies vary widely. The approaches and safety considerations of PA are crucial for patients with IBD . Future research can summarize the evidence from relevant studies to develop the best activity plan to guide the widespread adoption of PA. PA can cause changes in the body composition of patients with IBD, such as reductions in the levels of inflammatory biomarkers . A previous study used real-time detection of biochemical indicators in patients with IBD before and after PA to investigate the effects of PA on body composition. D'incà et al. assessed the intestinal motility, permeability, and antioxidant levels of six remission-stage patients with CD and a healthy control population after PA. The participants were asked to engage in continuous activity for 1 h at a maximum oxygen consumption of 60%. The findings revealed that patients with CD had an increased cecal transit time after exercise compared with the control group. However, the difference was not significant, and the amount of urinary zinc was substantially increased. Another study investigated the safety of moderate-intensity continuous and high-intensity intermittent activities in youth with CD. The study involved 30-min cycles with six cycles of 4 × 15 s at 50% and 100% peak mechanical power, respectively. Blood samples were collected at rest, midpoint during exercise, end of exercise, and 30 and 60 min after recovery to assess changes in inflammatory cells, cytokines, and growth factors. The findings indicated that neither activity led to exacerbation of acute inflammation. Seeger et al. conducted moderate-intensity autologous weight and endurance training on patients with CD during the remission and mild activity phases, revealing that PA can enhance upper and lower limb strength as well as grip strength. Furthermore, several studies have examined the long-term effects of PA in patients with IBD. In a randomized controlled trial conducted by Robinson et al. , 117 patients with CD were randomly assigned to either a control group or a low-intensity exercise program. Bone density of the hip joint and spine was measured using a dual-energy x-ray absorptiometer at baseline and after 12 months of exercise. After 12 months of follow-up, a significant relationship was observed between patient bone density and exercise frequency. Low-intensity exercise can enhance bone density in patients with CD, thereby reducing the risk of osteoporotic fractures. In a randomized controlled trial conducted by Jones et al. , 47 adults with CD in remission were randomly assigned to either the exercise or the control group. The exercise group underwent a 6-month resistance training program. At the end of the 6th month, the control group demonstrated substantial improvements in lumbar spine bone density, muscle strength, and physical performance compared with the exercise group. The intensity and duration of exercise programs carried out vary from study to study. In a study conducted by Jones et al. , the exercise group received a 6-month combined impact and resistance training program, involving three, 60-min sessions per week and a gradual tapering of supervision to self-management. Moderate endurance exercise training and muscle training were performed for 30 min three times per week for 12 weeks in the study conducted by Seeger et al. . In Ng's study , patients performed low-intensity walking at an interval of three times per week for a duration of 3 months, and each walking session lasted for 30 min. We can find that the exercise was mainly medium or low intensity, and the frequency was mostly three times a week, and each time was not more than 1 h. In addition, the independent relationship between results and exercise frequency confirms the “training effect” of fully compliant patients . However, some studies have unclear definitions of the duration and intensity of exercise interventions, as well as a lack of effective exercise supervision, which may lead to poor compliance among participants and dilute exercise effectiveness. Although current studies suggest the potential benefits of PA for IBD, the measurement of PA mostly relies on questionnaires, which may lead to recall bias. Wearable device–based accelerometers can more accurately measure PA. Future study needs to explore the best type of exercise and the most appropriate exercise time from an evidence-based perspective, build an exercise program suitable for IBD patients of different ages and disease degrees, and finally verify the effectiveness of the exercise program through randomized controlled trials. This review has several limitations that deserve transparent discussion. Some studies have revealed the potential benefits of exercise for people with IBD; however, exercise programs vary from study to study, and our review focused on exercise effects without summarizing the characteristics of these exercise intervention programs. It is still necessary to sort out these exercise programs and explore the optimal type of exercise for people with IBD in the future. In addition, our review did not summarize the differences in the impact of exercise on different age groups, which is not conducive to developing personalized exercise prescriptions. Patients with IBD have insufficient PA levels, and the correlation between PA and the incidence and disease activity of IBD remains controversial. Nevertheless, PA has demonstrated beneficial effects on clinical outcomes, particularly in reducing mortality, enhancing quality of life, and improving body composition. Future research should investigate various approaches, intensities, and volumes to determine the optimal activity plan for patients with IBD. In clinical practice, patients must undergo exercise health screenings and risk evaluations, considering patient goals, disease status, living conditions, and other relevant factors to tailor personalized exercise prescriptions for each patient. | Review | biomedical | en | 0.999996 |
PMC11699990 | Muscle stem cells, also called satellite cells (SCs) and defined by the transcription factor paired box 7 (Pax7), are responsible for skeletal muscle maintenance and repair. Once activated after injury, SCs enter the cell cycle, commit to myogenic progenitor cells (MPCs), differentiate into myoblast cells, and finally fuse into multinucleated myotubes. A subset of SCs undergoes asymmetric division, renewing the SC pool . However, endogenous SCs may be inefficient in responding to severe trauma or chronic degenerative diseases such as malnutrition, neuromuscular diseases, and sleep apnea [ 2 – 4 ]. When more than 20% of the muscle volume is missing, as in volumetric muscle loss (VML), endogenous self-repair is hindered . Moreover, SCs are found in very limited numbers in adult muscles and exhibit undesirable amplification activity in vitro ; thus, these SCs are not the appropriate stem cell pool for muscle repair. Dental pulp stem cells (DPSCs) are a type of mesenchymal stem cells (MSCs) that exhibit therapeutic potential for tissue regeneration . These cells are easily established from dental pulp and have several advantages compared to other types of MSCs: better proliferative potential and migratory capabilities , a simpler primary isolation method, a higher success rate in long-term in vitro culture , and long-term storage without losing their stemness and ability to differentiate . Kerkis et al. found the therapeutic effect of human DPSCs on Golden Retrievers with muscular dystrophy. Recently, DPSCs were shown to undergo myogenic differentiation (MyoD), and these myogenic lineage cells could treat the dystrophic muscles of mdx/SCID mice . However, the current myogenic efficiency is still quite low . Here, we searched for novel factors to develop appropriate MyoD systems for DPSCs. MyoD is regulated by genes such as MyoD1, myogenic factor 5 (Myf5), myogenic regulatory factor 4 (MRF4), Desmin, and myosin heavy chain (MyHC) to determine cell fate, including myoblast differentiation into myocytes, fusion into myotubes, and maintenance of myofibers . Factors, molecules, or stimuli can be applied to affect MSCs' fate and commitment , and biocompatible scaffolds are provided for the natural environment in the use of MSCs . A recent RNA-seq analysis identified that repressed bone morphogenetic protein (BMP) and activated Wnt signaling pathways are required for nascent somites during embryonic myogenesis . Wnt activation alone can induce pluripotent stem cells (PSCs) committed to myogenic progenitors. It further requires BMP inhibition to maintain progenitor fate, followed by the myogenic program to generate myofibers and associated Pax7 + cells . These results suggest that Wnt activation or BMP antagonism might be important mechanisms in myogenic regulation. Noggin, a secreted homodimeric glycoprotein, is promoted by Wnt-1 in medial somites and is required for embryonic somite and skeletal patterning . Then, Noggin was found to inhibit the actions of BMP-2, -4, -5, -7, -13, and -14, thus blocking Smad-dependent signaling . BMP signaling has been shown to play a role in controlling SC lineage progression during embryonic myogenesis , and these SC progeny secrete BMP antagonists, such as Noggin, for the differentiation of muscle progenitor cells . Noggin-null mice display defective skeletal muscle fibers . However, the effect of Noggin on the MyoD of DPSCs has not yet been reported. In the present study, Noggin was applied to MyoD systems for DPSCs. Then, we implanted these pretreated DPSCs combined with Matrigel as scaffolds into the mouse tibialis anterior muscle with VML. Here, we explore the DPSCs repair capacity in muscle injury. DPSCs were acquired as previously described and cultured in α -MEM (Gibco, NY, USA) with 10% fetal bovine serum (FBS, Gibco, NY, USA) and 1% penicillin-streptomycin (Gibco, NY, USA) at 37°C in the presence of 5% CO 2 and 95% air. Flow cytometric analysis was used to determine the cell surface markers present on DPSCs. DPSCs were identified with phycoerythrin (PE)-conjugated antibodies against human CD29 and FITC-conjugated antibodies against human CD90 from BD Biosciences (CA, USA), fluorescein isothiocyanate (FITC)-conjugated antibodies against human CD44 from eBioscience (CA, USA), and PE-conjugated antibodies against human CD34 and FITC-conjugated antibodies against human CD45 from BioLegend (CA, USA) and analyzed using FlowJo software (FlowJo, OR, USA). Cell cycle analysis was also performed after staining for 24 h with propidium iodide (PI) (Beyotime, Shanghai, China) according to the manufacturer's protocol using flow cytometry (ACEA NovoCyte, CA, USA). DPSCs were seeded on 6-well plates at a cell density of 4000 cells/cm 2 in an expansion medium consisting of α -MEM with 10% FBS for adherence. When the confluence reached 80%, the culture medium was replaced first with a myogenic induction medium containing the following for 24 h: IMDM (Gibco, NY, USA) + 2% FBS + 1 μM 5-Aza-2′-deoxycytidine (5-Aza) (an important trigger for myogenic commitment differentiation ) (Sigma-Aldrich, CA, USA) . IMDM + 2% FBS without 5-Aza served as the base control medium. Subsequently, cells were rinsed three times in phosphate-buffered saline (PBS, HyClone, UT, USA) and then transferred to the following differentiation medium: IMDM with 10% FBS (defined as Day 0) . The differentiation medium was supplemented with or without Noggin (100 ng/mL = 100N or 200 ng/mL = 200N) or 50 ng/mL BMP4. Noggin from Sino Biological (Beijing, China) and BMP4 from HumanZyme (IL, USA) were used. Cells were cultured for 3 weeks, the differentiation medium was changed every 3 days, and the cells were observed under a microscope to confirm the formation of myotubes. Animal protocols were approved by Cyagen IACUC (No. ACU20-A034). Male Balb/C mice (8 weeks old) were purchased from Shanghai Bikai Biotechnology. All animals underwent surgery to create a muscle defect by unilaterally resecting the tibialis anterior muscle under 2% pentobarbital sodium anesthesia. The thin layer of fascia covering the tibialis anterior muscle was dissected away, and a 2 × 5 mm area was stained with hematoxylin. After a blunt separation, a 2 mm-deep cut was made around the stained hematine, creating a 2 × 5 × 2 mm defect. This VML defect accounted for a loss of ~40% of the tibialis anterior muscle. DPSCs were cultured and differentiated by 5-aza and Noggin treatment, and Matrigel (Corning, NY, USA) was used to provide a scaffold for stem cell transplantation. Matrigel alone ( n = 6) or Matrigel combined with Noggin-treated ( n = 6) or untreated DPSCs ( n = 6) was used to fill the dissected area. If no immediate transplantation was performed ( n = 6), the incision was sutured closed. Mice were allowed to heal for 30 days. The tibialis anterior muscles were excised from mice in the four groups and fixed in ice-cold 4% PFA. After dehydration in a graded alcohol series, the tissue was embedded in paraffin and cut into 5 µm-thick sections by using a Leica RM2255 rotary microtome (Leica Microsystems, Mannheim, Germany). After deparaffinization in xylene and hydration through a graded alcohol series to ddH 2 O, hematoxylin, and eosin (Servicebio, Wuhan, China), staining was performed according to the manufacturer's protocols. Total RNA was extracted from the cells using TRIzol (Life Technologies, CA, USA). The RNA concentration was quantified using a spectrophotometer by measuring the OD260/280 ratio (1.80–1.95). DNA was removed by gDNase at 42°C for 3 min. cDNA was reverse transcribed using a FastQuant RT Kit (Tiangen, Beijing, China) at 42°C for 15 min and at 95°C for 3 min. Subsequently, SYBR Green Premix (Tiangen, Beijing, China) was used for the amplification of cDNA on a Light Cycler 96 system (Roche, Basel, Switzerland) with each primer at 0.6 mM and 150 ng of cDNA template. The thermocycling conditions were as follows: 95°C for 15 min, followed by 40 cycles at 95°C for 10 s and at 60°C for 32 s. Transcript levels were normalized using β -actin as a housekeeping gene and analyzed with the 2 −∆∆Ct method. The primer sequences can be found in Table S2 . Total protein was extracted in RIPA lysis buffer (Thermo Fisher Scientific, MA, USA) plus protease/phosphatase inhibitor cocktail . Lysate proteins (25 mg) were loaded onto SDS-polyacrylamide gels (7.5% or 10%) and subsequently transferred onto PVDF membranes. Target proteins on the membranes were incubated overnight at 4°C with primary antibodies diluted 1:1000–1:200 in 1 × TBS-Tween (TBS: 0.05 M Tris, 0.15 M NaCl [pH 7.5]; with 0.2% Tween-20). Antibodies against β -actin , Pax7 , and Myf5 from Abcam; MyHC IIB (BF-F3) and MyHC IIA (SC-71) from Developmental Studies Hybridoma Bank (DSHB, ID, USA); p-Smad 1/5/9 and Smad 1 from Cell Signaling Technology (CST, MA, USA); and MyoD , Desmin , MRF4 , Pax3 , Six1 , Eya2 , ID1 , and MSX1 from Proteintech (IL, USA) were used. After intensive washing, the membranes were incubated with HRP-conjugated secondary antibodies for 1–2 h. The membranes were visualized using Super Signal West Dura substrate (Thermo Scientific, MA, USA), and the bands were detected with an AI600 imager (GE Healthcare, IL, USA). Cells, myofibers, and sections were fixed with 4% paraformaldehyde and placed in PBS containing 0.25% Triton X-100 for 10 min. After the samples were blocked with 3% bovine serum albumin (BSA, Solarbio, Beijing, China) in PBS for 30 min at room temperature, they were then incubated with the primary antibody in PBS containing 2.5% BSA at 4°C overnight. In addition to the primary antibodies listed above, antibodies against Pax7 (PAX7, DSHB), CD34 , human Lamin A/C , human nucleoli (hNu) , and Laminin were used. After washing, the samples were incubated with anti-mouse Alexa-594 or Alexa-488, or antirabbit Alexa-594 or Alexa-488 (Jackson ImmunoResearch, PA, USA), and then with 4′,6-diamidino-2-phenylindole (DAPI). Images of the cells were captured using a microscope (Leica Microsystems, Mannheim, Germany) and analyzed with ImageJ. The cells were harvested and fixed in 70% precooled ethanol overnight at 4°C. After washing with PBS the next day, the cells were incubated with a solution containing RNase A and 50 µg/mL of PI for 30 min at 37°C. Then, the cell cycle analysis was examined using a Novocyte 2040R flow cytometer (ACEA Biosciences, Hangzhou, China) with a cell count of ~0.5 × 10 4 ~ 2.0 × 10 4 frequency of G1, S, and G2 phases using NovoExpress software version 1.4.1 (ACEA Novocyte). The results were graphed using GraphPad Prism software. We performed statistical analyses using GraphPad Prism 5.0. Data are expressed as the mean ± standard deviation. The protein quantities were analyzed using semi-quantitative analysis. Comparisons between groups were performed using the Mann‒Whitney U test or Kruskal‒Wallis H test with multiple comparisons. Statistical significance was defined as ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. We cultured and identified DPSCs and found that 5-Aza can induce MyoD in DPSCs by increasing myogenic markers but with low efficiency. To examine whether Noggin could affect the MyoD of DPSCs, we treated DPSCs with Noggin after 5-Aza induction . First, we found that Noggin had no significant effect on the proliferation of DPSCs but increased myotube formation in DPSCs . A more pronounced myotube morphology was observed after treatment with the Noggin protein for 21 days, and the differential index of multinuclear myotubes increased to ~20% . The protein expression levels of MyHC , such as MyHCIIA and MyHCIIB , were significantly increased compared to those in the control group. This finding implies that Noggin might facilitate the formation of myotubes in DPSCs. To evaluate the effect of Noggin treatment on the MyoD of DPSCs, we assessed the expression of myogenic genes such as MyoD, MRF4, and Desmin on Days 7, 14, and 21. Noggin increased the expression of MyoD and Desmin but had no effect on the mRNA expression of MRF4 on Day 7 . Immunofluorescence staining for MyoD and Desmin revealed elevated levels of MyoD and an increased quantity of long, spindle-shaped myotube-like cells in the Noggin-treated groups. Moreover, the protein expression levels of Desmin and MRF4 significantly increased upon treatment with Noggin on Day 14 . In our study, Noggin was shown to play an important regulatory role in accelerating the MyoD of DPSCs, which might be a novel factor in myogenesis. SCs are characterized by the expression of the paired box (Pax)3/7. Six1 and eya2 have been shown to activate SCs (Pax7 + ) and promote myoblast differentiation. Notably, we found that Noggin increased the protein levels of Pax7 in DPSCs cultured in a proliferation medium . To determine SCs generation through MyoD of DPSCs with Noggin, we measured the relative mRNA levels on Days 1, 3, and 7, as well as the protein expression levels of Six1, Pax3, Pax7, and Eya2 on Days 7, 14, and 21. The mRNA expression of Pax7 increased in a concentration-dependent manner under Noggin treatment, reaching its peak on Day 3 and remaining higher than that of the control on Day 7 . Consistent with the expression of Pax7, the mRNA expression of Pax3 increased on Day 3 and remained elevated on Day 7 . Additionally, the mRNA expression of Six1 and Eya2 increased on Day 7. Western blotting and immunofluorescence staining for Pax7 revealed that Noggin upregulated the protein expression of Pax7 on Days 7, 14, and 21 . The protein expression levels of Pax3 , Six1 , and Eya2 were also increased in the Noggin-treated groups on Day 14 and remained high on Day 21 . These results suggest that Noggin promotes the myogenic process in DPSCs by upregulating the expression of Six1/Eya2 in addition to Pax3/Pax7. To determine whether Noggin regulates the MyoD of DPSCs by regulating Smad signaling, we first simulated 3D protein structures using SWISS-MODEL and visualized them with PyMOL software, which showed that Noggin competitively inhibits the binding of BMP4 to BMP-receptor I A (BMPRIA) . Then, we blocked the effect of Noggin by adding the BMP protein. The protein levels of Pax7, Pax3, Eya2, and Desmin were observed . Compared with that of the Noggin treatment group (200N), their expression decreased in the Noggin + BMP treatment group. Among them, Pax7 was most prominent when compared with that in the 5-Aza or control groups . To further explore the downstream regulation of BMP signaling by Noggin, we determined the phosphorylation levels of members of the BMP/p-Smad pathway . The protein levels of p-Smad 1/5/9 increased following the activation of the BMP pathway by adding BMP4 . Noggin persistently eliminated the phosphorylation levels even after subsequent stimulation with BMP4 . Downstream effectors of the BMP/p-Smad pathway, such as inhibitors of DNA binding 1 (ID1) and msh homeobox 1 (MSX1), were also downregulated by Noggin treatment . Therefore, our results indicate that Noggin facilitates the MyoD of DPSCs by inhibiting BMP/Smad activation. To test the utility of DPSCs in repairing muscle injuries, we established a mouse VML injury model that exhibited a loss of ~40% of the tibialis anterior muscle . In the untreated VML-injured group, the volumetric defect remained . Shrinkage occurred due to the defect area. The muscle defect deformity exhibited the deposition of a thin layer of disorganized collagenous scar tissue . The injury defect exceeded the threshold and could not be restored by the endogenous regenerative potential of the skeletal muscle. This result indicates the success of VML modeling. The utilization of scaffolds can provide temporary mechanical support and the necessary growth environment for seed cell adhesion, growth, proliferation, and differentiation. Therefore, we used the thermosensitive hydrogel Matrigel to provide a scaffold for stem cell transplantation . We observed that the Matrigel group exhibited a reduced shrinkage area and decreased muscle fibrosis . In addition, we found some mononuclear cells infiltrating in the defect . To examine whether Noggin-pretreated DPSCs could better repair muscle injury than the controls, we transplanted DPSC bioconstructs into the defects of VML muscles . We implanted untreated DPSCs or Noggin-pretreated DPSCs reconstituted in Matrigel into the defects. Morphometric analysis of muscle cross-sections revealed that the Noggin-treated DPSCs showed a 73% decrease in the size of the defect and a 69% decrease in scar tissue . In contrast to the defects in the above two groups, which exhibited irreversible and robust fibrotic scars, the defects treated with DPSCs showed markedly reduced fibrotic tissue surrounded by cells of varying morphologies, such as fibrotic, inflammatory, and vessel-like cells, indicating the tissue repair process . In contrast, Noggin-treated DPSCs might have accelerated this process and facilitated the improved formation of muscle tissue, leaving little cell infiltration . To explore the contribution of grafted cells to muscle injury, we immunostained muscle cross-sections for Pax7 (SC marker), MyoD (activated SCs/myoblasts), hNu, and human LaminA/C (specific antibody to trace human cells), and Laminin (to identify position within the sarcolemma). Pax7/MyoD costaining revealed that stem cell transplantation increased the proportion of activated SCs compared to the sham or Matrigel groups . We also observed that hNu was integrated into the nucleus of regenerated tissue and was located on the Laminin-stained muscle sarcolemma , a phenomenon more readily discernible in the Noggin-treated DPSCs groups than in the DPSCs groups. Increased numbers of hLaminAC + /Pax7 + and hNu + /MyoD + cells were also found in the Noggin-treated DPSCs groups compared with the DPSCs groups, indicating an increase in donor-derived SCs . These results suggest that Noggin-treated DPSCs partially benefited the SC population. This benefit might provide support for muscle regeneration in VML injury. In our study, we found that Noggin could promote the MyoD of DPSCs and increase the generation of satellite-like cells by regulating the BMP/Smad signaling pathway. Then, we treated VML model mice with Noggin-pretreated DPSCs bioconstructs and found that Noggin pretreatment improved the repair of muscle injury. Our work implies that Noggin can promote the MyoD of DPSCs, rendering DPSCs an important source of muscle stem cells for muscle repair. Noggin, a secreted BMP antagonist, was promoted by Wnt-1 in medial somites and found to promote the expression of MyoD in embryonic tissues . Our results showed that Noggin could promote the MyoD of DPSCs and increase the number of Pax3 + /Pax7 + cells . Previous studies have concluded that Pax3-mediated myogenesis requires an environment where Six1 synergizes with Eya2 to activate the expression of MyoD . Our results showed that Noggin promoted the expression of Six1 and Eya2, suggesting that these Pax3 + /Pax7 + cells were in an environment conducive to subsequent MyoD. We also found that Noggin eliminated Smad phosphorylation, accompanied by decreased levels of MSX1 and ID1 . Consistent with our results, Noggin inhibited p-Smad1/5/8 and repressed ID1 and MSX1 expression, impeding myoblast differentiation [ 29 – 31 ]. Sustained release of BMP4 significantly decreased Pax7 + and MyoD + SC densities . When BMP4 was added to antagonize Noggin, we observed inhibition of the MyoD of DPSCs. Our data revealed that Pax3/7 might be related to the BMP/Smad pathway in the context of Noggin effects. Previous research has shown that activated Pax7 + cells coexpressed p-Smad1/5/8 in injured mouse skeletal muscle sections . In the competition for MSX1, the expression of downstream effectors of the BMP/Smad pathway, Pax3 or Pax7, was increased towards the myogenic fate . Knockout of Noggin leads to increased levels of p-Smad1/5/8, ID1, and MSX1, and a decreased population of mesenchymal Pax7 + muscle precursor cells . Muscle regeneration is ensured by Pax7 + SCs; when activated, SCs are able to divide asymmetrically, enabling self-renewal or myoblast differentiation . ESCs-derived Pax7 + cells can give rise to both muscle fibers and Pax7 + satellite-like cells when grafted in vivo, suggesting that these cells might behave like SCs . In our study, we found that Noggin expanded Pax7 + cells in DPSCs. After 21 days of differentiation in vitro, the expression levels of late myogenic marker genes (MyHC) and SC marker (Pax7) were both higher with Noggin treatment . These results revealed that Noggin could promote the generation of Pax7 + satellite-like cells from DPSCs . We then transplanted Noggin-pretreated DPSCs combined with Matrigel into defects of a VML injury model. In our study, Noggin-pretreated DPSCs bioconstructs decreased the size of the defect and increased the number of donor-derived hLaminAC + /Pax7 + and hNu + /MyoD + cells in mouse muscles . Some scholars transplanted ESC- or PSC-derived muscle progenitor cells into NSG-mdx4cv mice and found that donor-derived hLaminAC + /Pax7 + SCs contributed to the muscle stem cell pool in tissues . The successful use of Pax7 + satellite-like cells derived from ESCs in muscle injury may be attributed to their self-renewal capacity within the SCs pool . DPSCs are a type of MSCs from dental pulp, with easy access, long-term storage, and high proliferative and differentiation potential compared to other types of MSCs [ 9 – 12 ] and can serve as a proper stem cell source for muscle regeneration. SCs derived from MSCs were shown to give rise to differentiated muscle fibers . However, the contribution of 5-Aza-induced DPSCs engraftment might only be a paracrine effect instead of MyoD . In contrast to muscle-derived cells or ESCs, the transplant efficiency of MSCs was relatively low, with ~1%–2% as hybrid myofibers . We speculate that the low myogenic efficiency may be attributed to fewer SCs. Our results indicated that Noggin expanded Pax7 + satellite-like cells in DPSCs, which increased the percentage of donor-derived cells to ~15%–20% . This finding suggests that Noggin-treated DPSCs can partially benefit the muscle SC pool and promote myogenic repair in VML injury. In this study, we demonstrate that Noggin, a secreted BMP antagonist, is an important protein that promotes MyoD and increases the generation of Pax7 + satellite-like cells by modulating BMP/Smad signaling. These satellite-like cells combined with Matrigel can effectively repair mouse tibialis anterior muscle injury in a VML model, leading to increased repair size and decreased scar tissue. Noggin-treated DPSCs bioconstructs can benefit the Pax7 + SC pool and promote muscle regeneration. This finding suggests the importance of producing Pax7 + SCs and highlights the potential of these SCs for improved muscle regeneration. Noggin might enhance the generation of satellite-like cells from DPSCs to provide potential transplantable stem cells for muscle regeneration. Further investigations are needed to study the relationship between Pax7 and Smad/its downstreams in order to improve myogenic induction conditions and enhance the transplantation of DPSCs into damaged muscle tissue. | Study | biomedical | en | 0.999995 |
PMC11699991 | Atlantic salmon ( Salmo salar ) is the most important aquaculture species in Norway. In order to reduce feed costs and develop sustainable feed, high-energy diets and the replacement of marine-based ingredients with plant-based ingredients have been widely applied in Norwegian salmon feed in recent years . These strategies have resulted in an increase in the fat level, a decrease in the n-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) level, and decreases in the zinc (Zn) level and availability in feed . Zn is crucial for multiple physiological functions in fish, such as forming Zn-containing enzymes and promoting skeletal development . Feeding fish with Zn-deficient diets induced Zn-deficiency diseases, such as lens cataracts, skin erosion, and skeletal malformation [ 5 – 7 ]. Considering reductions in Zn level and availability in plant-based feeds, additional Zn must be provided to meet the Zn requirement in S. salar . However, Zn digestibility is lower than 40%, and Zn retention is less than 30% in S. salar , indicating that a high level of Zn in the feed would have a negative environmental impact. In this regard, the EU limited the upper Zn level in salmon feed to 180 mg kg −1 , and the EFSA has suggested further reducing it to 150 mg Zn kg −1 . Therefore, it is necessary to increase knowledge on enhancing Zn availability and utilization in salmon feeds. Even though fish can osmotically absorb aqueous minerals, feed remains the predominant resource to maintain body minerals homeostasis . In the case of Zn, the absorptive site is primarily the intestine . Similar to mammals, fish have two families of cellular Zn transporters: zips (Zrt/Irt-like proteins, transport Zn into cytosol) and znts (Zn transporters, transport Zn out of the cell) . In addition, because zip4 and znt1 are located at the apical and basolateral epithelium, respectively, they are regarded as two key proteins in intestinal Zn uptake and transport . After Zn enters the cell, metallothionein (mt) would bind with Zn to prevent Zn toxicity . If cellular Zn levels exceed the mt binding capacity, transcription factors such as metal regulatory transcription factor 1 (mtf1) would be upregulated, thus increasing the expression of znt1 and promoting the Zn efflux . However, very little is known about the regulation of Zn transporters in Atlantic salmon. Intestinal Zn uptake and transport are affected by several factors, including anti-nutrient factors (such as phytate), mineral interactions (with iron [Fe] and manganese [Mn]), chemical form of Zn (chelated vs. inorganic) . Furthermore, dietary component interaction might be another factor impacting intestinal Zn uptake and transport, such as fat level and n-3 LC-PUFA. In mammals, higher plasma and femur Zn levels were observed in rats fed with 3% fat diets compared to those in the 26% fat diet group . Also, rats fed with fish oil-based diet (rich in n-3 PUFA) obtained higher hepatic Zn levels than those fed with a lard-based diet (rich in saturated fatty acids [SFA]), indicating that n-3 PUFA may have a positive effect on Zn absorption . A recent study in Atlantic salmon also reported that fish fed with low-fat/high n-3 LC-PUFA diets obtained the higher whole-body Zn level compared to those fed with high-fat/low n-3 LC-PUFA diets . Due to the crucial physiological role and the upper legal limitations in feed, understanding the Zn uptake as affected by dietary components is important to the aquaculture industry. Furthermore, dietary Zn may impact intestinal fat transport. According to a previous study, rats fed Zn-deficient diets obtained lower triacylglycerol (TAG) absorption and abundant intestinal lipid droplets, demonstrating the importance of Zn on intestinal fat transport . However, the effect of dietary Zn on intestinal fat transport in fish is still unclear. In previous fish nutrition studies, samples were generally collected at fasting status or 24 h after the final meal. Worth mentioning, different postprandial sampling time points may obtain different plasma minerals and TAG levels, indicating the importance of the sampling time selected when investigating plasma parameters. However, reports on postprandial plasma profiles related to lipids and minerals in S. salar are still scarce. Therefore, the aim of this study was to investigate the combined effect of dietary fat level and Zn, and the combined effect of dietary n-3 LC-PUFA and Zn on postprandial plasma profiles in S. salar . In addition, intestinal gene expression related to Zn and lipid uptake and transport in selected postprandial time points were also analyzed. Diet formulations and analyzed nutritional compositions are shown in Table 1 . In trial 1, four experimental diets containing one of two different levels of fat (%) and Zn (mg kg −1 ) were formulated and referred to as follows (fat/Zn): HFHZ, 35/200; HFLZ, 35/120; LFHZ, 32/200; LFLZ, 32/120. In comparison, Norwegian S. salar commercial feeds for growing out contain 35%–39% fat level . Additionally, the EU restricts the maximum Zn in S. salar feed to 180 mg kg −1 , whereas 120 mg Zn kg −1 is a suboptimal level in S. salar plant-based feed . Fat levels in the feed were adjusted by varying the levels of fish oil and rapeseed oil, with wheat gluten, guar meal, and wheat meal used to balance the composition. Zn levels in the diet were modified by adding Zn sulfate at different doses. In trial 2, three experimental diets were used. The first two experimental diets (HFHZ and HFLZ) were the same as trial 1, while the third diet contained higher n-3 LC-PUFA (with high fat and Zn levels). Three diets were named as follows (eicosapentaenoic acid [EPA] + docosahexaenoic acid [DHA], % of total feed/Zn, mg kg −1 ): HFHZ, 2.0/200; HFLZ, 2.0/120; HPUHZ, 3.2/200 (average level of DHA + EPA in Norwegian commercial salmon feed is 2.2% . Different dietary n-3 LC-PUFA levels between HFHZ and HPUHZ were achieved by adjusting the ratio of rapeseed oil and fish oil. In addition, due to the lack of cholesterol (CHO) in rapeseed oil, additional CHO was provided in HFHZ, HFLZ, LFHZ, and LFLZ to obtain a similar CHO level as the HPUHZ diet. In both feeding trials, S. salar were fed a commercial feed (Skretting Norway) for 2 weeks to acclimate them to experimental conditions. In trial 1 , 280 S. salar with IW of 718 ± 58 g were randomly distributed to eight tanks , with 35 fish in each. Four experimental diets (HFHZ, HFLZ, LFHZ, and LFLZ) were randomly assigned to the tanks in duplicates. Fish were provided experimental diets twice per day until apparent satiation (9:00 and 16:00) for 4 weeks. During the nutritional intervention, the fish were held in full-strength seawater with the water temperature ranged from 8 to 9°C. In trial 2 , 225 fish with a weight of 667 ± 20 g were randomly distributed to nine tanks with 25 fish in each. Three experimental diets (HFHZ, HFLZ, and HPUHZ) were randomly assigned to nine tanks in triplicates. Further, fish were fed with one of three experimental diets twice daily until apparent satiety (9:00 and 16:00) for 4 weeks. During the nutritional intervention, the fish were held in full-strength seawater, and the water temperature ranged from 10 to 12°C. As shown in Figure 1 , at the termination of the feeding trial, fish were fasted for 48 h to completely empty their gastrointestinal tract, which was considered their preprandial status (0 h), and their blood and intestine were sampled. Next, fish were fed with respective diets for 2 h, and the postprandial time was calculated after this feeding. Similar samples (blood and mid-intestine) were collected at different postprandial time points. In trial 1, five fish were sampled from each tank (total 10 fish from each dietary treatment group) at 2, 4, 8, 14, 24, and 36 h after feeding. In trial 2, three fish from each tank (total nine fish from each dietary treatment group) were sampled at 4, 6, 10, 14, 26, 32, and 38 h after the feeding. During the sampling, fish were randomly and carefully removed from the tank to reduce stress and then euthanized with an overdose of tricaine mesylate (MS-222, Tricaine Pharmaq). The euthanized fish were measured for weight and length, following which blood was drawn from the caudal vein using a lithium heparin-coated vacutainer. The blood samples were centrifuged (14,200 g, 2 min, 4°C) to obtain supernatant (plasma), which was then frozen on dry ice for plasma parameters analysis. Afterward, the mid intestine (just behind the pyloric ceca) was collected after emptying the intestinal contents and immediately frozen in liquid nitrogen for further analysis. During the sampling, the state of feed remnants in the stomach was inspected and recorded, which served as the indicator of fish ingestion. The number of fish without pellets in their stomachs is presented in Table S1 . If the fish hadn't eaten, the sample was excluded from further analysis. The chemical composition in the feed was analyzed after homogenization. Moisture, protein, and ash were analyzed by drying to constant weight (105°C), the Kjeldahl method (N x 6.25), and muffle furnace (550°C), respectively; fat level in feeds was analyzed by acid hydrolysis and extraction with diethyl ether. FAs composition in feed was analyzed as described in . Briefly, after isolation of lipids from feed (Folch solution) and completely dried chloroform–methanol phase (using N 2 gas), residual lipids were trans-methylated overnight with 2′-2′-dimethoxy propane, methanolic HCl, and benzene at room temperature. Methyl esters were isolated and analyzed using a gas chromatograph equipped with a split injector, an SGE BPX70 capillary column (SGE Analytical Science), and a flame ionization detector. He 2 served as the carrier gas, with the injector and detector temperatures both maintained at 280°C. The oven temperature was initially increased from 50 to 180°C at a rate of 10°C per minute, then further increased to 240°C at a rate of 0.7°C per minute. Individual FA methyl esters were identified by comparison with previously characterized standards. Results were further analyzed using HP ChemStation software. FAs results were presented as a percentage of the total fatty acids in the feed, and the absolute amount of EPA + DHA per gram of feed was calculated using C23:0 methyl ester as the internal standard. Mineral contents (Zn, copper [Cu], Fe, Mn, and selenium [Se]) in diet and plasma were measured using inductively coupled plasma mass spectrometry (ICP-MS), as described in . Specifically, samples (0.2 g dry feed with 0.5 mL deionized water or 0.5 mL plasma) and 2 mL concentrated HNO 3 were added into test tubes and digested in a Milestone UltraWave Microwave Digestion System (Milestone Inc., USA). Further, digested samples were diluted to 25 mL by deionized water. Next, minerals were analyzed in ICP-MS (Thermo Scientific, USA) equipped with an autosampler (FAST SC-4Q DX, Elemental Scientific, USA). The eluate was introduced into the nebulizer tube of the ICP-MS, and minerals were analyzed in the KED reaction mode. To correct for instrumental drift during the analysis, a solution of germanium and rhodium was added online. The ICP-MS was tuned prior to analysis using a 1 ppb tuning solution B (Thermo Fisher, in 2% HNO3 and 0.5% HCl). Data collection and processing were carried out using Qtegra software (Thermo Scientific). An external calibration curve ranging from 10 to 500 ng mL −1 was utilized to quantify the minerals. Plasma lipids, including TAG, CHO, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and total protein (TP), were measured using Pentra C400 (HORIBA; Montpellier, France), following the instruction of the manufacturer. Intestinal samples for gene expression analysis were selected at three postprandial time points based on the plasma TAG profile: exogenous lipid had not entered circular system (trial 12 h; trial 2:4 h), exogenous lipid entered circular system (trial 1 and trial 2:14 h) and exogenous lipid abundantly entered circular system (trial 1:24 h; trial 2:26 h). Candidate genes for qPCR included two house-keeping genes ( β -actin and elongation factor 1 alpha [ef-1 α ]), five genes related to Zn transport (solute carrier family 39 member 4 [zip4], metallothionein A [mta], metallothionein B [mtb], solute carrier family 30 member 1 [znt1], mtf1) and 11 genes related to lipid transport and metabolism (Niemann-Pick C1-like 1 [npc1l1], cluster of differentiation 36 [cd36], fatty acid transporter protein 4 [fatp4], fatty acid transporter protein 6 [fatp6], fatty acid-binding protein 2 [fabp2], diglyceride acyltransferase 1 [dgat1], microsomal triglyceride transfer protein [mtp], apolipoprotein B [apob], carnitine O-palmitoyltransferase 1 [cpt1], monoacylglycerol O-acyltransferase 2 [mgat2], apolipoprotein A-IV [apoa4]). Total RNA from intestine samples was isolated using the Maxwell HT simplyRNA Kit (Promega, USA) and the Biomek 4000 automated liquid handler (Beckman Coulter, USA), following the instructions of the manufacturer. In addition, the quality and quantity of isolated RNA were measured in the Bioanalyzer and the spectrophotometer , respectively. Further, an inverse transcription kit (Thermo Fisher Scientific, USA) was used to synthesize cDNA, following the instructions of the manufacturer. Afterward, RT-PCR for target genes was quantified on the qPCR instrument (Bio-Rad, USA) with SYBR GREEN PCR Master Mix (Roche-Norge, Norway) , with the following program: 10 min preincubation at 95°C, followed by 40 cycles of 95°C for 30 s, 60°C for 30 s, 72°C for 30 s, and a melting curve. The expression level of the target gene was normalized on the CFX Maestro (Bio-Rad, USA) based on two house-keeping genes. The associated information of candidate genes is listed in Table 2 . Except for β -actin and ef-1 α , other primers were newly designed through NCBI online primer designing tool ( https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi? ). One-step RT-PCR (QIAGEN one-step RT-PCR kit) was used to evaluate the primer specificity, according to the instructions of the manufacturer. The normality and variance of the data homogeneity were analyzed under the Shapiro–Wilk test and Levene's test ( p > 0.05), respectively. In trial 1, the weight gain (WG) was analyzed by two-way ANOVA, with fat level and Zn as independent variables ( n = 2). In addition, results from other growth performance parameters (initial weight [IW], initial condition factor [ICF], final weight [FW], final condition factor [FCF]) were analyzed by “two-way nested ANOVA,” with fat level and Zn as independent variables (tank as a random factor). Furthermore, results from plasma minerals, plasma lipids, and intestinal gene expression were analyzed by three-way nested ANOVA, with fat level, Zn, and time as independent variables (tank as a random factor), and the significant results between “0h” and “selected time point” were analyzed by multiple comparisons with “Dunnett's test” (5 samples from each tank, total 10 samples from each dietary treatment). In trial 2, the WG was analyzed by t -test between the HFHZ and HFLZ (or HFHZ vs. HPUHZ) groups ( n = 3). In addition, results from other growth performance parameters (IW, ICF, FW, FCF) were analyzed by t -test nested between the HFHZ and HFLZ (or HFHZ vs. HPUHZ) groups (tank as a random factor). Furthermore, results from plasma minerals, plasma lipids, and intestinal gene expression were analyzed by two-way nested ANOVA, with diet (Zn or n-3 LC-PUFA) and time as independent variables (tank as a random factor), and the significant results between “0h” and “selected time point” were also analyzed by multiple comparisons with “Dunnett's test” (three samples from each tank, total nine samples from each dietary treatment). If the p -value was <0.05, results between groups were regarded as significant. All experimental results were presented as the mean ± standard deviation (SD). Statistical analysis was performed in the software GraphPad Prism 8 (Insightful Science, USA) and R , respectively. The growth performance of S. salar fed the different diets from trial 1 and trial 2 are presented in Table 3 . In trial 1, the WG of the fish ranged from 18.5% to 22.2%, while in trial 2, it ranged from 39.6% to 44.9%. In addition, the survival rates (SRs) of fish fed with different diets were 100% in trial 1 and 94%–98% in trial 2. However, 4-week dietary treatments did not impact the fish growth among all experimental groups in trial 1 and trial 2 ( p > 0.05). In trial 1, reduced Zn level in feed significantly reduced the postprandial plasma Zn level . Additionally, postprandial plasma Fe ( p < 0.01) and Cu ( p < 0.05) profiles were significantly impacted by the time change. Peak values of plasma Fe (14–24 h, p < 0.01) and Cu (2−4 h and 36 h, p < 0.05) appeared at specific postprandial time points. Further, the postprandial plasma Cu profile was significantly affected by Time x Fat ( p < 0.05). Compared to low-fat diet treatment, high-fat diet treatments reduced the plasma Cu level at postprandial 4 h but increased the plasma Cu level at postprandial 36 h. On the contrary, postprandial plasma profiles of Zn, Mn, Fe, and Se were not affected by dietary fat level ( p > 0.05). In trial 2, the postprandial plasma Zn profile was not affected by dietary Zn ( p =0.20) . In addition, HFHZ treatment significantly reduced the postprandial plasma Mn level compared to HFLZ treatment ( p < 0.05). In addition, fish fed with the high n-3 LC-PUFA diet obtained significantly higher postprandial plasma Mn level compared to those fed with the low n-3 LC-PUFA diet ( p < 0.05), which is attributed to different Mn levels in diets ( Table 1 ). However, postprandial plasma profiles of Zn, Fe, Cu, and Se were not affected by dietary n-3 LC-PUFA ( p > 0.05). The postprandial plasma TAG profile was significantly impacted by the time changes in trial 1 . After 14 h of feeding, the plasma TAG level had risen significantly ( p < 0.01) compared to that at preprandial (0 h). Further, the peak value of plasma TAG appeared at postprandial 24 h and was statistically elevated to 36 h ( p < 0.01). However, dietary fat level and Zn did not impact the postprandial plasma TAG profile ( p > 0.05). In trial 2, the postprandial plasma TAG profile was significantly affected by the time change , which agrees with the result in trial 1. Compared to preprandial status (0 h), plasma TAG level was significantly higher between 10 and 38 h after the meal ( p < 0.05). Also, plasma CHO was affected by postprandial time ( p < 0.05), and the result at postprandial 32 h was significantly higher than that at postprandial 6 h. However, the postprandial plasma TAG profile was not affected by dietary n-3 LC-PUFA and Zn ( p > 0.05). As shown in Figure 6 (trial 1, all results presented in Table S4 ), intestinal mRNA expression related to Zn uptake and transport was not affected by dietary Zn and fat level ( p > 0.05). However, gene expression related to Zn uptake and transport, including zip4 ( p < 0.01), znt1 ( p < 0.05), and mtf1 ( p < 0.01), were significantly affected by postprandial timing. Compared to the 2 h postprandial mark, zip4 , znt1 , and mtf1 were upregulated at 24 h. In trial 2, reduced Zn level in feed significantly downregulated the intestinal mRNA expression of mta and mtb postprandially . In addition, postprandial timing significantly impacted intestinal mRNA expression related to Zn uptake and transport, including zip4 ( p < 0.05), znt1 ( p < 0.01), and mtf1 ( p < 0.01). At 26 h postprandially, zip4 was upregulated, while znt1 and mtf1 were downregulated compared to the 4 h mark. In trial 1, intestinal mRNA expression related to lipid uptake and transport was not significantly affected by dietary Zn . However, the expression levels of dgat1 , mtp , and cpt1 were significantly influenced by the interaction between time and fat levels ( p < 0.05), with reduced fat level downregulating the expression of these genes at 14 h postprandially. Additionally, most mRNA expressions related to lipid uptake and transport were significantly impacted by postprandial timing ( p < 0.05). Compared to postprandial 2 h, CHO uptake gene ( npc1l1 ) was downregulated at 14 h, and genes related to TAG uptake and transport ( cd36 , fatp4 , fatp6 , mgat2 , dgat1 , mtp , apob ) and fatty acid β -oxidation ( cpt1 ) were downregulated at 24 h. In trial 2, dietary Zn did not impact intestinal mRNA expression related to lipid uptake and transport , consistent with the results observed in trial 1. However, postprandial timing had a significant impact on most of these gene expressions ( p < 0.01). At 26 h postprandially, genes related to lipid transport and metabolism, including npc1l1 , cd36 , fatp4 , fatp6 , fabp2 , dgat1 , mtp , apob , and cpt1 , were downregulated compared to the 4 hr mark. Increasing knowledge on the postprandial absorption kinetics of Zn and its dietary impact could improve the understanding on dietary Zn availability. Different from reports in rainbow trout and hybrid striped bass , there did not appear a significant absorptive peak in the postprandial plasma Zn profile in the present two trials. In the above-mentioned studies, one or more of the diets contained dietary Zn levels that were deficient or very low, resulting in very low basal plasma Zn concentration and hence inducing a visible peak in plasma Zn immediately after a meal. In trial 1, although postprandial plasma Zn concentration was correlated with dietary Zn level, as reported in other fish at fasting status [ 29 – 31 ], mRNA expression related to intestinal Zn uptake ( zip 4 ) and transport ( znt 1 ) was not affected by dietary Zn. These results are due to the passive uptake mechanism in the intestine. It is well known that active uptake mechanisms in the intestine, primarily DMTs, ZIPs, and ZnTs, are upregulated at limiting dietary Zn concentrations, whereas at optimal or high dietary Zn levels, passive uptake mechanisms are dominating . Apart from intestinal Zn absorption, other Zn metabolic processes, such as storage and excretion, are also attributed to the regulation of plasma Zn status in vertebrates . In contrast with trial 1 (8–9°C), the postprandial plasma Zn was not affected by dietary Zn in trial 2 (10–12°C), which is due to the different water temperatures. As reported by Bervoets, Blust, and Verheyen , increased temperature would increase metal diffusion and reaction rates, resulting in increased metal accumulation in tissues. Also, increased temperature and dietary Zn level interactively increased hepatic Zn accumulation in Pelteobagrus fulvidraco . In trial 2, increased Zn level in feed increased the intestinal mt ( mta and mtb ) expression, indicating that more exogenous Zn is temporarily stored in the intestinal epithelium by binding with mt before transport into plasma , and thus the postprandial plasma Zn level was not affected by dietary Zn. Temperature-dependent intestinal Zn transport was also reported in other fish studies [ 37 – 39 ]. These results suggest the importance of the regulatory effect of the intestine on plasma Zn . Therefore, a mismatch between Zn absorptive markers in the intestine and plasma Zn status at the different time-points can be attributed to the differential regulative mechanisms. Besides the differential Zn regulation of absorptive epithelia and systemic regulation, marine fish present different intestinal Zn adaptations when subjected to different dietary Zn levels . In the two present trials, intestinal mRNA expression related to Zn influx ( zip 4 ) and efflux ( mtf 1 and znt 1 ) were not affected by dietary Zn, indicating the dynamic balance between intestinal Zn influx and efflux. In comparison, Sparus aurata (marine fish) fed with Zn-deficient diets (7.9 mg Zn kg −1 ) increased intestinal Zn influx but reduced intestinal Zn efflux to maintain intestinal Zn homeostasis, whereas fish fed optimum Zn diet (64.7 mg Zn kg −1 ) maintained the dynamic balance between intestinal Zn influx and efflux . These results indicate that suboptimal Zn supplementation in S. salar feed (120 mg Zn kg −1 ) does not impair the dynamic balance between intestinal Zn influx and efflux after 1-month feeding trial. In addition, increased n-3 LC-PUFA or reduced fat level in feed increased whole-body Zn content in S. salar . However, intestinal Zn uptake and transport were not affected by dietary n-3 LC-PUFA and fat level in the present study. Also, the postprandial plasma Zn profile was not affected by fat level or n-3 LC-PUFA. These results suggest that dietary fat level and n-3 LC-PUFA might impact other Zn metabolism processes (such as storage and excretion) rather than intestinal absorption, resulting in impacting whole-body Zn status. Apart from Zn, postprandial plasma levels of other divalent trace minerals were also affected in the studies. In trial 1 (8–9°C), the postprandial plasma peak values of Fe and Cu appeared later than the report in Oncorhynchus mykiss (17.5°C) , which is due to different experimental temperatures. Increased water temperature in an appropriate range is generally associated with an increased passage rate of chyme through the gastrointestinal tract of fish . In addition, two discontinuous peak values of postprandial plasma Cu were observed in trial 1, which is similar to the report in O. mykiss . It can be explained that the Cu absorption site in S. salar occurs in the stomach, mid-intestine, and posterior intestine, similar to the report in O. mykiss . Furthermore, increased fat level in feed reduced plasma Cu level at postprandial 4 h but increased plasma Cu level at postprandial 36 h, indicating increased dietary fat level depressed Cu absorption in the stomach but promoted Cu absorption in the intestine. Increased fat level in feed significantly increased the Cu utilization in rats (2% vs. 8% fat level) . However, knowledge on the effect of fat level on Cu availability in animals is still scarce. In addition, mineral interaction is another important factor impacting mineral status in fish . In trial 2, postprandial plasma Mn level was negatively correlated with dietary Zn level, which may be due to the competitive effect between Zn and Mn during absorption. In the case of Mn, its active transport during absorption is mediated by the divalent metal transporter 1 (DMT1), which also transports other divalent metals, such as Zn, Fe, and Cu . Also, previous studies reported that high Zn treatment significantly reduced intestinal Fe and Cu absorption in O. mykiss . In the present study, postprandial plasma Fe and Cu were not affected by dietary Zn, indicating that S. salar fed with diets ranging from 120 to 200 mg Zn kg −1 did not impact Fe and Cu absorption. The pyloric ceca and mid-intestine are the two sites for lipid digestion and absorption in S. salar . In the present study, the plasma TAG began to significantly increase at postprandial 14 h (trial 1, 8−9°C) /10 h (trial 2, 10−12°C), indicating the time point when chyme started to enter the pyloric ceca and the exogenous lipid enters the circulation system . Although a small proportion of plasma TAG may be attributed to hepatic VLDL secretion, lipid metabolism in the liver was not analyzed in the current study. In addition, the peak value of plasma TAG appeared at postprandial 24−36 h (trial 1, 8–9°C) and 32 h (trial 2, 10 −12°C), respectively, indicating the majority of chyme had reached the pyloric ceca and mid-intestine. FAs are taken up and transported by the intestine through membrane-associated proteins, although a small proportion is performed by passive diffusion . In the two present trials, intestinal mRNA expression related to lipid uptake and transport was not affected by dietary Zn. Additionally, postprandial plasma TAG level was not affected by dietary Zn. In previous studies, Zn-deficient diet treatment increased intestinal fat accumulation in rats , and it is correlated with inhibition of intestinal lipoprotein formation . Also, Zn-deficiency impaired intestinal lipid transport in S. aurata . Therefore, these results suggest that the 120 mg Zn kg −1 in S. salar feed is not low enough to impact intestinal lipid transport (compared to the 200 mg Zn kg −1 diet). In trial 1, the postprandial plasma TAG was not affected by dietary fat level, suggesting that dietary fat level (32%–35%) did not alter the intestinal TAG transport. Similarly, a recent S. salar study reported that increased fat level in the feed from 16% to 25% did not impact the mRNA expression of the key lipoprotein assembly gene ( mtp ) . However, in trial 1, reduced fat level in feed upregulated intestinal mRNA expression related to TAG re-esterification ( dgat1 ), lipoprotein assembly ( mtp ), and β -oxidation ( cpt1 ) at postprandial 14 h, when the chyme entered the pyloric intestine. These results may be linked to the higher wheat gluten content in the low-fat diet compared to the high-fat diet (17.5% vs. 10.2%). A previous study showed that increasing dietary wheat gluten from 15% to 30% elevated the intestinal gene expression associated with lipid metabolism and transport in S. salar . However, it is important to note that mRNA expression may not directly correspond to protein synthesis levels due to regulatory processes such as mRNA editing, modification, and degradation . In trial 1, the downregulation of dgat1 , mtp , and cpt1 genes at postprandial 24 h compared to 14 h suggests possible mRNA degradation. Future studies could provide more comprehensive insights by simultaneously quantifying mRNA and protein expression. In the two present trials, the postprandial plasma CHO level was not affected by the dietary Zn, fat level, and n-3 LC PUFA, which is attributed to the similar CHO level among different experimental diets, as reported in other fish studies . Similarly, changing the dietary fat level (31% vs. 38% fat level) or fatty acid profiles did not impact the plasma CHO level in S. salar . Also, suboptimal supplementation of Zn in feed did not alter the plasma CHO level in other fish [ 63 – 66 ]. To sum up, increased Zn level in feed significantly increased the postprandial plasma Zn level in S. salar (8 −9°C). Analyzed markers related to intestinal Zn uptake and transport were not affected by dietary fat level (32%–35% of feed) and n-3 LC-PUFA (2%−3.2% EPA + DHA of feed). Since dietary fat and n-3 LC-PUFA affect whole-body Zn content in Atlantic salmon, further study is needed to explore how these components influence other Zn metabolism processes, such as retention and excretion. Additionally, markers related to intestinal fat transport were unaffected by Zn levels ranging from 120 to 200 mg kg −1 in the feed. | Study | biomedical | en | 0.999996 |
PMC11700017 | In numerous countries, individuals with brain injuries are a primary focus of rehabilitation, and driving assessments play an important role in occupational therapy. Post-stroke evaluations encompass off-road tests, such as neuropsychological examinations and driving simulators, and on-road tests involving actual driving alongside physical function assessments . The on-road test is considered the gold standard for examining real-world driving skills among these assessments . Because various studies have aimed to determine the predictability of on-road test results , it is an essential factor to ensure the reliability and validity of on-road tests. While some on-road tests for brain-injured individuals have been developed globally, only a few have been validated with a high degree of reliability and validity. A systematic review identified a scarcity of on-road tests demonstrating general reliability and validity . This scarcity suggests that a few on-road tests may partially exhibit reliability and validity. A subsequent study by Bellagamba et al. reinforced these findings, concluding that currently available on-road tests lack sufficient reliability and validity. Consequently, the reliability and validity of on-road tests remain inadequately established. Despite uncertainties regarding the reliability and validity of the on-road tests themselves, past efforts have focused on predicting driving skill assessment outcomes using off-road tests. Studies have attempted to formulate prediction equations using multiple off-road tests to predict actual on-road evaluation outcomes . However, this methodology cannot inherently verify the validity of the on-road test, leaving the authenticity of the results unverified. Consequently, these results are utilized as predictive tools without confirming sufficient validity. Alternatively, exploring alternative conceptual frameworks that involve both on-road and off-road tests introduces the crucial aspect of discriminant validity. Discriminant validity, a statistical concept, assesses whether a measurement captures unique attributes distinct from other related measurements . Several previous studies have used discriminant validity to demonstrate the validity of driving evaluations, supporting the validity of these assessments . Instead of relying on outcome predictions derived from past on-road test results as observed in previous studies, it is more beneficial to authenticate whether the on-road test effectively encapsulates the essence of driving skills, embodying discriminant validity. This methodology enhances the assurance of on-road test validity. Taking an alternative perspective, the legal and road conditions in different countries contribute to the complexity of on-road testing. On-road tests evaluate several key components of driving, such as cognitive abilities (e.g., attention, decision-making, and visual perception), motor skills (e.g., pedal and steering control), and environmental awareness (e.g., responding to vehicles and pedestrians, adherence to traffic rules) . Eligibility criteria for these assessments often include a confirmed diagnosis of brain injury, sufficient physical ability to operate a vehicle, and adequate cognitive functioning to manage driving-related tasks. However, the specific criteria for determining eligibility for driving assessments vary across countries or institutes, reflecting differences in legal regulations, cultural contexts, and healthcare systems. Existing on-road tests are primarily conducted on actual roads, exposing brain-injured patients to various hazards such as other vehicles and road users. In contrast, Japan conducts some on-road tests in a closed course at driving schools under instructor guidance. Legal regulations in Japan may restrict brain-injured survivors from driving on public roads until they pass an aptitude test from the local public safety commission . These legal constraints necessitate the assessment of on-road tests for brain-injured individuals solely on closed courses within driving schools . In other situations, other countries have difficulty conducting on-road tests under actual road conditions due to legal regulations . The inclusion criteria were as follows. 1) Patients referred by a physician for driving assessment at the collaborating hospital, 2) patients who were primarily diagnosed with a stroke, and 3) patients who will undergo an on-road assessment. The exclusion criteria were 1) patients with severe cognitive impairments that prevent understanding of the research content, 2) patients with aphasia that would interfere with the assessments, and 3) patients who do not provide consent for participation in this study. Although vision is a factor that influences driving, it was not included as a criterion in the present study. Instead, the focus was placed on examining correlations with the off-road tests utilized in previous driving studies. In this study, several off-road tests were adopted as alternative conceptual frameworks for discriminant validity. The off-road tests utilized in this study are the Mini-Mental State Examination Japanese Version (MMSE-J), Trail Making Test Japanese Version (TMT-J), Kohs Block Design Test (KBDT), Rey-Osterrieth Complex Figure Test (ROCF), Frontal Assessment Battery (FAB), and Stroke Drivers’ Screening Assessment Japanese Version (J-SDSA), which have been commonly employed in previous research for guessing driving skills in individuals with brain injuries . However, these assessments are conducted to evaluate the cognitive functions (higher brain functions) of stroke patients and do not measure the actual driving skills. Since on-road tests are often compared with evaluations by driving instructors, their correlation with off-road tests can help establish discriminant validity. The study group consisted of 108 stroke patients, 83 men and 25 women, with ages ranging from 34 to 80 years (mean: 50.0 years, SD: 10.2). The participants' diagnoses were as follows: 62 individuals were diagnosed with cerebral infarction, 26 with cerebral hemorrhage, 10 with subarachnoid hemorrhage, three each with traumatic brain injury, encephalitis, and brain tumor, and one individual with hydrocephalus. The average driving experience of the participants was 36.3 (SD: 11.0) years. The means and standard deviations of the off-road test are shown in Table 2 . There were two missing values in the FAB, and the optional items of SOAD have some missing values (60 for item 9, 102 for item 12, 87 for item 14, and 26 for item 23). Several correlations were observed between the items of the SOAD and various off-road tests, though most of these correlations were not statistically significant (Table 3 ). Among the off-road tests, the J-SDSA dot time showed the highest number of significant correlations with six items (maintain the appropriate posture for safe driving, operate a gear change appropriately, position in the lane appropriately, maintain appropriate distance, slow down appropriately, check safety when turning left), followed by the MMSE-J with four items (operate brakes appropriately, reverse park appropriately, check rearview, check safety using head/body movement), and the TMT-J with three significant items (TMT-A: operate a gear change appropriately, follow yield situation appropriately, check safety using head/body movement, TMT-B: operate indicators appropriately, slow down appropriately, check safety when starting to drive). The results of this study revealed generally weak correlations between SOAD and the various off-road assessments, particularly in evaluations of practical driving-related skills. This pattern supports the discriminant validity of SOAD by demonstrating its ability to evaluate unique driving capabilities that are not assessed by off-road tests. Discriminant validity refers to the degree to which a test or measurement distinguishes between the construct it aims to measure and other, potentially related constructs. It indicates that the assessment effectively captures distinct attributes, ensuring the measured skills are distinct from those in similar evaluations . In the field of rehabilitation, discriminant validity is frequently established by calculating the correlations between assessments of distinct conceptual constructs . In this study, we examined the discriminant validity by calculating correlations between off-road and on-road tests. The on-road test evaluates actual, multifaceted driving skills, while the off-road test assesses specific cognitive functions, each targeting fundamentally different constructs. Therefore, this methodology is appropriate, as it enables a clearer distinction between the specific capacities assessed by each test. Correlated items suggest areas where SOAD aligns well with existing neuropsychological assessments (e.g., TMT and SDSA) , while uncorrelated items highlight the unique aspects of SOAD that are not assessed by conventional off-road tests. These findings underscore the complementary role of SOAD in driving evaluations and emphasize the importance of integrating both on-road and off-road assessments for a comprehensive evaluation of driving skills. This finding aligns with prior studies, which suggest that off-road assessments primarily focus on cognitive domains such as visual processing and attention but fail to fully represent the complex skills required for safe driving . For instance, some off-road tests measure isolated skills (e.g., visual scanning or attention switching), which may not encompass real-world driving demands, such as maneuvering in traffic, which requires continuous integration of sensory input, executive functioning, and motor response . This fact suggests that items 34 and 35, which require advanced visual confirmation, had more significant correlations with off-road tests. Consequently, off-road tests may miss critical components of driving skills that SOAD captures, emphasizing the unique relevance of the on-road test within rehabilitation settings. The wide age range of participants (34-80 years) could be considered a potential source of variability in the study. However, age-related declines in both neuropsychological performance and driving skills are demonstrated, and these changes likely occur in parallel . Therefore, the influence of age on the correlation between neuropsychological tests and driving skills may not significantly impact the validity of the discriminative analysis. This study showed that there was a slight correlation between the off-road test and the SOAD in stroke individuals. This result indicates that the SOAD has minimal overlap with off-road cognitive assessments and highlights its discriminative validity. It demonstrates that the SOAD provides a valuable tool for assessing driving skills in a controlled environment. This study emphasizes its role as a reliable tool for evaluating the driving abilities of stroke patients, particularly in settings with legal constraints, such as Japan. | Study | biomedical | en | 0.999995 |
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