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Autoimmune liver disease and sickle cell anemia in children: a report of three cases. PURPOSE We wish to alert clinicians to the possible association between Sickle cell anemia (SCA) and autoimmune liver disease (AIL). METHODS AIL was diagnosed by serologic, histologic and/or cholangiographic studies in patients with known SCA. RESULTS Three schoolaged children with SCA were investigated for increased levels of transaminases and/or worsening jaundice. All were diagnosed to have AIL. Two patients had autoimmune hepatitis and have responded to immunosuppressive therapy, and I had primary sclerosing cholangitis and is stable with therapy. CONCLUSIONS Autoimmune hepatitis and sclerosing cholangitis are rare liver disorders of childhood, but may occur with greater frequency in patients with SCA. Early diagnosis and treatment is essential to prevent progression to liver cirrhosis. This association may provide a clue to the etiology of AIL and warrants further investigation.
Primary Burkitt's lymphoma of the liver: report of a case with long-term survival after surgical resection and combination chemotherapy. PURPOSE A case of primary Burkitt's cell lymphoma of the liver is reported. PATIENT A 14-year-old Chinese boy presented with a 10-day history of postprandial epigastric pain and weight loss. RESULTS Preoperative imaging studies revealed a large solid mass confined to the right lobe of the liver; there was no evidence of involvement of other sites. There was serological and immunohistochemical evidence of asymptomatic hepatitis B virus infection. Complete removal of the mass was achieved by right hepatic lobectomy. Histological examination revealed a small noncleaved cell lymphoma of Burkitt's type that immunostained positively for B-cell markers. The patient remains well, with no evidence of disease > 8 years after surgical resection and combination chemotherapy. CONCLUSIONS Only five cases (two children and three adults) of primary small noncleaved cell lymphoma of the liver have been reported. We believe this is the second reported case of childhood hepatic lymphoma with long-term disease-free survival. Further work is needed to elucidate the relationship between hepatitis B virus infection and the development of a primary small noncleaved lymphoma of the liver.
Bleeding disorders in Noonan syndrome: three case reports and review of the literature. PURPOSE Noonan syndrome (NS) is a congenital disorder characterized by various phenotypic features and congenital anomalies. Bleeding disorders are among the more serious, common, yet poorly defined complications associated with NS. As a means of focusing on these complications, we report three patients with stigmata of NS, each of whom had a combination of different hemostatic disorders, and review the literature on bleeding disorders in NS. PATIENTS AND METHODS The clinical course and hemostatic abnormalities in three patients with NS were studied, and a literature review on NS was undertaken. RESULTS The three patients we report had decreased coagulation factor levels (factors XI and II), von Willebrand disease, various levels of thrombocytopenia, and abnormal platelet function. The literature review on NS discloses multiple types of hemostatic abnormalities and a wide range of clinical presentations. A low level of coagulation factor XI is the most frequently described; thrombocytopenia and abnormal platelet function are also common. CONCLUSIONS The existence of various types of bleeding disorders within one syndrome is unusual and requires further investigation. Recognition of this common complication in children with NS would aid both clinical management and understanding of the spectrum, the frequency, and perhaps even the basis of the hemostatic defects in this syndrome. We recommend performing coagulation screening tests in every patient with NS.
Impact of morning versus evening schedule for oral methotrexate and 6-mercaptopurine on relapse risk for children with acute lymphoblastic leukemia. Nordic Society for Pediatric Hematology and Oncology (NOPHO). PURPOSE To study the risk of non-B-cell acute lymphoblastic leukemia (ALL) relapse in relation to the routines of administration of oral methotrexate (MTX) and 6-mercaptopurine (6MP) and to the erythrocyte (E) levels of the intracellular cytotoxic metabolites, that is, MTX polyglutamates and 6-thioguanine nucleotides (E-MTX and E-6TGN). PATIENTS AND METHODS E-MTX and E-6TGN levels were measured at least three times (medians, eight and nine) in 294 children with non-B-cell ALL during oral MTX and 6MP therapy. For each patient, we registered (a) the individual circadian schedule of drug administration and (b) the coadministration of food, and (c) calculated a mean (m) of all E-MTX and E-6TGN measurements and (d) the product of mE-MTX and mE-6TGN (mE-MTX*6TGN), due to their synergistic action. RESULTS A total of 42 patients were on a morning schedule, 219 were on an evening schedule, and 33 had miscellaneous routines. A total of 149 patients took the drugs with meals, 106 took the drugs between meals, and 39 had varying routines. With a median follow-up of 78 months, ALL has recurred in 66 patients. The patients on an evening schedule had a superior outcome [probability of event-free survival (pEFS) = 0.82 +/- 0.03 vs. 0.57 +/- 0.08; p = 0.0002], whereas the coadministration of food did not significantly influence outcome. Patients with a mE-MTX*6TGN < 813 [product of median mE-MTX (4.7 nmol/mmol Hb) and mE-6TGN (173 nmol/mmol Hb)] had an inferior outcome (pEFS = 0.70 +/- 0.04 vs. 0.85 +/- 0.03; p = 0.003), even if only patients on an evening schedule were analyzed. Thus, 109 patients on the MTX/6MP evening schedule with an mE-MTX*6TGN < or = 813 (nmol/mmol Hb)2 had a pEFS of 0.89 +/- 0.03 and a probability of continuous hematopoietic remission of 0.91 +/- 0.03. CONCLUSIONS An evening schedule should be recommended for oral MTX/6MP maintenance therapy. The value of individual dose adjustments by E-MTX and E-6TGN remains to be determined in prospective randomized trials.
Sensorimotor neurotoxicity associated with high-dose deferoxamine treatment. PURPOSE We report a reversible sensorimotor neurotixicity that developed in two beta-thalassemic patients treated with high-dose deferoxamine (DFO) for iron overload. METHODS Two patients were treated with high-dose (120 mg/kg/day) intravenous DFO for iron overload. RESULTS Sensorimotor toxicity developed after 5 and 6 months of treatment, respectively. The development of the neurotoxicity did not correlate with the serum ferritin or the ratio of DFO dose to serum ferritin. Symptoms resolved in both patients with discontinuation of DFO treatment. In 1 patient, symptoms recurred with resumption of DFO treatment. CONCLUSIONS These cases demonstrate that a reversible sensorimotor neurotoxicity, a previously unreported toxicity, may complicate DFO therapy, this complements the previously reported auditory and visual neurotoxicity associated with DFO therapy. Discontinuation of therapy at the time of onset of neurotoxicity is recommended, with possible resumption at lower doses.
Disseminated Burkitt's lymphoma after kidney transplantation: a case report in a boy with Drash syndrome. PURPOSE We discuss the clinical, laboratory findings and treatment of a boy who developed Burkitt's lymphoma (BL) after renal transplant and some issues about lymphoproliferative disorders after transplantation. METHODS A 6-year-old boy with Drash syndrome developed disseminated Burkitt's lymphoma 38 months after transplantation for renal failure. Immunosuppressive therapy had consisted of prednisolone and cyclosporine. Polychemotherapy was initiated. RESULTS Polychemotherapy induced rapid and complete remission of the disease without major side effects despite the renal transplant allograft and prolonged immunosuppression. CONCLUSIONS A child with posttransplantation B-cell high-grade lymphoma can be successfully treated with the same chemotherapy regimen used for ordinary cases.
Aging and the mechanisms underlying head and postural control during voluntary motion. The quality of sensory information that is necessary for balance and postural stability will depend to a great extent on head stability as the body moves. How older persons coordinate head and body motion for balance during volitional activities is not known. The purposes of this article are to present a basis for understanding the influence of aging on head control during voluntary motion and to discuss some data that demonstrate how elderly people might control head movement to improve gaze and the quality of vestibular inputs. A "top-down" or "head-first" control scheme is proposed as the mechanism that elderly people without disabilities use to maintain head position during self-initiated motion. This type of control ensures that the angular position of the head in space remains relatively constant--through the use of a head-stabilization-in-space (HSS) strategy--regardless of the magnitude or direction of displacements in the body's center of force. The HSS strategy is thought to reduce potential ambiguities in the interpretation of sensory inputs for balance and is derived primarily from a geocentric (orientation to the vertical) frame of reference. Egocentric (orientation of the head with respect to the body) or exocentric (orientation to objects in the environment) frames of reference, however, refine the control of head stabilization. Preliminary research suggests that elderly people use the HSS strategy to control head pitch during difficult balance tasks. These findings, if supported by more definitive studies, may be useful in the treatment of patients with balance disorders. The treatment of patients with balance dysfunction is discussed within the conceptual framework of a "head-first" organization scheme.
Locomotion in patients with spinal cord injuries. Following central motor lesions, two forms of reorganization can be observed that lead to improved mobility: (1) the development of increased muscle tone and (2) the activation of spinal locomotor centers induced by specific treadmill training. Tension development is different from normal during spastic gait and appears to be independent of exaggerated monosynaptic stretch reflexes. Exaggerated stretch reflexes are associated with an absence or reduction of functionally essential polysynaptic reflexes. Based on observations of the locomotor capacity of the spinal cat, recent studies have indicated that spinal locomotor centers can be activated and trained in patients with complete or incomplete paraplegia when the body is partially unloaded. The level of electromyographic activity in the gastrocnemius muscle, however, is considerably lower in patients with central motor lesions than in persons without neurological impairments. During the course of a daily locomotor training program, the amplitude of gastrocnemius muscle electromyographic activity increases during the stance phase and inappropriate tibialis anterior muscle activity decreases. Such training programs can improve the ability of patients with incomplete paraplegia to walk on stationary surfaces. This article reviews the pathophysiology and functional importance of increased muscle tone and the effects of treadmill training on the locomotor pattern underlying new attempts to improve the mobility of patients with paraplegia.
Helicobacter pylori gastritis in a child with sickle cell anemia and recurrent abdominal pain. PURPOSE Recurrent abdominal pain is a common complaint in children with sickle cell disease. Helicobacter pylori gastritis has recently been described in association with recurrent abdominal pain in children. PATIENTS AND METHODS A case report is given of a 16-year-old black male with hemoglobin SS disease presenting with recurrent abdominal pain and hematemesis. RESULTS Endoscopic exam of the upper gastrointestinal tract revealed gastritis, and biopsy confirmed H. pylori infection. Serology studies demonstrated increased anti-H. pylori antibody titers. The young man responded well to treatment, with resolution of his symptoms. CONCLUSION Helicobacter pylori infection is a new diagnostic consideration for children with recurrent abdominal pain and should be included in the differential diagnosis of children with sickle cell disease, especially when abdominal pain is recurrent and accompanied by vomiting. Larger case studies will be necessary to determine the true incidence of H. pylori in children with sickle cell disease and recurrent abdominal pain.
Light touch contact as a balance aid. Canes and crutches are commonly used mobility aids, and most studies of their use have focused on issues equating support with the resulting decrease in force required of the affected limb. Clinicians, however, often observe patients with poor balance control using light touch of surrounding objects and surfaces to stabilize themselves while standing and walking. A series of studies have shown that sensory input to the hand and arm through contact cues at the fingertip or through a cane can reduce postural sway in individuals who have no impairments and in patients without a functioning vestibular system, even when contact force levels are inadequate to provide physical support of the body. This article summarizes these results, which have implications for design considerations of rehabilitation aids. Mobility devices or rehabilitation aids that provide feedback about applied force or enhance existing resolution of applied force changes across the skin surface may lead to new rehabilitation techniques.
The role of limb movements in maintaining upright stance: the "change-in-support" strategy. Change-in-support strategies, involving stepping or grasping movements of the limbs, are prevalent reactions to instability and appear to play a more important functional role in maintaining upright stance than has generally been appreciated. Contrary to traditional views, change-in-support reactions are not just strategies of last resort, but are often initiated well before the center of mass is near the stability limits of the base of support. Furthermore, it appears that subjects, when given the option, will select these reactions in preference to the fixed-support "hip strategy" that has been purported to be of functional importance. The rapid speed of compensatory change-in-support reactions distinguishes them from "volitional" arm and leg movements. In addition, compensatory stepping reactions often lack the anticipatory control elements that are invariably present in non-compensatory stepping, such as gait initiation. Even when present, these anticipatory adjustments appear to have little functional value during rapid compensatory movements. Lateral destabilization complicates the control of compensatory stepping, a finding that may be particularly relevant to the problem of falls and hip fractures in elderly people. Older adults appear to have problems in controlling lateral stability when stepping to recover balance, even when responding to anteroposterior perturbation. Increased understanding and awareness of change-in-support reactions should lead to development of new diagnostic and therapeutic approaches for detecting and treating specific causes of imbalance and falling in elderly people and in patients with balance impairments.
A feasibility, toxicity, and early response study of etoposide, ifosfamide, and vincristine for the treatment of children with rhabdomyosarcoma: a report from the Intergroup Rhabdomyosarcoma Study (IRS) IV pilot study. PURPOSE The purpose of this study was to determine the feasibility, toxicity, and early response of patients with clinical group III rhabdomyosarcoma (RMS) to a chemotherapy regimen of etoposide (ETOP), ifosfamide (IFOS), and vincristine (VCR) with hyperfractionated radiation therapy (XRT). PATIENTS AND METHODS Sixty-eight patients aged < 21 years, previously untreated, with clinical group III RMS or undifferentiated sarcoma with normal organ function were eligible for this study. Chemotherapy was as follows: weeks 0-8: IFOS 1.8 g/m2/day X 5 days every 3 weeks X 3 (with mesna), ETOP 100 mg/m2/day X 5 days every 3 weeks X 3, and VCR 1.5 mg/m2/week X 9; weeks 9-16: hyperfractionated XRT (except patients with parameningeal tumors with meningeal extension, who received XRT on day 0), IFOS/mesna weeks 9, 12, 16, and VCR weeks 9, 10, 11, 12, 16; weeks 20-99; IFOS/mesna q 3 weeks X 2, ETOP q 3 weeks X 2, and VCR weekly X 6 weeks. Four drug cycles were repeated every 9 weeks, beginning at week 29. In January 1991, the duration of therapy was reduced to 12 courses due to emerging evidence of IFOS-induced renal tubular dysfunction. RESULTS Of the 62 patients evaluable for response, 45 (73%) achieved a complete response. There were three fatal toxicities due to infection. Life-threatening neutropenia was seen in 55 of 60 patients, and life-threatening infections occurred in 27 of 60 patients. Twenty-five patients (42%) developed some degree of neurotoxicity from vincristine. Eleven patients (18%) developed nephrotoxicity, 7 cases of which were severe; 6 of the 11 patients who developed nephrotoxicity were < 2 years old. CONCLUSIONS This pilot study had toxicity and response rates comparable to the other two Intergroup Rhabdomyosarcoma Study (IRS)-IV pilot trials of vincristine-actinomycin-cyclophosphamide and vincristine-actinomycin-ifosfamide and is, therefore, being evaluated in the current IRS randomized trial. Due to the high incidence of life-threatening neutropenia and infections, the use of growth factors is now routine. Five of 11 patients who developed nephrotoxicity did so after more than eight courses of IFOS; therefore, the current randomized trial limits IFOS to a total of eight courses.
Postural perturbations: new insights for treatment of balance disorders. This article reviews the neural control of posture as understood through studies of automatic responses to mechanical perturbations. Recent studies of responses to postural perturbations have provided a new view of how postural stability is controlled, and this view has profound implications for physical therapy practice. We discuss the implications for rehabilitation of balance disorders and demonstrate how an understanding of the specific systems underlying postural control can help to focus and enrich our therapeutic approaches. By understanding the basic systems underlying control of balance, such as strategy selection, rapid latencies, coordinated temporal spatial patterns, force control, and context-specific adaptations, therapists can focus their treatment on each patient's specific impairments. Research on postural responses to surface translations has shown that balance is not based on a fixed set of equilibrium reflexes but on a flexible, functional motor skill that can adapt with training and experience. More research is needed to determine the extent to which quantification of automatic postural responses has practical implications for predicting falls in patients with constraints in their postural control system.
Hepatoblastoma metastatic to brain: prolonged survival after multiple surgical resections of a solitary brain lesion. PURPOSE Pulmonary metastases of hepatoblastoma confined to the lung have been cured using therapy that included radical surgical resection. We report the case of a child with a hepatoblastoma metastatic to brain that was successfully treated with multiple surgical resections, irradiation, and chemotherapy. The case demonstrates that such an approach, employing aggressive surgery, can produce durable remission of an extrapulmonary metastasis with hepatoblastoma. PATIENTS AND METHODS A 17-month-old girl presented with a hepatoblastoma that remained unresectable after chemotherapy and irradiation and underwent orthotopic liver transplantation 14 months after diagnosis. After twice undergoing surgical resections of pulmonary metastases 22 and 31 months from diagnosis, 1 month later (32 months from diagnosis), she developed a solitary metastatic right brain lesion that later recurred twice in the same location, 5 and 6 years from initial diagnosis. Each time she underwent surgical resection of the brain lesion and received local irradiation after the first two resections and chemotherapy after the third. At the last surgery, resection was continued until histologically negative tumor margins were obtained. RESULTS The child is currently without evidence of disease or neurological deficit 10.5 years from initial diagnosis. CONCLUSION The durable remission achieved after multiple resections of the recurrent solitary cerebral metastasis in this child demonstrates that an aggressive surgical approach to extrapulmonary metastases in such a setting can contribute to prolonged survival, just as has been shown with isolated metastatic pulmonary disease.
Rehabilitation of balance in two patients with cerebellar dysfunction. The treatment of two patients with cerebellar dysfunction is described. One patient was a 36-year-old woman with a 7-month history of dizziness and unsteadiness following surgical resection of a recurrent pilocystic astrocytoma located in the cerebellar vermis. The other patient was a 48-year-old man with cerebrotendinous xanthomatosis (CTX) and diffuse cerebellar atrophy, and a 10-year history of progressive gait and balance difficulties. Each patient was treated with a 6-week course of physical therapy that emphasized the practice of activities that challenged stability. The patient with the cerebellar tumor resection also performed eye-head coordination exercises. Each patient had weekly therapy and performed selected balance retraining exercises on a daily basis at home. Measurements taken before and after treatment for each patient included self-perception of symptoms, clinical balance tests, and stability during selected standing and gait activities; for the patient with the cerebellar tumor resection, vestibular function tests and posturography were also performed. Both patients reported improvements in symptoms and demonstrated similar improvements on several kinematic indicators of stability during gait. The patient with the cerebellar tumor resection improved on posturography following treatment, whereas the patient with CTX improved on clinical balance tests. This case report describes two individualized treatment programs and documents functional improvements in two patients with different etiologies, durations, and clinical presentations of cerebellar dysfunction. The outcomes suggest that patients with cerebellar lesions, acute or chronic, may be able to learn to improve their postural stability.
Transient erythroblastopenia of childhood (TEC) presenting as leukoerythroblastic anemia. PURPOSE The diagnosis of transient erythroblastopenia of childhood (TEC) is usually straightforward, with temporary cessation of red blood cell production resulting in normocytic normochromic anemia, reticulocytopenia, and bone marrow erythroblastopenia. We describe here a case of TEC presenting with features of leukoerythroblastic anemia. To our knowledge, this is the first such report for TEC. PATIENT AND METHODS The case of a 1-year-old girl is described who had a leukoerythroblastic anemia. Bone marrow examination and clinical course indicated that the anemia had a benign etiology-TEC. RESULTS The patient presented with anemia, leukocytosis, and a left shift, with metamyelocytes, myelocytes, myeloblasts, and nucleated red cells in the circulation. There was no apparent viral etiology, and the bone marrow aspirate findings were consistent with a recovering marrow. After transfusion, the patient had an uneventful recovery from TEC. CONCLUSION TEC can cause leukoerythroblastic anemia. TEC with this presentation is clinically similar to TEC without leukoerythroblastosis, but other causes of leukoerythroblastosis need to be excluded.
Balance retraining after stroke using force platform biofeedback. Balance is a somewhat ambiguous term used to describe the ability to maintain or move within a weight-bearing posture without falling. Balance can further be broken down into three aspects: steadiness, symmetry, and dynamic stability. Steadiness refers to the ability to maintain a given posture with minimal extraneous movement (sway). The term symmetry is used to describe equal weight distribution between the weight-bearing components (eg, the feet in a standing position, the buttocks in a sitting position), and dynamic stability is the ability to move within a given posture without loss of balance. All of these components of balance (steadiness, symmetry, and dynamic stability) have been found to be disturbed following stroke. Balance testing of patients with hemiparesis secondary to stroke has revealed a greater amount of postural sway during static stance, asymmetry with greater weight on the nonparetic leg, and a decreased ability to move within a weight-bearing posture without loss of balance. Furthermore, research has demonstrated moderate relationships between balance function and gait speed (r = -.67 and .42, respectively), independence (r = .62), appearance (defined as "significantly abnormal," "slightly abnormal," and "nearly normal") (r = .50), dressing (r.55-.69), wheelchair mobility (r = .51), and reaching (r = .49-.78). Thus, a principal construct within physical therapy practice is the reestablishment of balance function in patients following stroke. Recent advances in technology have resulted in the commercial availability of numerous force platform systems for the retraining of balance function in patient populations, including patients with stroke. These systems are designed to provide visual or auditory biofeedback to patients regarding the locus of their center of force (COF) or center of pressure (COP), as well as training protocols to enhance stance symmetry, steadiness, and dynamic stability. Typical force platform biofeedback systems consist of at least two force plates to allow the weight on each foot to be determined, a computer and monitor to allow visualization of the COF or COP, and software that provides training protocols and data analysis capabilities. Some units allow auditory feedback in addition to the visual feedback in response to errors in performance.
Disseminated intrathoracic desmoplastic small round-cell tumor: a case report. PURPOSE Recently recognized as a distinct clinicopathologic entity, desmoplastic small round-cell tumors typically affect young men. These aggressive tumors usually arise in the abdomen; other sites of primary disease have been described only rarely. We report the case of an extraabdominal primary tumor with widespread dissemination, including the subcutaneous tissue, a previously unrecognized metastatic site. PATIENT AND METHODS We describe the case of a 16-year-old boy with a primary extraabdominal metastatic desmoplastic small round-cell tumor. RESULTS Our patient had a primary intrathoracic desmoplastic small round-cell tumor and widespread dissemination involving the subcutaneous tissue, kidney, liver, bone, and lymph nodes. Histopathologic analysis found intense desmoplasia and polyphenotypic expression of neural, muscle, and epithelial markers. Reverse transcriptase-polymerase chain reaction analysis of fresh tumor tissue confirmed the characteristic EWS-WT1 transcript. CONCLUSIONS Broader than originally anticipated, the clinical spectrum of desmoplastic small round-cell tumors continues to evolve. Primary intrathoracic tumors with soft-tissue dissemination and polyphenotypic expression should prompt suspicion of this malignancy. Molecular analysis of fresh tumor tissue is an important adjunct to diagnosing this rare neoplasm.
Activation of the contact system by filtration of platelet concentrates with a negatively charged white cell-removal filter and measurement of venous blood bradykinin level in patients who received filtered platelets. BACKGROUND Several recent reports have described hypotensive transfusion reactions in patients receiving platelet concentrates (PCs) filtered through white cell-reduction filters. It is well known that a negatively charged surface activates the contact system, consisting of factor XII, prekallikrein, and high-molecular-weight kininogen. STUDY DESIGN AND METHODS To clarify the mechanisms of these hypotensive reactions, the possibility that white cell-reduction filtration activates the contact system was examined. Venous blood plasma bradykinin levels were also measured in patients receiving PC transfusions through filters. RESULTS None of the measured values were changed by filtration through a positively charged filter. However, filtration through a negatively charged filter resulted in a decrease in the amounts of prekallikrein and an increase in the amount of bradykinin generated, which indicated the activation of the contact system. The bradykinin level was inversely related to the activity of angiotensin-converting enzyme (ACE) in the PCs and was elevated by addition of an ACE inhibitor. Although the venous blood plasma bradykinin level did not change in two patients with a normal ACE activity during PC transfusion through the negatively charged filter, two patients who had decreased ACE activity, showed a significant increase in bradykinin during the transfusion. CONCLUSION These results suggest that the generation of a large amount of bradykinin by filtration of PCs through a negatively charged filter might cause hypotensive reactions in patients with decreased ACE activity. The clinical significance of bradykinin generation requires further study.
A point score system for predicting the likelihood of blood transfusion after hip or knee arthroplasty. BACKGROUND Given the high cost of autologous blood donation for elective surgery, it would be desirable to predict which patients are most likely to benefit from the procedure. The purpose of this study was to develop a point score system for predicting the likelihood of blood transfusion in hip and knee arthroplasty. STUDY DESIGN AND METHODS A database of 599 patients undergoing elective surgery at a teaching hospital was used for the analysis. Variables were analyzed to determine their univariate association with postoperative blood transfusion. Significant factors were entered into a multiple logistic regression model, and a point score system was developed on the basis of the regression coefficients. Four strata of transfusion risk were constructed. RESULTS Factors independently associated with blood transfusion included preoperative hemoglobin, type of arthroplasty, primary versus revision surgery, autologous donor status, and patient weight. Four factors were used to create a point score system with four strata. The likelihood of blood transfusion for patients in the four risk strata was 1.7, 11.0, 40.0, and 78.3 percent. The calculated area under the receiver operating characteristic curve was 0.86. CONCLUSION The likelihood of a postoperative blood transfusion can be predicted by using this simple point score system. Autologous blood donation can subsequently be targeted to the high-risk patients.
Generation of interleukin 8 in stored apheresis platelet concentrates and the preventive effect of prestorage ultraviolet B radiation. BACKGROUND Several recent studies have reported both the generation of cytokines, including interleukin (IL)-1 beta, IL-6, tumor necrosis factor alpha (TNF-alpha), and IL-8, in the supernatants of stored platelet concentrates (PCs) and the implications of this generation in febrile nonhemolytic transfusion reactions. Prestorage filtration is regarded as highly effective in the prevention of cytokine generation. STUDY DESIGN AND METHODS Studies evaluated 1) the levels of these cytokines in apheresis PCs during storage, 2) the effects of white cell inactivation by ultraviolet B or gamma-radiation on the generation of cytokines, and 3) the effects of poststorage filtration on cytokine levels. The apheresis PCs were treated by either ultraviolet B radiation (20,000 J/m2), gamma-radiation (30 Gy), or filtration. Samples were collected sequentially on various days after storage. Cytokines were determined by enzyme-linked immunosorbent assay. RESULTS The average white cell count in 15 PCs tested was 2.58 +/- 0.7 x 10(6) per mL (range, 0.7-10 x 10(6)/mL). A detectable level of IL-8 was found at 3 days of storage, and the levels of this cytokine increased progressively with increasing storage time, ranging from 1.6 to 35,280 pg per mL on Day 5 and from 2.7 to 83,601 pg per mL on Day 8. Reverse transcriptase-polymerase chain reaction analysis showed that the level of IL-8 paralleled the expression of IL-8 transcripts. The levels of IL-1 beta, IL-6, TNF-alpha, and monocyte chemotactic protein-1 were very low, even on Day 8. Ultraviolet B-radiated PCs failed to generate IL-8, even at 8 days of storage, whereas levels of IL-8 in gamma-radiated PCs were similar to those in nonirradiated PCs. Poststorage filtration of PCs with a negatively charged polyester filter, but not with a positively charged one, markedly reduced the levels of IL-8. CONCLUSION Of the cytokines tested, IL-8 had the most evident generation in apheresis PCs during storage. Prestorage inactivation of white cells by ultraviolet B radiation, but not by gamma-radiation, was effective in preventing the generation of cytokines during the storage of PCs.
Improvement of platelet storage conditions by using new polyolefin containers. BACKGROUND Storage of pooled platelet concentrates (PCs) with yields above 3.0 x 10(11) platelets per unit in a 1-L PL-732 polyolefin container for 5 days often results in a drop in pH to below 6.0. Recently, new oxygen-permeable platelet containers (1-L PL-2410, 1-L and 1.5-L Compoflex) have been developed. The maximal platelet storage capacities of the new containers and the PL-732 were compared. STUDY DESIGN AND METHODS Large platelet pools (n = 27) with platelet concentrations between 1.2 and 1.4 x 10(11) per L were made from 3 to 5 PCs prepared from buffy coats. The pools were divided in equal volumes among the PL-732 and the three new platelet containers. Platelet counts in the PCs ranged from 1.0 to 5.0 x 10(11) per unit. All PCs were stored on a flatbed shaker at 22 +/- 2 degrees C and evaluated on Days 1, 3, 5, and 7 by measuring platelet count, pH, pO2, pCO2, HCO3-, glucose, lactate, platelet swirling, and soluble p-selectin. RESULTS Day 7 storage of PCs (n = 6) with yields between 3.0 and 4.0 x 10(11) platelets in PL-732 showed mean +/- SD pH values of 5.93 +/- 0.05 and lactate values of 32.3 +/- 7.9 mmol per L; in 4 of these 6 PCs, pH was below 6.0. In contrast, storage of these PCs in 1-L PL-2410 and 1.5-L Compoflex containers and of 2 of these 6 PCs in 1-L Compoflex containers showed pH values above 6.8. Lactate values were 15.5 +/- 1.3, 15.3 +/- 1.8, and 19.5 +/- 4.7 mmol per L, respectively (p < 0.001 vs. PL-732). The platelet storage capacity of the new containers with platelet yields between 4.0 and 5.0 x 10(11) per unit (n = 6) was evaluated. Day 7 storage of these PCs in the 1.5-L Compoflex showed an average pH value of 6.74 +/- 0.20; in 2 of 6 PCs, pH was below 6.8. The average pH value in the PL-2410 was 6.38 +/- 0.31, and in all PCs, pH was below 6.8. Average lactate values were 17.8 +/- 5.7 and 25.8 +/- 5.6 mmol per L (p < 0.05), respectively. Soluble p-selectin values on Day 7 of storage increased approximately twofold in all PCs. CONCLUSION The new oxygen-permeable containers showed platelet quality comparable to that with the PL-732 and for longer storage periods and at higher platelet counts.
High-yield platelet concentrates attainable by continuous quality improvement reduce platelet transfusion cost and donor exposure. BACKGROUND Donor exposure risk and cost in platelet transfusion practice can be limited by increasing the recovery of platelets from donor units. STUDY DESIGN AND METHODS This study presents results of continuous quality improvement efforts in platelet production and compares the in vivo therapeutic efficacy of currently produced platelet concentrates (PCs) with that of apheresis platelets. Production quality improvement measures included optimization of instrument performance (rotor speed trials), process (massaging whole-blood units, using cup liners, limiting spin-expression time, and refining plasma expression technique), and staff (intensive training with observation and ongoing quality control data feedback). Corrected count increments and increments per kg were calculated for transfusions of 4 pooled PCs and apheresis platelets over a 30-day period. RESULTS The mean number of platelets per PC increased from 5.5 x 10(10) in 1975 to 9.69 x 10(10) in 1994. The mean platelet dose was 3.78 x 10(11) for 4 PCs and 4.17 x 10(11) for apheresis platelets. A total of 34 pooled PCs and 17 apheresis platelets was transfused to 21 patients. The mean increment, the increment per kg, and the corrected count increment were, respectively, 31 x 10(3) per microL, 4.8 x 10(2) per microL, and 14,700 for 4 PCs and 35.4 x 10(3) per microL, 5.4 x 10(2) per microL, and 14,700 for apheresis platelets. Differences were not significant. CONCLUSION Therapeutic efficacy comparable to that of apheresis platelets can be obtained with 4 high-yield PCs.
A multicenter evaluation of the routine use of a new white cell-reduction apheresis system for collection of platelets. BACKGROUND Residual white cells (WBCs) cause serious side effects in platelet transfusion. An in-line WBC-reduction system based on fluidized particle bed technology was recently developed as a modification of an existing plateletpheresis system. STUDY DESIGN AND METHODS In an investigational phase, three flow profiles were evaluated using prototype software in five centers, each using their standard conditions. In the confirmatory phase, the released software was tested in three centers. WBCs were counted in two full Nageotte grids (dilution 1-in-5). RESULTS With the prototype software, WBC levels were always below 1 x 10(6) per procedure (median, 25,000/procedure; n = 314). One profile proved to be superior to the other two with respect to platelet yield and residual WBCs, and it was incorporated in the released WBC-reduction system, together with a built-in process control. Median residual WBCs in these WBC-reduction system components not rejected by the process control were 19,000 per procedure (n = 211/225 total), with 99.5 percent of the platelet components having less than 1 x 10(6) WBCs. CONCLUSION The protocol selected in the initial phase, now available as a WBC-reduction system, results in platelet concentrates with very low residual WBC levels. This satisfies even the most stringent criteria for WBC reduction in platelets, without the platelet loss typically seen with conventional fiber filtration.
Differentiation of anti-D, -C, and -G: clinical relevance in alloimmunized pregnancies. BACKGROUND The differentiation of anti-D, -C, and -G specificities is seldom considered clinically important in pretransfusion testing. However, distinguishing these antibody specificities in alloimmunized pregnancies may be essential. The clinical prognosis as well as Rh immune globulin prophylaxis depends on the accurate identification of these antibodies. CASE REPORT A pregnant woman, para 1 gravida 4, who had received Rh immune globulin at appropriate intervals during her previous pregnancies was reported to have anti-D (titer = 4) and anti-C (titer = 32). Differential adsorption and elution studies showed that the patient had anti-C and anti-G, but not anti-D. This case prompted retrospective examination of the sera from six other women with anti-D and anti-C who were referred to a high-risk pregnancy clinic. Of six pregnant women reported to have anti-D and anti-C; two had anti-D, -C, and -G; three had anti-D and -G, but not anti-C; and one had anti-C and -G, but not anti-D. This last is similar to the index case. CONCLUSION Cases of pregnant women with anti-C and -G, but not anti-D, are not infrequent. Studies to differentiate anti-D, -C, and -G should be performed on alloimmunized pregnant women presumptively identified as having anti-D and anti-C when the medical history (Rh immune globulin prophylactic therapy) and/or titer values (e.g., anti-C titer higher than anti-D titer) suggest that anti-D may not actually be present. Rh immune globulin has not failed in these patients, and they should receive this therapy during pregnancy to prevent immunization to D.
Removing IgG antibodies from intact red cells: comparison of acid and EDTA, heat, and chloroquine elution methods. BACKGROUND To accurately phenotype red cell from patients with a positive direct antiglobulin test (DAT), nonlytic elution procedures were assessed for their ability to dissociate IgG from antibody-coated red cells without altering red cell antigen expression. STUDY DESIGN AND METHODS Antibodies coating red cells that were sensitized in vivo (warm-reactive autoantibodies: 8 patients) or in vitro (42 alloantibodies) were eluted by using glycine-HCl and EDTA (acid/ EDTA), heat (56 degrees C, 10 min), or chloroquine method. RESULTS Acid/EDTA elution gave the best results, reducing DAT positivity to microscopic levels or rendering the DAT negative in 48 of 50 instances, whereas 4 samples remained resistant to heat elution and 24 to chloroquine. Standard DAT agglutination scores demonstrated that both acid/EDTA and heat elution were superior to the chloroquine method (p < 0.0001). With the gel low-ionic-strength saline indirect antiglobulin test, acid/ EDTA was superior to heat (p < 0.001). Overall, acid/ EDTA elution dissociated more antibodies than heat (p < 0.0001), especially for Kell system (K, k, Kpa, Kpb) alloantibodies. Common red cell antigens, other than Kell system antigens, were unaffected by acid/EDTA elution. In contrast, the expression of most blood group antigens was diminished after heat elution. However, it was possible to type red cell antigens by using gel low-ionic-strength saline indirect antiglobulin tests or tube agglutination methods. CONCLUSION Although heat elution may be used on a limited basis, the acid/EDTA method appears to be the procedure of choice for typing red cell coated with warm-reactive IgG alloantibodies or autoantibodies.
Detection and differentiation of platelet-specific antibodies by flow cytometry: the bead-mediated platelet assay. BACKGROUND Platelet-reactive antibodies cause a number of clinical disorders. The detection and differentiation of these antibodies are prerequisites for the adequate treatment of these disorders. The bead-mediated platelet assay described here enables the detection and differentiation of platelet-bound antibodies by the use of flow cytometry. STUDY DESIGN AND METHODS The bead-mediated platelet assay is based on the isolation of human platelet glycoproteins by using flow cytometric standardization beads after the incubation of typed platelets with human sera. The specificity and sensitivity of this assay were tested with five sera, each containing a known platelet-reactive antibody. The monoclonal antibody-specific immobilization of platelet antigens assay was used as a reference test. RESULTS The bead-mediated platelet assay was able to determine the glycoprotein specificity of the antibody without cross-reactions in every case. In serial dilution tests, the bead-mediated platelet assay was able to detect the antibodies at higher dilutions than the monoclonal antibody-specific immobilization of platelet antigen assay. Total test time was 3.5 hours. CONCLUSION The bead-mediated platelet assay is a fast and reliable method for the detection and differentiation of platelet-reactive antibodies.
Factors affecting mobilization of CD34+ cells in normal donors treated with filgrastim. BACKGROUND Multiple days of apheresis are required for some normal peripheral blood progenitor cell (PBPC) donors, to ensure a sufficient collection of CD34+ cells for allografting. It would be of practical value to be able to identify the patients with poor mobilization on the basis of simple pretreatment clinical or hematologic variables. STUDY DESIGN AND METHODS Clinical characteristics and laboratory data for 119 normal PBPC donors who underwent apheresis on Days 4 to 6 of treatment with granulocyte-colony-stimulating factor (filgrastim) were analyzed for correlations with CD34+ cell yield from the first day of apheresis. RESULTS The CD34+ cell yield was significantly lower in donors who were more than 55 years of age, who underwent apheresis on Day 4 of filgrastim therapy, or who were not obese. There were weak direct correlations between CD34+ cell yield and the baseline white cell count, preapheresis white cell count, and preapheresis mononuclear cell count, and there was a weak inverse correlation with age. Twenty-one donors (18%) were considered to have poor mobilization (< 20 x 10(6) CD34+ cells/L blood processed). In the multivariate analysis, the only significant factor was age greater than 55 years, which conferred a 3.8 times greater risk (95% CI, 1.1-13.7) of poor mobilization (p = 0.04). However, poor mobilization occurred in all age groups, so the predictive value of the model was low. CONCLUSION Donor variables correlated with CD34+ cell yield only weakly, so no particular clinical characteristic can be used to exclude an individual as a PBPC donor if he or she is otherwise suitable for the apheresis procedure.
Transmission of human immunodeficiency virus through blood transfusion: the use of lookback and traceback approaches to optimize recipient identification in a regional population. BACKGROUND The purpose of this study was to define the epidemiologic features of the transmission of human immunodeficiency virus (HIV) by blood transfusion in a region of Canada between 1980 and 1985 and the results of intensive recipient-identification practices. STUDY DESIGN AND METHODS Lookback (notification of all recipients of blood from an HIV-infected donor) and traceback (identification of the HIV-infected source donor, after an HIV-infected recipient of blood cites transfusion as a risk for infection) programs were established linking (with patient consent) a transfusion service and an HIV clinic to identify HIV-infected donors and the recipients of their blood. RESULTS Twenty-two cases of documented HIV infection and 26 cases of presumed infection were found in local blood recipients. Twenty-eight recipients have died of causes unrelated to HIV. Twelve recipients have developed AIDS. Six of the seven living recipients have yet to develop an AIDS condition. These 48 infections have been linked to 11 donors who have subsequently tested positive for HIV infection. Six donors were found on subsequent blood donation. Five donors were found by traceback. CONCLUSION Forty-eight recipients of blood from donors who subsequently tested positive for HIV were identified in a low-prevalence area. Active lookback and traceback programs linking a transfusion service and an HIV clinic were successful in identifying infected recipients.
Transmission of parvovirus B19 by coagulation factor concentrates exposed to 100 degrees C heat after lyophilization. BACKGROUND Double inactivation by solvent/detergent treatment plus heating at 100 degrees C for 30 minutes after lyophilization has been adopted to improve viral safety of factor VIII and factor IX concentrates, particularly with respect to non-lipid-enveloped viruses. The aim of this study was to evaluate the safety of concentrates exposed to these virucidal methods. STUDY DESIGN AND METHODS Twenty-six previously untreated hemophiliacs, 19 with factor VIII deficiency and 7 with factor IX deficiency, were investigated in a prospective multicenter study over a 12-month follow-up period by the use of serologic and virologic markers for lipid- and non-lipid-enveloped viruses (human immunodeficiency virus types 1 and 2; hepatitis A, B, and C viruses; B19 parvovirus antibodies; and B19 DNA). Overall, 270,000 U of factor VIII and 102,000 U of factor IX concentrate were administered during the study period. RESULTS None of the 26 patients seroconverted for human immunodeficiency virus or hepatitis C virus. Hepatitis B virus markers remained negative in the 10 unvaccinated hemophiliacs. No hepatitis A virus seroconversion occurred among 17 susceptible patients. B19 seroconversion (IgM) and B19 viremia were observed within 2 weeks of the first concentrate infusion in 8 of 15 susceptible patients, 5 of 11 treated with factor VIII and 3 of 4 with factor IX concentrate. CONCLUSION This prospective study indicates that very high temperatures applied to lyophilized concentrates appear to prevent the transmission of hepatitis A virus to hemophiliacs. However, B19 parvovirus still contaminates concentrates despite the use of this robust virucidal method.
Autologous donation error rates in Canada. BACKGROUND Although certain transfusion risks are eliminated by the use of autologous blood, clerical errors may still occur. In addition, because of differences in donor selection criteria and donor-patient expectations, the consequences of certain errors may be different in autologous and allogeneic donations. STUDY DESIGN AND METHODS In January 1996, autologous donation error rates in Canada from 1989 to November 1995 were estimated by 1) a detailed questionnaire sent to hospitals supplied by the Canadian Red Cross, Blood Services, Transfusion Center of Quebec at Montreal autologous donation program (n = 31), 2) a review of that institution's quality assurance non-compliance reports, and 3) a detailed questionnaire sent to other Canadian Red Cross centers with autologous donation programs (n = 16) and hospital-based autologous programs in Canada (n = 3). The total number of autologous donations collected was determined from Canadian Red Cross annual reports and information supplied by hospital-based programs. RESULTS There were 113 errors reported for 16,873 units collected by the Montreal center (1/149 units) based on collection center and hospital data. The most frequent errors were the late receipt of units for surgery (25% of errors) or the receipt of units in the wrong hospital (23%). Other Canadian programs reported 166 errors for approximately 53,500 units collected (1/322 units). However, this figure was based mainly on collection center, and not hospital, data. The most frequent errors were in labeling (48%) and component preparation (25%). One unit of autologous fresh-frozen plasma was transfused to the wrong recipient. Errors were more frequent if components were produced, if units were drawn in hospitals for interhospital transfer, or it units were shipped between Red Cross centers. CONCLUSION Errors are not infrequent in autologous donation programs. Autologous transfusion should not be considered as being without risk.
Relationship of the time of storage and transfusion reactions to platelet concentrates from buffy coats. BACKGROUND Transfusion reactions to platelet concentrates prepared from buffy coats (BC-PCs) were reviewed to determine the effect of some variables of BC-PC preparation and storage: time of BC storage before BC-PC preparation (1-2 days); time of BC-PC storage before transfusion (1-5 days); no white cell reduction versus laboratory and bedside BC-PC white cell reduction. STUDY DESIGN AND METHODS A multiple linear logistic regression model was used by which the relative effect of one variable is expressed as the relative risk of transfusion reaction against a baseline level (1-day storage, no white cell reduction). RESULTS During the 14 months of study, a total of 2707 BC-PC transfusions were given to 192 patients; 37 reactions (1.4%) were reported in 25 patients (13%). The transfusion reactions were febrile, nonhemolytic in 23 cases; allergic in 5; febrile and allergic in 2; and other in 7. The relative risk of transfusion reaction to BC-PCs prepared from BCs stored for 2 days was 1.98 times that to BC-PCs prepared from BCs stored for 1 day (p = 0.07). The relative risk of transfusion reaction of 5-day-old BC-PCs was 10.7 times that of 1-day-old BC-PCs (p = 0.001). The relative risk of transfusion reactions of BC-PCs white cell-reduced in the laboratory and at the bedside were 0.65 (p = 0.3) and 1.87 (p = 0.1) times, respectively, that of non-white cell-reduced BC-PCs. CONCLUSION Time of storage seems to be an important variable associated with BC-PC transfusion reaction.
Comparison of the Q-switched alexandrite (755 nm) and Q-switched Nd:YAG (1064 nm) lasers in the treatment of benign melanocytic nevi. BACKGROUND A variety of pigmented lesions have been shown to be effectively treated with several pigment-specific laser systems currently available. There has been recent evidence to indicate that they may also be useful in the treatment of melanocytic nevi. OBJECTIVE To compare the clinical and histologic effects of the Q-switched (QS) alexandrite (755 nm) and Nd:YAG (1064 nm) lasers in the treatment of melanocytic nevi. METHODS Eighteen patients received three QS alexandrite and Nd:YAG laser treatments to either half of a large nevus or to two small adjacent nevi. Tissue biopsies were obtained for histologic examination. Degree of clinical improvement was determined by comparative photographic global assessment scores. The amount of melanin present within the nevi before and after laser irradiation was measured by reflectance spectrometry. RESULTS Clinical global assessment scores were significantly reduced in all QS alexandrite and QS Nd:YAG laser-treated nevi after three treatments. Melanin reflectance spectrometry scores improved after the first laser treatment only. Histologically, a significant reduction in epidermal pigmentation and melanocytes were observed following laser irradiation with either QS system. CONCLUSION Both the QS alexandrite and Nd:YAG laser systems resulted in significant improvement (lightening) of treated nevi. The QS alexandrite laser produced slightly better results using the parameters outlined.
Carbon dioxide laser treatment vs subcutaneous resection of axillary osmidrosis. BACKGROUND Axillary osmidrosis is a distressing disorder characterized by unpleasant odor, profuse sweating, and occasional staining of clothes that may handicap those affected both socially and in the work place. Various types of surgical procedures have been developed for the treatment of axillary hyperhidrosis and osmidrosis. OBJECTIVE Our purpose is to seek a more effective surgical procedure than preexisting various subcutaneous resection techniques for axillary osmidrosis. METHODS After single transverse incision in the center of one axilla, undermining was performed from the incision edges to make a wide subcutaneous tunnel and then apocrine glands and subcutaneous fats were vaporized with a CO2 laser. Subcutaneous resection technique was performed on the opposite axilla in the same patient. A total of 20 patients have been evaluated for 4 months to 1 year, with an average of 8 months. RESULTS The results from commonly used surgical procedures can be improved upon by the use of the CO2 laser. The frequency of complications and the mean duration of suture removal were diminished on the laser-operated side. CONCLUSIONS CO2 laser vaporization in osmidrosis produces significant patient-benefit during the postoperative course. We believe that this laser-assisted combined surgical procedure can be a viable option for the treatment of axillary hyperhidrosis and osmidrosis.
Debulking of skin cancers with radio frequency before cryosurgery. BACKGROUND The purpose of prior debulking of skin cancers, by curettage, is to prepare the lesion for cryosurgical treatment. The main inconveniences of that procedure are the nearly always inevitable hemorrhage and the time necessary to control it. METHODS We performed a study consisting of debulking skin tumors with radiosurgery, prior to cryosurgery, on a series of 38 patients (31 basal cell carcinomas, six squamous cell carcinomas, and one Bowen's disease), meeting the classical indications for cryotherapy. CONCLUSIONS The advantages of the latter procedure over the traditional debulking by curettage are: the time needed to perform a biopsy, with easy and fast control of the bleeding, is considerably shortened; the whole procedure can be easily performed in one single session; the required surgical material also is greatly reduced; and the treatment can be performed on an out-patient regimen.
Perineural and neural involvement in skin cancers. BACKGROUND Malignant skin tumors rarely spread along nerves. Complete resection of involved nerves is often unsuccessful. OBJECTIVE In the treatment of tumors with perineural invasion, surgeons should accurately estimate the extent of distant spread. METHODS We report six cases of skin cancers, including two basal cell carcinomas, two squamous cell carcinomas, and two neurotropic malignant melanomas, that invaded nerve or perineural spaces. RESULTS In three of the cases, the tumors developed on the face and involved the infraorbital nerves or its branches. Two patients suffered from tumors on old burn scars of lower legs. Branches of posttibal nerves were involved in both cases. In the last case, tumor invasion of a branch of the greater occipital nerve was detected. CONCLUSION The extent of surgical excision should include the area of skin supplied by the affected nerve, which must be resected in continuity.
Malignant metastatic eccrine poroma. Proposal for a new therapeutic protocol. BACKGROUND Malignant metastatic eccrine poroma is a very rare cutaneous neoplasm, and consequently the references in the literature regarding the treatment of this tumor, known also as porocarcinoma, are very poor. OBJECTIVE To call attention to a new therapeutic protocol in the treatment of metastatic porocarcinoma, as well as to underline an antineoplastic efficacy of vitamin A analogues. METHODS The results are presented on the basis of the clinical case of a malignant eccrine poroma with metastatic regional lymph nodes. RESULTS With our new chemotherapeutic protocol, arrest of the metastatic progression was achieved after 3 months and the remission was maintained until the 10th month of therapy. CONCLUSIONS A new chemotherapy protocol consisting of isotretinoin and interferon alpha has confirmed the advantages of polychemotherapy in the treatment of metastatic malignant eccrine poroma. On the basis of the considerably long, although incomplete, remission with good drug tolerance in spite of the high doses used as well as the undoubtedly major antineoplastic strength of the latest generation of synthetic retinoids, we feel that these findings could be a good starting point for further experimental verifications of the therapy of this aggressive cutaneous neoplasm.
Ambulatory phlebectomy of the foot. Review of 75 patients. BACKGROUND Review of 75 patients on whom ambulatory phlebectomy of the foot was performed as part of their varicose vein treatment. OBJECTIVE To demonstrate that ambulatory phlebectomy is an effective modality of treatment for varicosities of the foot. METHODS Ambulatory phlebectomies were performed on an outpatient basis under local anesthesia. RESULTS The overall satisfactory result of ambulatory phlebectomy of the foot employed in the 75 patients in this study revealed the procedure to be very effective with few complications resulting and with a high degree of patient satisfaction. CONCLUSIONS Ambulatory phlebectomy of the foot has proven to be a most satisfactory procedure for the treatment of varicose veins of the foot.
Postsclerotherapy pigmentation. Is serum ferritin level an accurate indicator? BACKGROUND Human beings have suffered and sought treatment for disease of veins as early as the recordings of the old testament. The use of irritating sclerosing agents have been and are widely used today to treat varicose veins and telangiectasia. One of the most common and cosmetically significant side effects of sclerosing agents is varying degrees of hyperpigmentation. It has been reported that elevated serum ferritin level plays a role in this postsclerotherapy pigmentation. OBJECTIVE To support or negate the possibility of a direct correlation between serum ferritin levels and pigmentation postsclerotherapy using for our investigation a patient with hemochromatosis. METHODS A patient with hemochromatosis having a serum ferritin level of 1200 was treated for spider veins. Clinical and histologic studies were performed pretreatment and posttreatment. RESULTS There was no clinically apparent hyperpigmentation noted on the patient after sclerotherapy over a 6-month period. Histology reports revealed macrophagic pigmentation both pretreatment and posttreatment. CONCLUSION Our results do not confirm the theory that lab values of elevated serum ferritin correlate with pigmentation postsclerotherapy. Further study of the correlation between postsclerotic pigmentation and serum ferritin levels are needed. One would anticipate that if a true correlation existed, then an extreme case such as this would clearly support this theory.
Durometer measurements of skin induration in venous disease. BACKGROUND The degree of skin induration (lipodermatosclerosis) around venous ulcers has prognostic significance; however, objective measurements are needed to assess the induration. The durometer, an engineering instrument used to measure the hardness of metals and plastic, has recently been adapted to assess skin induration. OBJECTIVE The purpose of this study was to measure skin induration and its relationship to skin ulceration by the use of a durometer, and to determine the influence of edema, if any, on durometer measurements. METHODS The degree of skin induration on the medial leg was determined in six sequential, nonselected patients with lipodermatosclerosis and leg ulcers, and in five normal volunteers by using a blinded observer's clinical score (0 = normal to 3 = maximal induration) and a hand-held Type 0 durometer. In addition, durometer readings in 14 patients with edema and eight control subjects were taken on the tibia, the dorsum of the foot, and behind the ankle. RESULTS Durometer readings in patients with leg ulcers and lipodermatosclerosis diminished as one measured from the superior edge of the ulcer to the knee (r = 0.925). The higher the clinical skin score the higher were the durometer readings (P = 0.0062). The presence of edema did not influence durometer measurements. CONCLUSION The durometer is an effective and reliable instrument for measuring the degree of skin induration in venous ulceration and its readings are not affected by edema. Ulcers occur in skin most affected by lipodermatosclerosis.
Bilevel tumescent anesthetic infiltration for hair transplantation. BACKGROUND A dual level of tumescent anesthetic infiltration has been found to expedite hair graft or strip harvesting for hair transplantation, reducing bleeding, and increasing the exit angle of the hairs, thereby promoting increased survivorship of hair shafts and bulbs. OBJECTIVE To acquaint the readership with this variation of anesthetic tumescent technique in hair transplantation. METHODS Patients were utilized for this study during international teaching exchanges. Larger multiport infiltrators were used for the deep infiltration; smaller and more narrow infiltrators were used for the more superficial anesthetic administration. RESULTS Subcutaneous compression and a bilevel vasoconstriction in both donor and recipient sites resulted in an increased upwardly angled exit angle of the hairs to be harvested, an increased separation of individual hair shafts, and reduced bleeding. Placement of the correct incisional angle of instruments inserted into the donor site was facilitated. CONCLUSION Utilization of a bilevel tumescent anesthetic infiltration technique is a superior method of anesthesia for hair transplant harvesting and transplanting. Bleeding at both occipital donor and frontal recipient sites was negligible.
Use of a modified binding model for the investigation of affinity dependence on antibody concentration in immunoassay systems. Affinity parameters obtained from standard calibration curves of radioimmunoassays (RIA) have been shown to decrease with increasing antibody concentration. A modified binding model was introduced in which not only binding capacity but also affinity was influenced by the antibody concentration. This allows a consistent overall description of RIA standards and titration curves for the characterization of binding assays.
Use of the acute phase serum amyloid A2 (SAA2) gene promoter in the analysis of pro- and anti-inflammatory mediators: differential kinetics of SAA2 promoter induction by IL-1 beta and TNF-alpha compared to IL-6. A cytokine responsive construct, pGL2-SAA2pt, was generated by cloning the acute phase promoter of human serum amyloid A2 (SAA2) upstream of a luciferase reporter gene. The construct responds to the inflammatory mediators MoCM, IL-1 beta, TNF-alpha, and IL-6 in a manner that closely mimics the response of the endogenous SAA2 gene to such stimuli: i.e. single treatments induce transcriptional activation by IL-1 beta and TNF-alpha to a greater extent than by IL-6 at 12-24 h. However, timecourse experiments show that the kinetics of induction generated by IL-1 beta and TNF-alpha are quite distinct from IL-6, IL-6 having a much greater effect at 3-6 h. IL-1 beta and TNF-alpha synergize with IL-6 to give a 10-fold increase in transcriptional readout over single cytokine treatments. The kinetics of this synergistic response resembles that generated by IL-6 alone. The IL-1 receptor antagonist, hIL-1ra, can specifically block the IL-1 beta driven transcriptional activation of pGL2-SAA2pt, but not that driven by TNF-alpha or IL-6. Furthermore, in synergistic cytokine combinations, it blocks only the IL-1 beta driven component indicating that the effect is biological and not attributable to toxicity. Consequently assays utilizing pGL2-SAA2pt will be useful both for the investigation of the kinetics of inflammatory signalling in a cytokine specific manner, and for the evaluation of the pro- and anti-inflammatory properties of novel natural and synthetic molecules.
Baculovirus cDNA libraries for expression cloning of genes encoding cell-surface antigens. We describe a method for the production of baculovirus-based cDNA libraries. By staining with monoclonal antibodies, single positive cells can be sorted and the virus encoding for the surface epitope can be isolated by limiting dilution. We have used this method to isolate cDNAs encoding several cell-surface antigens.
The use of computer-assisted video image analysis for the quantification of CD8+ T lymphocytes producing tumor necrosis factor alpha spots in response to peptide antigens. Enzyme-linked immunospot (ELISPOT) analysis is a sensitive technique for the detection and quantification of single T lymphocytes forming cytokine spots after antigen contact in vitro. Herein computer-assisted video image analysis (CVIA) was applied to automatically determine the number and size of tumor necrosis factor alpha (TNF-alpha) spots formed by single blood-derived CD8+ T cells after contact with peptide-loaded target cells. With CVIA and TNF-alpha ELISPOT analysis we quantified CD8+ T cells responsive to HLA-A2.1-binding tyrosinase and influenza matrix peptides in healthy donors. We followed the course of the virus-specific T cell response in two HLA-A2-positive patients with reactivation of latent cytomegalovirus (CMV) infection during immunosuppressive therapy. The test proved sufficiently sensitive to detect in the blood of both patients a temporary expansion of CD8+ T lymphocytes reactive with a known immunogenic HLA-A2.1-binding peptide from glycoprotein B of CMV. Reactivity to peptide antigens was not only reflected by numeric increases of spot formation, but also by the appearance of larger spot areas, presumably formed by strongly peptide-reactive CD8+ T cells. We conclude that the combined use of the TNF-alpha ELISPOT assay and CVIA allows reliable monitoring of the T cell responsiveness to peptide antigens in peripheral blood.
The Fab region of IgG2 human myeloma proteins does not bear the streptococcal protein G-specific determinant. Seven out of ten Fab (F(ab')2/Fab') preparations derived from purified human myeloma IgG showed a substantial binding to protein G-Sepharose. Subclass analysis revealed that the 7 protein G-reactive Fabs included 3 IgG1, 2 IgG3 and 2 IgG4 Fabs, whereas the remaining 3 which were not adsorbed were IgG2 Fab. Incubation of protein G-Sepharose with non-saturating amounts of 4 Fab preparations, representative of all IgG subclasses, showed that gamma 1, gamma 3 and gamma 4 Fabs adsorbed from 26 to 28.3%, whereas 80% of gamma 2 Fab was left in the supernatant after adsorption. These results indicate that human IgG2 lack PG-specific Fab-associated reactive site(s).
An improved, ultrasensitive method for the detection of IgM oligoclonal bands in cerebrospinal fluid. A new, 10-fold more sensitive method, based on an improved immunofixation technique, has been devised to detect oligoclonal IgM bands in unconcentrated cerebrospinal fluid (CSF). Using agarose gel electrophoresis, 5 microliters of an unconcentrated sample containing oligoclonal bands was separated and blotted on to a polyvinyldifluoride membrane. To visualise the pattern, a peroxidase-labelled double-antibody technique was used. No prior concentration of CSF was needed and the process required only 5 h. The technique may prove very useful in diagnosing an early intrathecal immune response.
An ELISA for selectins based on binding to a physiological ligand. Members of the selectin family of adhesion receptors, consisting of L-, P- and E-selectin, mediate the initial interaction between leukocytes and endothelium during leukocyte trafficking from the blood into tissue sites. These receptors have attracted great attention in recent years due to their participation in a number of acute and chronic inflammatory diseases. We describe here a new ELISA that measures the binding between selectin-IgG chimeras and a physiological ligand for L-selectin and can be used to screen selectin inhibitors. The ligand used is a mucin-like glycoprotein known as GlyCAM-1, which is derived from high endothelial venules in secondary lymphoid organs. We demonstrate binding of all three selectins to GlyCAM-1 and demonstrate that the binding interactions satisfy a number of important criteria. The advantage of this ELISA over previous assays is that a macromolecular physiological ligand is employed, rather than a fortuitous or simplified carbohydrate ligand. Thus, the protein-carbohydrate interactions, as well as other interactions contributing to ligand recognition, can be investigated. The assay is suitable for high-throughout screening of compounds and may find use in the identification of selectin antagonists with anti-inflammatory potential.
Flow-cytometric screening for the modulation of receptor-mediated endocytosis in human dendritic cells: implications for the development of an in vitro technique for predictive testing of contact sensitizers. The aim of this study was to explore the usefulness of human blood dendritic cells (DC) in the development of an in vitro model for predictive testing of contact sensitizers. A method was established to monitor the influence of chemicals on the intracellular targeting of antibody-crosslinked MHC class II molecules after their uptake by human DC. Using a three-colour flow-cytometric technique, freshly prepared DC were distinguished from other MHC class II-bearing cell types such as B-cells and monocytes in unseparated mononuclear cell suspensions of healthy volunteers. The assay is based on the pH-sensitivity of internalized fluorescein-coupled MHC class II specific antibodies. Quenching of fluorescence intensity due to internalization into acidic intracellular compartments was observed with untreated DC whereas internalization into less acidic structures following stimulation with strong contact sensitizers ensured that the fluorescence intensity was conserved. The usefulness of this approach for predictive testing of the preservatives MI/MCI, imidazolidinyl urea, methyl-4-hydroxy-benzoate and 2-phenoxyethanol in comparison to the strong allergen DNFB and the irritants sodium lauryl sulphate and dithranol was explored. Whereas low concentrations of MI/MCI resembled the strong allergen DNFB, high concentrations of imidazolidinyl urea were required for a moderate response. Methyl-4-hydroxy-benzoate and 2-phenoxyethanol as well as the irritants SLS and dithranol failed to induce a significant effect in this assay. The non-responsiveness to the latter compounds reflected their minor or absent capacity to induce contact hypersensitivity in humans, whereas DNFB, MI/MCI and imidazolidinyl urea are well established contact sensitizers. These data suggest that the capacity of a chemical to modulate endocytotic mechanisms in dendritic cells in vitro seems to reflect the probability of that substance acting as a hapten in vivo.
A rapid method for semiquantitative analysis of the human V beta-repertoire using TaqManR PCR. Analysis of the V beta-repertoire of antigen-reactive T cell populations can be approached using either flow-cytometry or PCR-based techniques. While the former method requires a complete set of V beta-specific monoclonal antibodies (mAbs) and large cell numbers for analysis, the latter is both time-consuming and labour-intensive. To circumvent the drawbacks of both these methods we have employed the recently developed technique of TaqManR PCR to analyse the V beta-usage of human T cell populations. TaqManR PCR is based on the 5'-->3' nuclease activity of Taq polymerase. During PCR amplification an internal oligonucleotide probe, that is labelled with a fluorescent reporter and a quencher dye, is cleaved by Taq polymerase. After cleavage, quenching of the reporter dye is lost and reporter fluorescence can be detected with a fluorescence plate reader. Using one C beta-specific fluorogenic probe and a panel of V beta-specific primers, we show that fluorescence-detected amplification of TCR beta cDNA is V beta-specific and linear within a 2-3-log range of template concentration. The sensitivity of TaqManR PCR is comparable to conventional detection of PCR-products by agarose gel staining, while processing time is reduced. Furthermore, superantigen-induced skewing of the V beta-repertoire of human T cells is readily detected with this method. Thus TaqManR PCR is a reliable and fast method for semiquantitative analysis of the V beta-repertoire of human T cell populations.
Combined immunomagnetic cell sorting and ELISPOT assay for the phenotypic characterization of specific antibody-forming cells. A combination of immunomagnetic cell sorting and ELISPOT techniques has been evaluated to permit enrichment and characterization of antibody-secreting cells (ASC). Cell suspensions containing putative ASC were first incubated with magnetic microbeads coated with antibodies specific for a given cell surface marker. After separation of bead-cell clusters and free cells, the resulting cell populations were examined for the presence of ASC by an ELISPOT assay. As a model system, the expression of selected cell differentiation markers by human circulating ASC has been evaluated after parenteral tetanus vaccination and during the course of a Leishmania infection. Prior treatment of blood MNC with beads coated with antibodies to CD38, HLA-DR or CD19 permitted the isolation of virtually all blood ASC. Further, prior immunomagnetic removal of T (CD2+) cells from blood MNC, followed by isolation of CD38+ cells facilitated the detection of Leishmania major-specific ASC in all six patients examined, whereas parasite-specific ASC among unfractionated blood mononuclear cells could only be detected in 3 out of these six patients. Simple and rapid, this approach provides not only accurate estimates of the frequency of ASC within a given B cell population or subpopulation, but can also efficiently enrich functional ASC from complex cell suspensions and thus should be particularly useful in situations where ASC are present at low frequencies.
Stability of a perturbed Leslie model. A small perturbation introduced in the stochastic Leslie model leads to a long-lived stable population in a region or parameter space where the stochastic model predicts the demise of the population. This somewhat surprising result is understood by considering a related model with a density-dependent sex ratio which exhibits similar features.
The evolution of genomic imprinting: two modifier-locus models. We present two autosomal two-locus models in which the primary locus, A, may be imprinted according to the alleles present at the second, modifier locus, M. In the first model, the modifier is cis-acting, which assumes that imprinting occurs late in gametogenesis: whether or not A is imprinted depends only on the M allele in the (unfertilized) egg. We examine three cases in which polymorphism at A is maintained by a mutation-selection balance or heterozygote advantage. We show that a newly arising modifier allele without direct fitness effects can increase at a rate only of the order of the mutation rate at the A locus. This result mirrors that found in two-locus models of the evolution of dominance modifiers. Modifiers that also alter fitnesses, however, may spread quickly. In the second model, a monomorphic primary locus, A, is imprinted according to the mother's genotype at the second, diallelic modifier locus, M. The model is therefore trans-acting, which assumes imprinting occurs early in gametogenesis: whether or not A is imprinted depends on both of the mother's M genes. We show that a newly arising modifier will increase in frequency via selection if either imprinting is advantageous and the modifier increases the proportion of imprinted gametes or imprinting is disadvantageous and the proportion is decreased. Both of these factors-the selective effect of imprinting and the proportion of gametes imprinted-affect the rate of modifier evolution. Selectively maintained polymorphism at the modifier locus is unlikely unless the alleles interact in a nonadditive fashion.
Postinflammatory medial canal fibrosis. OBJECTIVE Our objective was to describe the diagnosis and treatment of postinflammatory medial canal fibrosis by reviewing a large series. STUDY DESIGN This study was conducted via retrospective chart review. SETTING This study was conducted at a tertiary otologic referral center. PATIENTS The 24 patients had a clinical diagnosis of postinflammatory medial canal stenosis. Only one of the 16 females and eight males was under 18 years of age, and the mean age for the group was 50.5 years (range 5-78). fourteen patients had bilateral diseases. INTERVENTION Surgical therapy was performed in 14 ears (11 patients), and medical therapy was performed in nine. RESULTS For patients undergoing surgical treatment, mean pure-tone average hearing threshold improved from 37 dB preoperatively to 26 dB postoperatively. The air-bone gap improved from 24 dB to 15 dB. There were three recurrences of disease in the surgery group. CONCLUSION Postinflammatory medial canal stenosis is a rare disorder resulting from chronic external otitis that required surgical intervention to correct the resulting conductive hearing loss.
Development of tympanosclerosis: can predicting factors be identified? HYPOTHESIS The etiological hypothesis is that there might be factors triggering an immunological chain reaction that eventually leads to tympanosclerosis formation. BACKGROUND Tympanosclerosis is a condition leading to a calcification process in the middle ear and, occasionally, also to the lining of the inner ear. This sometimes leads to hearing loss due to fixation of the middle ear ossicles. In severe cases. deafness may occur as a result of the inner ear impairment. Surgery is the treatment offered, often with poor long-term results, and, alternatively, prescription of hearing aids. Some patients develop tympanosclerosis after mild inflammatory otitis media processes whereas some heal without tympanosclerosis after more aggressive infections. This difference may be due to individual variations in the inflammatory response. The biological mechanism of calcification in tympanosclerosis is probably similar to that occurring in other calcifying tissues due to diseases. METHODS The present investigation was performed to develop methods for immunohistochemical analyses of this delicate tissue consisting of both hard bone and the very thin tympanic membrane. Sprague-Dawley rats were inoculated with a suspension of Streptococcus pneumoniae, type 3, into the middle ear and sacrificed after 1 week up to 6 months. A new technique was elaborated where the whole specimen was prefixed briefly and then en bloc incubated with the primary antibodies and after that decalcified in edetic acid (EDTA). Primary antibodies against macrophages were used for the immunohistochemical staining. RESULTS Acute otitis media was successfully induced in the rats and myringosclerosis was seen in 30% of the animals, often localized close to the bony frame where macrophages could also be detected. CONCLUSIONS Acute otitis media and myringosclerosis were introduced in the animals. Conventional immunological techniques were tested on this delicate tissue. A new method for immunohistochemical staining was elaborated in which specimens were stained en bloc before decalcification and sectioning were performed. Expression of macrophages was demonstrated in the tympanic membrane.
Clinical importance of the Korner's septum. OBJECTIVE To evaluate the clinical importance of the petrosquamosal lamina (Korner's septum [KS]), which is not only a bony plate dividing the mastoid cells at the level of antrum, but is also a lamina starting from the posterior aspect of the glenoid fossa that extends above the middle ear cavity and courses in an inferior direction lateral to the facial canal and proceeds to the mastoid apex. STUDY DESIGN AND SETTING A retrospective review of 688 mastoidectomies performed in University Hospital from 1987 to 1992. PATIENTS The study group consisted of 389 males and 299 females (mean age 30.85 +/- 12.80, the youngest being 8 and the oldest being 67 years of age). MAIN OUTCOME MEASURES The main outcome measures were the prevalence of KS encountered during mastoidectomies and comparison of prevalence of retraction pockets (RPs) or retraction and/or adhesion of the whole tympanic membrane (R/A-TM) between ears with KS and without KS. RESULTS The prevalence of KS was 30.4% among the ears with RP or R/A-TM, 6.58% in normal ears, and 17.4% in ears with chronic otitis media without RP or R/A-TM. CONCLUSIONS KS is an important anatomic handicap predisposing the individual to chronic otitis media, particularly when it is characterized by attic retraction pockets and cholesteatoma, and adhesive otitis media, because KS contributes to attic blockage. This statement is in accordance with the original articles written by Cheatle (1910, 1923) and Williams (1966), and recently published data related to supratubal recess and the cog (Tono et al., 1996).
Tensor fold and anterior epitympanum. HYPOTHESIS The aim of this study was to investigate the anatomy and pathology of the anterior epitympanum and of the tensor fold. BACKGROUND Early studies reported data that are primarily still relevant, but contemporary reports present conflicting data, including several erroneous concepts. METHODS Fifty-one temporal bones were dissected, and the anatomic details were photographed in 42 normal and nine infected bones. Histology was documented from seven serially sectioned bones, five normal and two infected. RESULTS The tensor fold formed the frontal wall of the anterior epitympanum between tensor tendon and attic bony wall, the anterior insertion consisting of composite connective and fatty tissue with some bone trabeculae. The transverse crest was posterior to it and extended from the anterior tympanic spine to the facial canal. The tensor fold angle in 78% of the specimens was between 45 degrees and 80 degrees, seldom horizontal, and the size of the supratubal recess (or space) increased as the fold angle increased. In 14 ears (27%) the fold had a membrane defect connecting the two spaces. Blockade of the tympanic isthmus caused inflammatory obliteration of the anterior epitympanum when the tensor fold was intact. CONCLUSIONS The anterior epitympanum, a closed space around the anterior half of the head of the malleus, is normally closed by an intact tensor fold, but about one fourth of ears may show membrane defects. Aeration occurs via the tympanic isthmus through a constriction formed by the head of the malleus with the medial attic wall. In surgery for ears with epitympanal pathology, incus transposition should be combined with resection of the thin portion of the tensor fold for safeguarding permanent attic aeration.
Structural variations of the supratubal recess: the anterior epitympanic space. HYPOTHESIS We sought to classify the shape and structure of the anterior epitympanic space (AES) and produce measurements of its dimensions together with its relationships with the facial nerve and geniculate ganglion. BACKGROUND The AES is limited by the middle cranial fossa superiorly, zygoma root anteriorly, cog posteriorly, chorda tympani laterally, facial nerve medially, and tensor tympanic semicanal inferiorly. METHODS The AES was examined in 30 human temporal bones using two different methods. Twenty bones were cut vertically and a modified radical mastoidectomy was performed in the other 10 bones. RESULTS The AES showed two types in the vertically cut bones according to its shape and structure. Type I, found in 17 (85%) of the bones, showed two cavities that were separated by a bony landmark and the tensor tympanic fold. The name "supratubal ridge" is suggested for this bony landmark. In type II, which was seen in three (15%) of the bones, there was only one cavity. In the mastoidectomy group, again two types of AES were found: eight (80%) were type I and two (20%) were found to be type II. If we combine these findings with the vertically cut bones, we find that 25 (83.3%) possess an AES type I, whereas five (16.7%) are type II. CONCLUSIONS These variations in the structure of the AES and its close relationships with a number of vital structures such as the facial nerve, cochlea, and middle fossa dura must be taken into account during the surgical management of middle ear disease.
A new adhesive bonding material for the cementation of implantable devices in otologic surgery. BACKGROUND Presently, there are no U.S. Food and Drug Administration (FDA)-approved adhesive bone cements for the surgical fixation of prosthetic materials in the middle ear. A promising new cement, 4-META/MMA-TBB opaque resin, has shown remarkable adhesive properties as a bone cement in vivo. The cement is composed of 4-methacryloyloxyethyl trimellitate anhydride (4-META) and methyl methacrylate (MMA) as monomers and tri-n-butyl borane (TBB) as an initiator. METHODS An electromagnetic semiimplantable hearing device presently under development was implanted into the middle ear of six cats using 4-META/MMA-TBB resin to cement a titanium-encased magnet to the incus. The animals were subsequently killed (at a mean of 9.6 months) to assess the (temporal bones and specifically the magnet-incus complex in each animal. RESULTS The titanium-encapsulated magnet was firmly adherent to all incuses without any failure of the cement-bone interface. Histopathologic examination of the implanted temporal bones demonstrated lack of middle ear inflammation. Transmission electron microscopy of the incuses demonstrated a unique "hybrid layer" in the bone-side subsurface of the bone-cement interface that elucidates the mechanism of interfacial adhesion. CONCLUSIONS Our investigation highlights the special biomechanical properties as well as the biocompatibility of 4-META/ MMA-TBB resin that make it an attractive bone-bonding agent for use in otologic surgery, including its potential usefulness during ossicular reconstruction.
Effect of magnetic resonance imaging on a new electromagnetic implantable middle ear hearing device. OBJECTIVE A 1.5-T magnetic resonance imager has been shown to be contraindicated for use in patients with pacemakers, cochlear implants, and neurostimulators. Our semi-implantable middle ear device uses a new adhesive bone cement. 4-META/MMA-TBB, for cementation of a 29-mg titanium-encased neodymium-iron-boron (NdFeB) magnet to the incus. METHODS Five NdFeB magnets and four solid titanium cylinders were cemented onto the incus of five preserved human temporal bones and two cadaver heads. They were all inserted into a magnetic resonance imager and evaluated for possible disruption. RESULTS Owing to the magnetic torque, the three magnets on the temporal bone were disrupted from the incus. The two cylinders on the temporal bones and the two cylinders and two magnets on the whole heads were not affected. The magnetic resonance imaging field did not affect the coercive force of the NdFeB magnets. CONCLUSION The large torque produced by a magnetic resonance imager may disrupt the magnet-cement and cement-incus interfaces, causing dislodgement. We postulate that patients with implantable magnets on the incus should not undergo magnetic resonance imaging testing.
Audiological findings of sensorineural deafness associated with a mutation in the mitochondrial DNA. OBJECTIVE The aim of this study was to investigate audiologic features and the lesion site of sensorineural deafness with mitochondrial DNA mutation at position 3243. STUDY DESIGN Case review. SETTING The study was conducted at the Kochi Medical School. PATIENTS A case of sensorineural deafness in a patient who had a mitochondrial DNA mutation was presented. The incidence of deafness and diabetes mellitus (DM) was very high in the patient's family, but she did not have DM. MAIN OUTCOME MEASURES The patient's mitochondrial DNA was examined. Furthermore, the pure-tone audiogram, the Bekesy audiogram, an auditory brain stem response, and the electrocochleogram were analyzed. RESULTS The patient's mitochondrial DNA had a point mutation at codon 3243 (A-->G). The pure-tone audiogram showed moderate sensorineural deafness. An auditory brain stem response showed normal latencies. The electrocochleogram showed an enhanced negative summating potential. CONCLUSIONS It was speculated that the lesion site of the auditory system was the inner ear. The possible sites in the inner ear were hair cells, the stria vascularis, and the endolymphatic sac.
Facial nerve stimulation after Nucleus 22-channel cochlear implantation. OBJECTIVE To review the clinical features, radiographic findings, and programming strategies used in our population of patients who developed facial nerve stimulation after cochlear implantation. STUDY DESIGN AND SETTING Patients referred to our nonprofit, outpatient facility were studied prospectively. PATIENTS The study consisted of 14 patients with facial nerve stimulation after placement of the Nucleus 22-channel cochlear implant. INTERVENTIONS Records were reviewed retrospectively, and patients were studied with three-dimensional computed tomographic scanning techniques. Electrical testing was performed, and various cochlear implant programming strategies were evaluated. MAIN OUTCOME MEASURES Important clinical features were reviewed. The radiographic and anatomical relationships of the facial nerve to the cochlea were evaluated, and the programming strategies used to effectively control facial nerve stimulation were reviewed. RESULTS Prevalence of facial nerve stimulation in our population was 7%. The most common cause was otosclerosis. Anatomical data confirmed the close proximity of the basal turn of the cochlea and the labyrinthine segment of the facial nerve. There was a high correlation between the electrodes causing symptoms and those found radiographically to be closest to the labyrinthine segment of the facial nerve. We were able to control facial nerve stimulation in all patients through programming mode changes. CONCLUSIONS Otosclerosis appears to be a risk factor for developing facial nerve stimulation after cochlear implantation, and the site of stimulation appears to be the labyrinthine segment of the facial nerve. Familiarity with more elaborate programming techniques is critical to managing patients with this complication.
Open-set speech perception in congenitally deaf children using cochlear implants. OBJECTIVE To assess and document the development of open-set speech recognition in congenitally deaf children implanted with the Nucleus multichannel cochlear prosthesis at < 5 years of age. STUDY DESIGN The study group consisted of 38 consecutively chosen children in whom the decision to proceed with implantation had already been made. PATIENTS AND SETTING Congenitally profoundly deaf children were implanted with the Nucleus multichannel cochlear implant at < 5 years of age and followed at NYU Medical Center for a period of 1-5 years. MAIN OUTCOME MEASURES Open-set speech perception was evaluated preoperatively and postoperatively using the following: the Glendonald Auditory Screening Procedure (GASP) word subset, the GASP sentence subtest, Phonetically Balanced Kindergarten monosyllabic word lists, Common Phrases test, Multisyllabic Lexical Neighborhood test, and Lexical Neighborhood test. RESULTS Correlation coefficients were calculated between scores at each interval and age at implantation; one-way analyses of variance were performed independently. Results showed that all subjects had significant open-set speech recognition at the time of the last postoperative evaluation. Thirty-seven of the children use oral language as their sole means of communication. CONCLUSIONS Multichannel cochlear implants provide significant and usable open-set speech perception in congenitally deaf children given implants at < 5 years of age.
Efficacy of antimigrainous therapy in the treatment of migraine-associated dizziness. OBJECTIVE To assess the effects of antimigrainous therapy on migraine-associated dizziness/vertigo. We hypothesized that a medication's ability to ameliorate dizziness/vertigo in this patient population would be directly correlated with its efficacy in improving headache symptoms. STUDY DESIGN Patient survey. SETTING Patients were entered into the study from the University of California, San Diego (UCSD) Headache Clinic, a tertiary care referral clinic. PATIENTS All patients presenting to the UCSD headache clinic are entered into its comprehensive database. Patients who identified dizziness or vertigo as symptoms were entered into this study and were surveyed. MAIN OUTCOME MEASURES Patients were surveyed as to the nature of their vestibular symptoms, and the therapeutic response of these symptoms and their headaches to various antimigrainous medications. Patients were asked to rank therapeutic efficacy utilizing a numeric scale. These results were then subjected to statistical analysis (Spearman rank correlation) to identify any correlation between the efficacies of the medications in improving headache and dizziness/vertigo. RESULTS The efficacy of the medications in treating migraine-associated dizziness was directly correlated with their ability to alleviate headaches. CONCLUSION We conclude that antimigrainous therapy may offer specific treatment to patients suffering from the spectrum of migraine-associated vestibular disorders. This would include the entity known alternately as vestibular Meniere's disease, benign recurrent vertigo, or recurrent vestibulopathy. Given the potential benefits that may be derived from this therapy, clinicians should be sensitive to a history of migraines in patients complaining of dizziness, particularly in those complaining of recurrent episodic vertigo.
Sound-evoked activity in primary afferent neurons of a mammalian vestibular system. HYPOTHESIS Some primary vestibular afferents in the cat respond to sound at moderately intense sound levels. BACKGROUND In fish and amphibians, parts of the vestibular apparatus are involved in audition. The possibility was explored that the vestibular system in mammals is also acoustically responsive. METHODS Microelectrodes were used to record from single afferent fibers in the inferior vestibular nerve of the cat; some acoustically responsive fibers were labeled intracellularly with biocytin. RESULTS Vestibular afferents with regular spontaneous activity were unresponsive to sound, whereas a sizable fraction of vestibular afferents with irregular activity were acoustically responsive. Labeling experiments demonstrated that acoustically responsive afferents innervate the saccule, have cell bodies in Scarpa's ganglion, and project to central regions both inside and outside the traditional boundaries of the vestibular nuclei. Acoustically responsive vestibular afferents responded to sound with shorter latencies than cochlear afferents but had higher thresholds (> 90 dB sound pressure level) and responded only in the range 0.1-3.0 kHz. In contrast to cochlear afferents, efferent stimulation excited background activity and proportionately increased sound-evoked responses in these vestibular afferents, that is, there was centrally mediated enhancement of gain (gain = spike-rate/motion). CONCLUSIONS The evolutionary conservation of a saccular auditory pathway in mammals suggests that it confers survival advantages. Recent evidence suggests that acoustically responsive saccular afferents trigger acoustic reflexes of the sternocleidomastoid muscle, and hence measurement of such reflexes may provide a relatively simple test for saccular dysfunction.
Evaluation of the nonorganic hearing loss suspect. OBJECTIVE To examine the utility of distortion-product otoacoustic emissions (DPOAEs) in the assessment of the nonorganic suspect because DPOAE analysis constitutes an objective test of hair-cell function that yields audiometriclike data. STUDY DESIGN Retrospective study of clinical findings. SETTING Audiology outpatient clinic of our university's medical center. PATIENTS The study cohort comprised 30 patients who presented with a profile of suspicion for nonorganic hearing loss. Most cases were found, with the aid of DPOAE testing, to be nonorganic or to have nonorganic overlays to organic hearing loss. INTERVENTIONS Interventions were diagnostic only. MAIN OUTCOME MEASURES Observed audiometric findings and changes thereof. RESULTS Statistically significant decreases in thresholds occurred in subgroups of those cases deemed to be truly nonorganic in origin. CONCLUSIONS Especially considering test efficiency, the results support the inclusion of DPOAE analysis in the diagnostic management of the suspect-nonorganic patient.
Perspectives on a state enacted hearing screening assessment program in the newborn population. OBJECTIVE A review of the early performance of Ohio's statewide infant hearing screening program was performed to provide insight as to the impact of the current medical and socioeconomic climate on its implementation. BACKGROUND In March 1988, the State of Ohio enacted a law that required universal screening of newborn children for hearing loss through a program known as the Infant Hearing Screening and Assessment Program (IHSAP). The program design consisted of a universally applied high-risk questionnaire followed by a screening auditory assessment for those who fail. Although the value of such a program engendered little early public debate, the institution of such a program represented a significant challenge from a public health perspective. STUDY DESIGN The program performance was analyzed using data from the index population of 160,000 live births per annum and hospital surveys. RESULTS The questionnaires were found to be failing twice the number of newborns as originally projected, whereas completion rates and compliance were excellent. The assessment arm was plagued with poor compliance rates and limited resources. Lack of resources for effective data management has prevented an accurate evaluation of the program's sensitivity and specificity. CONCLUSION IHSAP performance is being hampered by poor assessment follow-up and resource limitations, both in terms of screening equipment and habilitative follow-up services for infants identified as hearing impaired. The reasons for these problems are discussed in relation to existing legislative guidelines and medicoeconomic realities.
Rare lesions of the posterior fossa with initial retrocochlear auditory and vestibular complaints. OBJECTIVE To catalog a series of rare lesions of the posterior fossa that appeared with unusual initial retrocochlear symptoms and signs and to make the reader more aware of these unusual lesions with a view to improving initial assessment and treatment planning. STUDY DESIGN The study was a retrospective case review of seven patients. SETTING Multidisciplinary team evaluation in a tertiary hospital referral center. PATIENTS Patients with unusual lesions of the cerebellopontine angle and posterior fossa with initial retrocochlear symptoms and signs were included. INTERVENTIONS Diagnostic and therapeutic. MAIN OUTCOME MEASURES Hearing preservation and balance function. RESULTS The rare lesions presented include two aneurysms of the anterior inferior cerebellar artery, one giant basilar artery aneurysm, and one each of the following neoplasms: endodermal cyst, choroid plexus papilloma, cavernous angioma, and ependymoma. CONCLUSIONS A close working relationship among the otolaryngologist, neurotologist, neurosurgeon, and neuroradiologist is necessary to accurately evaluate these unusual cerebellopontine angle lesions and effect the best treatment outcome.
Endoscopically assisted prevention of cerebrospinal fluid leak in suboccipital acoustic neuroma surgery. HYPOTHESIS The purpose of this prospective study was to determine if direct inspection of air cells using endoscopy could reduce the occurrence of cerebrospinal fluid (CSF) leak in suboccipital acoustic neuroma surgery. BACKGROUND Cerebrospinal fluid leak remains one of the most common complications after acoustic neuroma surgery. The suboccipital approach for excision of acoustic neuromas has been used increasingly since gadolinium-enhanced magnetic resonance imaging has improved the ability to diagnose smaller tumors. Suboccipital approaches are reported to have CSF leak rates of as high as 27% with an average rate of 12%, most presenting as rhinorrhea. Ideally, this complication could be avoided by careful closure of all air cells exposed during the approach, especially those commonly found in the posterior wall of the internal auditory canal and in the retrosigmoid area. Packing these cells with a variety of materials has been done but often indirectly, as visualization of all cells by the conventional operating microscopes may not be possible. Failure to recognize patent cells because of limited visualization may be an important cause of postoperative CSF leak. METHODS This study compared CSF rhinorrhea rates of 38 consecutive suboccipital acoustic neuroma operations, in which conventional techniques were used to pack the temporal bone defect around the internal auditory canal, with the succeeding 24 consecutive operations, in which endoscopes were used to visualize all exposed air cells directly. After locating all patent air cells endoscopically, they were specifically sealed with bone wax, and then a small fat graft harvested from the wound margin was used to fill the remaining defect. RESULTS Postoperative CSF rhinorrhea occurred in 7 of 38 (18.4%) operations in which no endoscopic technique was used and in 0 of 24 operations in which endoscopes were used. CONCLUSIONS The use of endoscopes to visualize the temporal bone air cells that cannot be directly observed otherwise appears to reduce the incidence of postoperative CSF leak in suboccipital acoustic neuroma surgery.
Uncommon lesions presenting as tumors of the internal auditory canal and cerebellopontine angle. OBJECTIVE The aim of this study was to identify distinguishing characteristics of uncommon lesions of the cerebellopontine angle (CPA) and internal auditory canal (IAC) in order to attain the correct diagnosis and thus formulate an appropriate therapeutic protocol. STUDY DESIGN A retrospective chart analysis was performed on all patients with surgically managed lesions of the IAC and CPA referred to neuropathology from January 1985 to April 1996. SETTING All patients were treated by New York University faculty at a tertiary referral center. PATIENTS Among 426 surgical cases identified, 384 patients (90.1%) with acoustic neuromas and 18 patients (4.2%) with meningiomas were excluded. The remaining 24 cases, involving 17 women and seven men with a median age of 34 years, were analyzed. INTERVENTION Most patients underwent audiovestibular evaluations, as well as magnetic resonance imaging (MRI) and computed tomographic (CT) scanning, and all patients underwent neurotologic surgery as part of their management protocol. MAIN OUTCOME MEASURES Correlating patient presentation, preoperative imaging, and surgical findings often identified distinguishing characteristics of unusual CPA and IAC lesions. RESULTS Unusual lesions identified at the CPA and IAC included: four epidermoids, four lipomas, two facial neuromas, two arachnoid cysts, two choroid plexus papillomas, two metastatic adenocarcinomas, one metastatic neuroblastoma, one ependymoma, one lymphoma, one cholesterol cyst, one angioleiomyoma, one venous hemangioma, one cavernous angioma, and one pontine glioma. CONCLUSIONS Preoperative tumor differentiation based on the patient history, physical examination, audiovestibular testing, CT, and MRI help the surgeon to formulate an appropriate treatment protocol.
Meningiomas intrinsic to the geniculate ganglion. OBJECTIVE To present the clinical, surgical and histopathological manifestations of meningioma intrinsic to the geniculate ganglion. STUDY DESIGN Retrospective study of outcome. SETTING Three private tertiary and one university (otology/neurotology) referral centers. PATIENTS Six patients with cranial nerve VII paresis underwent magnetic resonance imaging and/or high-resolution computed tomography for subsequently histologically proven intrinsic meningioma of the geniculate ganglion. An additional six cases were identified in the literature. Most patients were female and ranged in age from 5 to 40 years. INTERVENTION Total tumor removal via middle fossa and mastoid exposures followed by cable graft VII-VII neuroanastomosis. MAIN OUTCOME MEASURE Meningioma can occur intrinsic to the geniculate ganglion and produces gradual VIIth nerve paresis as its first symptom. Other sites of predilection may occur extrinsically within the temporal bone or along intracranial venous sinuses at sites of arachnoid villi. RESULTS Hearing was maintained in each patient, and postoperative House-Brackmann grade III-V facial nerve function was achieved. CONCLUSIONS Intrinsic meningiomas of the geniculate ganglion rarely occur. However, this entity should be included in the differential diagnosis of a slowly progressive VIIth nerve paresis, especially in young females. Surgical removal and cable graft VII-VII neuroanastomosis is the treatment of choice. Long-term follow-up should be maintained because of the potential for von Recklinghausen's disease.
Vagal nerve monitoring: a comparison of techniques in a canine model. HYPOTHESIS An optimal technique exists for intraoperative, electrophysiologic vagal nerve monitoring. BACKGROUND Analogous to facial nerve monitoring during lateral skull base surgery, vagal nerve monitoring may be used at surgery involving the jugular foramen, the posterior cranial fossa, the infratemporal fossa, the parapharyngeal space, and the thyroid gland to decrease the incidence of iatrogenic injuries. Laryngeal electromyography (EMG) is an accurate test of vagal nerve function: four applications have been described for use intraoperatively. The purpose of this study was to compare the sensitivities of these techniques in a canine model in order to identify the optimal method of intraoperative vagal nerve monitoring. METHODS Four techniques of EMG vagal nerve monitoring were studied in dogs. The thyroarytenoid muscle (TA) was monitored directly in three techniques. Two methods used bipolar hookwire electrodes (L.A. Diagnostics, Los Angeles, CA) inserted in the TA percutaneously through the cricothyroid membrane or via direct laryngoscopy (DL). The third TA monitoring technique involved the use of an EMG endotracheal tube (Xomed-Treace, Jacksonville, FL). The fourth technique used a laryngeal surface EMG electrode (RLN Systems, Jefferson City, MO), laryngoscopically placed in the postcricoid space. After placing each monitoring device, the vagus nerve was identified bilaterally in the neck. The nerves were sequentially stimulated at a constant current of 4.1 Hz with increasing intensity (starting at 0.05 mAmps) to determine the minimum thresholds to stimulate vocal cord contraction. A positive response at the vocal cord was defined as a train of four contractions of > or = 50 mV. The lowest threshold for each technique in each dog was recorded. RESULTS A positive response was obtained in 27 of 32 possible cases using a maximum boundary of 0.5 mAmps for stimulus intensity. Survival analysis was then used to generate Kaplan-Meier survival curves, allowing a comparison of the mean time needed to obtain a response. Log-rank chi statistics showed that the survival curves are inhomogenous (degrees of freedom [df] = 3, chi = 15.58, p < 0.001). The laryngeal surface electrode appears to offer the most sensitive method for vagal nerve monitoring. CONCLUSIONS Four techniques of intraoperative, EMG vagal nerve monitoring were compared in a canine model. The results suggest that EMG recordings can be obtained successfully through a variety of techniques and that the laryngeal surface electrode appears to be the most sensitive technique in the canine model.
Preoperative electroneuronography and facial nerve outcome in acoustic neuroma surgery. OBJECTIVE To determine whether preoperative electroneuronography (ENoG) predicts facial nerve outcome in patients undergoing acoustic neuroma surgery. STUDY DESIGN Prospective, consecutive patients undergoing surgery for acoustic neuroma. SETTING A private tertiary otology and neurotology practice. PATIENTS One hundred consecutive patients presented for surgical removal of an acoustic neuroma between May 1992 and September 1993. The mean patient age was 49 years (range 17-77). Forty-three percent were male and 57% were female. The mean tumor size was 1.77 cm (range 3 mm to 5 cm). The tumors were removed by a translabyrinthine approach in 59% of patients, via the middle fossa in 40%, and retrosigmoid in 1%. The facial nerve was anatomically intact at the conclusion of the operation in all but one patient. INTERVENTION Preoperative ENoG in all patients undergoing surgical removal of their acoustic neuromas. MAIN OUTCOME MEASURES Facial nerve outcome was measured using the House-Brackmann scale immediately after the operation. 5-7 days after surgery, and > 1 year after surgical resection. RESULTS Preoperative ENoG had no predictive value in determining immediate or eventual facial nerve outcome. CONCLUSIONS ENoG has no value in predicting the facial nerve outcome in acoustic neuroma patients. The results of this study are similar to reports with smaller series in the literature. Preoperative ENoG has proven useful, in another study from this institution, in predicting the possible presence of a facial nerve neuroma. This test may be helpful in determining the possible etiology of an intracanalicular mass.
Morpholino and phosphorothioate antisense oligomers compared in cell-free and in-cell systems. Morpholino and phosphorothioate (S-DNA) antisense oligos were compared in both cell-free and in-cell translation systems. In the most stringent test of specificity in the cell-free system, a globin-targeted S-DNA oligo was found to inhibit its target sequence at concentrations of 10 nM and above, but the sequence-specific component of this inhibition dropped below 50% at concentrations of 100 nM and above. A corresponding Morpholino oligo achieved even higher inhibition at 10 nM, but in contrast to the S-DNA, with the Morpholino, the sequence-specific component of this inhibition remained above 93% at a concentration of 3000 nM. In this same cell-free test system, several S-DNA oligos exhibited substantial undesired nonantisense effects at concentrations of 300 nM and above, whereas corresponding Morpholino oligos exhibited little or no nonantisense activity through a concentration of 3000 nM. In scrape-loaded HeLa cells, both globin-targeted and HBV-targeted S-DNAs (both antisense and control oligos) generally failed to achieve significant translational inhibition at extracellular concentrations up to 3000 nM. In contrast, the Morpholino oligos achieved effective and specific translational inhibition at extracellular concentrations ranging from 30 nM to 3000 nM.
A helix 1-extended hairpin ribozyme exhibits altered cleavage behavior in vitro. The catalytic domain of a hairpin ribozyme was incorporated at the 3'-end of a 254-base antisense RNA directed against the RNA of human immunodeficiency virus type 1 (HIV-1), generating a hairpin ribozyme with a largely extended helix 1. In parallel, a catalytic antisense RNA based on a hammerhead ribozyme was directed toward the same cleavage motif in the HIV-1 target. Both ribozymes were expected to create identical cleavage products. Cleavage analysis in vitro confirmed that the hammerhead ribozyme delivered the expected cleavage products. However, the helix 1-extended hairpin ribozyme catalyzed additional RNA cleavage at several unexpected sites, which were mapped. Some of the 3' cleavage products had other nucleotides than G at their 5'-terminus, indicating that the helix 1-extended hairpin ribozyme was able to cleave bonds other than NpG+1. Inspection of the sequence context of the different cleavage sites suggested that unconventional helices 2 in combination with an asymmetric loop A consisting of up to 32 unpaired nucleotides in the substrate strand were formed. A second variant of a helix 1-extended hairpin ribozyme that differed in two nucleotides gave consistent results.
Comparative pharmacokinetics, tissue distribution, and tumor accumulation of phosphorothioate, phosphorodithioate, and methylphosphonate oligonucleotides in nude mice. The goals of this study were to systematically compare the pharmacokinetics and tissue distribution of phosphorothioate (PS), methylphosphonate (MP), and phosphorodithioate (PS2) oligonucleotide analogs; 15-mers of sequence d-TAC GCC AAC AGC TCC (5'-3') complementary to the AUG region of K-ras were radiolabeled with carbon-14. Oligomers were administered as a single dose in the tail vein of nude mice harboring a K-ras-dependent human pancreatic tumor (CFPAC1). The kinetics of PS, PS2, and MP oligomer availability in the bloodstream was followed. Concentration versus time profiles for all oligomers were biphasic, indicative of a two-compartment model. A rapid distribution phase with t1/2 alpha values of 1 minute or less and an elimination phase with average t1/2 beta values of 24-35 minutes were observed. Volumes of distribution (Vd) were 3.2, 4.8, and 6.3 ml for PS2, MP, and PS, respectively, in comparison to 3.6 ml for sucrose, a fluid-phase marker. Relative tissue drug levels obtained at 1 and 24 hours after administration were kidney > liver > spleen > tumor > muscle. Total kidney and liver oligonucleotide accumulation was approximately 7%-15% of the initial dose, with tumor accumulating 2%-3%. Intact compound was recovered from all tissues, including tumor, as assessed by high-pressure reversed-phase HPLC coupled to radiometric detection. Integrity of the oligonucleotides ranged from 73% in blood to 43%-46% in kidney and liver. Kidney and liver appear to be the primary sites of metabolism. These results demonstrate widespread tissue availability of these compounds and suggest their development as potential antitumor agents.
Characterization and evaluation of NGF antisense oligonucleotides: inhibition of NGF synthesis in transfected COS cells. We present a system for the assessment of the inhibiting capacity of antisense oligonucleotides. The aim of this study was to identify an oligonucleotide that can inhibit chicken nerve growth factor (NGF) synthesis. Five antisense chicken NGF phosphorothioate oligonucleotides, AS1-5, were designed and were tested for their capacity to inhibit NGF expression in COS cells. COS cells that transiently expressed chicken NGF were treated with the oligonucleotides, and NGF expression was analyzed using a bioassay and Western blotting for NGF protein. Two oligonucleotides, AS 1 and AS 5, were more capable than the others of inhibiting expression compared with nonsense oligonucleotide, and they targeted the translational initiation and stop sites. The chicken NGF is expressed at a high level from an adenovirus major late promoter, and AS 1 was capable of inhibiting more than 80% of the NGF expression as determined using the bioassay and Western blotting. Expression of another member of the NGF gene family, neurotrophin-4, was not affected by treatment of the antisense oligonucleotides. A 10-fold lower concentration of the AS 1 oligonucleotide could be used to inhibit NGF synthesis if the cellular uptake was facilitated using lipofectin compared with addition of oligonucleotide directly to the culture medium. The amount of oligonucleotide taken up by the cells was similar in the lipofectin-treated cells as in the cells treated by a 10-fold higher concentration of medium-supplemented nucleotide. This system based on COS cells can facilitate evaluation of the capacity of inhibiting antisense oligonucleotides, particularly targeting those genes in which endogenous products are present in low levels and are difficult to analyze.
Effects of S-dC28 on vascular smooth muscle cell adhesion and plasminogen activator production. We sought to examine phosphorothioate oligodeoxynucleotide (PS oligo) non-G-quartet, nonsequence specific effects on smooth muscle cell (SMC) adhesion by using S-dC28, a 28-mer cytidine homopolymer that lacks contiguous guanosine residues. Human aortic SMC were incubated with vehicle or various doses of S-dC28, and the number of SMC adhered to noncoated plates was determined using a Coulter Counter. S-dC28 significantly inhibited SMC adhesion. SMC adhesion dramatically improved with S-dC28 in fibronectin-coated plates. When laminin-coated plates were used, the inhibition of SMC adhesion by S-dC28 was completely reversed. Replacement of serum-free medium with 5% fetal bovine serum medium for SMC cultured on non-coated plates also greatly attenuated inhibition of adhesion by S-dC28. Human SMC TPA, UPA, and PAI-1 antigen levels were determined by ELISAs. PDGF significantly induced SMC TPA antigen production measured by an ELISA. Coincubation of PDGF with S-dC28 significantly attenuated SMC TPA antigen levels. SMC UPA antigen levels after coincubation with PDGF and S-dC28 were significantly greater than the values observed in SMC incubated in medium containing PDGF alone. SMC PAI-1 antigen levels were not altered by the addition of S-dC28. We conclude that S-dC28 inhibits human SMC adhesion and that this inhibition can be diminished by fibronectin or laminin coating of culture plates or by the presence of serum in the culture medium. Furthermore, S-dC28 attenuates SMC TPA production, thereby inhibiting SMC migration, whereas S-dC28 augments SMC UPA production, thus diminishing SMC cellular adhesion.
Antisense oligonucleotides to p53 tumor suppressor suppress the induction of apoptosis by epidermal growth factor in NCI-H 596 human lung cancer cells. Apoptosis has become a basic tool in developing cancer research and establishing new cancer strategies. However, the molecular mechanism of apoptosis is not well understood. Recently, the authors found that epidermal growth factor (EGF) induces apoptosis in various cancer cells and that there is a novel signal pathway mediated through p53 in signal transduction of EGF. The effect of antisense gene therapy to p53 tumor suppressor on EGF-dependent apoptosis was investigated in cultured NCI-H 596 human non-small cell lung cancer cells with a wild-type p53 gene. Results showed that EGF plus p53 sense oligonucleotides induced EGF-dependent and p53-dependent apoptosis in NCI-H 596 cells within 8 hours. On the other hand, antisense gene therapy using antisense oligonucleotides to p53 tumor suppressor suppressed the induction of EGF-dependent and p53-dependent apoptosis. Mutated p53 antisense-containing mutated CG dinucleotides had the same effect as that of p53 antisense on suppression of apoptosis in NCI-H 596 cells. We found that a new nucleic acid drug, another mutated p53 antisense-containing mutation at three bases immediately 5' and 3' from the CG dinucleotides, potentiated the induction of apoptosis and failed to suppress the induction of EGF-dependent apoptosis. These results suggest that gene therapy using antisense oligonucleotides to the p53 tumor suppressor is effective on EGF-dependent apoptosis of NCI-H 596 human non-small cell lung cancer.
Identification of an oligodeoxynucleotide sequence motif that specifically inhibits phosphorylation by protein tyrosine kinases. Protein tyrosine kinases (PTKs) have central roles in cellular signal transduction. We have identified a sequence motif (CGT[C]GA) in phosphorothioate-modified oligodeoxynucleotides (ODNs) that specifically inhibits the enzymatic activity of recombinant or immunoprecipitated PTK in vitro. Hexamer ODNs containing this motif block both substrate and autophosphorylation of at least four different PTKs but have no apparent effect on the enzymatic activity of a serine/threonine protein kinase. These data suggest possible new applications for ODNs and have implications for the design and interpretation of experiments using antisense or triplex ODNs.
Triplex-forming oligonucleotides with unexpected affinity for a nontargeted GA repeat sequence. We examined the affinity and the specificity of triplex formation for different purine ODNs directed against two portions of a purine sequence derived from the mouse fli-1 gene. As expected, the ODNs antiparallel to the purine strand of their target can form triplex DNA. One parallel ODN showed binding to its target sequence. We explain this unusual binding by an interaction of the ODN with a GA repetition present in the sequence. We further examined the interaction of this ODN with a target composed of 14 GA repetitions. Unexpectedly, one ODN shows higher affinity for a partially complementary GA target relative to its completely complementary target. For another ODN, the binding to the GA target is weaker and might involve skipping of bases in a way that resembles alternate strand triplex formation.
PCR-amplified cDNA probes for verification of differentially expressed genes. Differential display has proven to be a powerful technique for the detection and isolation of differentially expressed genes. By generating reproducible cDNA expression patterns, it is possible to compare gene expression by two or more cell types, developmental stages or tissues and to isolate as yet unknown differentially expressed genes. A sensitive method is necessary to verify the differential expression of the isolated cDNAs. Here we describe the use of adaptor-ligated. PCR-amplified total cDNA of the two cell types compared as a probe for Southern hybridizations with the isolated cDNAs.
Agglutination-inhibition assay for the detection of recombinant proteins tagged with peptide epitopes. We have demonstrated that the expression of recombinant proteins labeled with an immunoreactive epitope can be rapidly assessed and quantitated using a modified haemagglutination inhibition assay in enzyme-linked immunosorbent assay (ELISA) trays. The agglutination of erythrocytes from a droplet of whole blood provided a simple visual assay. The additional reagents required for the assay were a recombinant anti-human erythrocyte Fab fragment fused to a peptide epitope and a bivalent antibody with specificity to the same epitope. In this report, we found that a convenient and sensitive epitope was the octapeptide FLAG in conjunction with the M2 anti-FLAG antibody, which had affinity to FLAG incorporated either at the C-terminus or N-terminus of the recombinant protein. The agglutination-inhibition (AI) assay was configured to detect as little as 1 mg/L of soluble recombinant protein in a 30-min assay. Since the AI assay was substantially more rapid and convenient than dot-blot or Western blot analyses, our laboratory now uses this method routinely for the assay of FLAG-labeled recombinant products following protein expression and subsequent small- and large-scale purification procedures.
Transient selection during vaccinia virus recombination with insertion vectors without selectable markers. Isolation of recombinant viruses after DNA transfection remains the most time-consuming and labor-intensive feature in the production of vaccinia virus (VV) recombinants. This procedure has been aided by the design and incorporation of both positive and negative selectable markers into the insertion vectors. Use of these selectable markers is not always feasible or desirable. In the absence of selectable markers, individual plaque characterization is required. A method of transient selection during transfection and recombination is described that does not require that any selectable marker be present in the insertion vector sequence. This method relies on the co-transfection of a guanine phosphoribosyl transferase (gpt) selectable marker on an unlinked plasmid into VV-infected cells pretreated for gpt selection. Following recombination, the output virus can be plated on cells without any further selection and screened for recombinant virus. While the total recombinant virus population is slightly reduced, the total progeny virus is reduced even further, resulting in more than sevenfold enhancement in the frequency of recombinant virus, which allows for more efficient identification.
Cloning DNA fragments between two adjacent/overlapping restriction sites using a "positive stuffer". Here we describe a solution to a common problem encountered in recombinant DNA cloning when directional cloning of a DNA fragment into a predetermined plasmid requires the use of restriction enzymes with adjacent or overlapping recognition sites. In preparing the double-digested plasmid, only one enzyme will often cut, whereas the second will not because of the lack of a sufficiently long stretch of double-stranded DNA at its recognition site. The problem can be solved by construction of a "user-friendly" intermediary plasmid in which the desired restriction sites are separated by a positively selectable stuffer with resistance to neomycin. This approach is particularly useful in cases where the choices of restriction sites are severely limited, for example, when it is necessary to clone an additional piece of DNA into a complex vector already containing multiple gene cassettes.
Making hybrids of two-hybrid systems. Two-hybrid systems are powerful tools to find new partners for a protein of interest. However, exchange of material between two-hybrid users has been handicapped by the various versions of two-hybrid systems available and by the widely accepted idea that they are not compatible. In the present paper we show that, contrary to the dogma, the most often used two-hybrid systems may be combined by either transformation or mating assays. The protocol to be followed in each case is provided. This will greatly increase the prospects of the growing network of interacting proteins, by reconciling the "two-hybrid systems" and the "interaction trap".
Adaptation of a beta-1,3-glucanase assay to microplate format. A method is described for performance of a beta-1,3-glucanase assay in microplates. The adaptation to microassay format has been made possible through the availability of heat-stable microplates. Assay samples containing beta-1,3-glucanase (cell extracts) and the substrate are mixed in 96-well, autoclavable microplates. Incubation of the enzyme-substrate mixture results in the release of reducing sugars by the action of beta-1,3-glucanase. The levels of these sugars are colorimetrically quantified through the addition of "copper reagent" and neocuproine, incubation at 100 degrees C for 10 min and the resulting reduction of Cu+2 to Cu+. A standard curve of glucose concentrations within the same plate allows for assessment of internal variance. Twenty samples can be assayed in one microplate in 1 h, compared to the 96 test tubes and 4 h needed for traditional assay methods. The net savings in reagents used with the microplate format is substantial. The measurement of optical density is performed rapidly for all of the samples, eliminating the problem of oxidization of Cu+ to Cu+2 during quantification. This method represents a significant savings in time, chemicals and cost.
Association of enzyme inhibition with methods of museum skin preparation. Enzyme inhibition is commonly encountered when using molecular biological techniques on museum-prepared animal skin samples, and this problem is exacerbated by a lack of information on how particular skins have been prepared for preservation. This report: (i) demonstrates that while some methods of museum preparation inhibit both proteinase K digestion and the PCR, others do not; (ii) describes a change in buffer conditions that reduces proteinase K enzyme inhibition during tissue digestion: and (iii) uses electron-dispersive X-ray microanalysis (EDXA) to show that the preparation methods for museum-preserved skin are often more complex than the treatment description provided with samples and also suggests that some of these descriptions are incorrect.
Detection of mycoplasma infection of mammalian cells. Mycoplasma infection was detected in cultures of COS cells with a novel, simple assay that detects the conversion of arginine to citrulline by the enzyme, arginine deiminase, specific to all species of mycoplasma. Transfection of COS cells was inhibited in mycoplasma-infected cells, a phenomenon that was readily reversed by treatment with a mycoplasma removal agent. Cultures of cells used for transfection should be regularly monitored for evidence of mycoplasma by assay of arginine deiminase activity or by other methods.
An iterative and regenerative method for DNA sequencing. This paper presents, to our knowledge, the first iterative DNA sequencing method that regenerates the product of interest during each iterative cycle, allowing it to overcome the critical obstacles that impede alternative iterative approaches to DNA sequencing: loss of product and the accumulation of background signal due to incomplete reactions. It can sequence numerous double-stranded (ds) DNA segments in parallel without gel resolution of DNA fragments and can sequence DNA that is almost entirely double-stranded, preventing the secondary structures that impede sequencing by hybridization. This method uses ligation of an adaptor containing the recognition domain for a class-IIS restriction endonuclease and digestion with a class-IIS restriction endonuclease that recognizes the adaptor's recognition domain. This generates a set of DNA templates that are each composed of a short overhang positioned at a fixed interval with respect to one end of the original dsDNA fragment. Adaptor ligation also appends a unique sequence during each iterative cycle, so that the polymerase chain reaction can be used to regenerate the desired template-precursor before class-IIS restriction endonuclease digestion. Following class-IIS restriction endonuclease digestion, sequencing of a nucleotide in each overhang occurs by template-directed ligation during adaptor ligation or through a separate template-directed polymerization step with labeled ddNTPs. DNA sequencing occurs in strides determined by the number of nucleotides separating the recognition and cleavage domains for the class-IIS restriction endonuclease encoded in the ligated adaptor, maximizing the span of DNA sequenced for a given number of iterative cycles. This method allows the concurrent sequencing of numerous dsDNA segments in a microplate format, and in the future it can be adapted to biochip format.
Alternative system for detection and mapping of activation domains. The ZEBRA protein is a transcriptional activator that induces expression of viral lytic genes in cells harboring latent Epstein-Barr virus (EBV). In this report it is shown that a derivative of ZEBRA that cannot activate transcription (Zd) can be used to detect and characterize activation domains. Three expression vectors that allow the fusion of putative activation regions in any reading frame were constructed using Zd. These vectors were used to demonstrate the activity of different classes of activation domains using a chloramphenicol acetyltransferase (cat) reporter gene construct containing seven ZEBRA response elements (Z7). The Zd/Z7 system effectively detected proline-rich, glutamine-rich and acidic activation domains in a variety of cell lines and cell types. Using a bioassay unique to the EBV Zd/Z7 system, fusion constructs can also be tested for the ability to activate gene expression directly from a chromatin structure, the EBV genome. These studies indicate that the Zd/Z7 system is an alternative to GAL4 and can be a useful tool for identifying heterologous activation domains.
Optimization of cellular ELISA for assay of surface antigens on human synoviocytes. The cellular enzyme-linked immunosorbent assay (CELISA) permits assay of cell surface antigens on intact fixed cells. Using a monolayer of cells as the solid phase, the CELISA offers an inexpensive alternative to flow cytometry. In addition, this protocol has the decided advantages of miniaturization (small numbers of cells) and ease of replication (the 96-well cluster plate format). In efforts to optimize CELISA for detecting surface antigens on human fibroblastlike synoviocytes, the authors found that cell number, serum proteins, choice of culture plate, pipetting technique and fixatives may all impact the results of the CELISA.
Microplate assay for measurement of histamine release from mast cells. A microplate-format assay for measurement of histamine release by activated rat peritoneal mast cells, with accompanying microplate-format spectrofluorometric assay of histamine, is demonstrated. Reproducibility and linearity of the histamine assay are comparable to the historical large format assay. Transfer of samples in the 96-well format, from histamine release through spectrofluorometric assay, greatly expedites handling and provides for better independence of each replicate. A sample experiment is included to show a typical size experiment easily done in this format.
Simultaneous detection of multiple point mutations using fluorescence-coupled competitive primer extension. We report the development of a method for the simultaneous genotyping of several distinct nucleotide positions by means of fluorescence-coupled competitive primer extension. We demonstrate the application of this method for the simultaneous detection of three point mutations in the human mitochondrial genome, at nucleotide positions 3460, 11778 and 14484, which account for about 90% of cases with Leber's hereditary optic neuropathy. mtDNA fragments encompassing these nucleotide positions are initially amplified in a multiplex PCR assay. Genotyping is then carried out by a simultaneous primer extension assay using wild-type-specific (FAM-labeled) and mutant-specific (JOE-labeled) oligonucleotides. Primer extension products are separated on a 6% polyacrylamide/8 M urea gel on a fluorescence DNA sequencer. Patients' genotypes can be derived from the peak color of the different-sized extension products. As little as 10% mutant DNA can be detected in heteroplasmic mixtures of wild-type and mutant mtDNA, a degree that is sufficient for routine clinical practice.
Collagen-coated Ba(2+)-alginate microcarriers for the culture of anchorage-dependent mammalian cells. Several types of microcarriers suitable for large-scale cultivation of mammalian cells are commercially available. However, many of these carriers have disadvantages, e.g., the need for enzymatic digestion for cell harvesting, size limitations and insufficient biocompatibility. These limitations have been overcome by the development of collagen-coated Ba(2+)-alginate microcarriers. Ba(2+)-alginate microspheres, made with the air-jet droplet generator technique, were collagen-coated by incubation in a 0.5% collagen solution, with subsequent gelling of the collagen layer around the alginate microspheres. Human chang liver (CCL-13) and mouse fibroblast (L929) cell lines were cultivated in stationary, unstirred cultures as model systems. After a lag phase of nearly 24 h, the cells grew rapidly on these microcarriers and reached confluence after 3 days. The microcarrier cultures were stable for an additional 4-9 days and longer. Cells were harvested either by trypsinization or by dissolution of the alginate matrix using 5 mM EDTA. The main advantages of this new microcarrier system are that the preparation procedure is easy and can be accomplished on demand with standard laboratory equipment.
Agarose-based system for separation of short tandem repeat loci. Short tandem repeats (STRs) are traditionally analyzed on large polyacrylamide electrophoresis gels. We demonstrate in this study that a small (10-cm-long, 1-mm-thick) agarose gel is sufficient for analysis of multiplexed samples for several commonly used STR loci. A system was developed using a high-resolution agarose, MetaPhor. Within an hour of electrophoresis, sufficient resolution was obtained to allow discrimination of triple-multiplexed STR loci. We show that this agarose is capable of resolving di- as well as tetranucleotide ladders. Using PCR conditions similar to those routinely used with sensitive detection systems, we found that direct staining of gels with SYBR Green I stain was comparable with silver staining, autoradiography or fluorescently tagged primers for sample detection and was considerably easier. This procedure significantly simplifies STR analysis and is much faster than many standard protocols.
The PerFect lipid optimizer kit for maximizing lipid-mediated transfection of eukaryotic cells. The transfection efficiencies of a panel of eight uniquely different lipid reagents has been evaluated with two other commercially available lipids for use in transfecting a diversity of eukaryotic cell lines. The PerFect lipids are available individually or together in an optimization panel format that can be tested in any given cell line, enabling one to evaluate the optimal lipid for transfecting each individual cell line. Our results demonstrate that no single lipid is optimal for plasmid transfection over a broad range of cell types, thus emphasizing the need for multiple unique lipid reagents and a simple format for testing their transfection efficiency on a given cell type.
Risk of breast cancer associated with atypical hyperplasia of lobular and ductal types. Epidemiological studies using the histological classification of Page for benign breast disease consistently demonstrate a positive association between atypical hyperplasia and the subsequent development of breast cancer. However, atypical hyperplasia is of either lobular or ductal types, and breast cancer risk in relation to type of atypical hyperplasia has not been studied extensively. Thus, we investigated prospectively the risk of breast cancer associated with histological subtypes of benign proliferative breast disease, including the types of atypical hyperplasia, among participants in the Nurses' Health Study who had biopsy-confirmed benign breast disease. Women who subsequently developed breast cancer were matched by year of birth and year of biopsy to participants who were free from breast cancer. Benign biopsy slides were classified according to the criteria of Page. Odds ratios (ORs) of breast cancer and 95% confidence intervals (CIs), adjusted for the matching variables and other breast cancer risk factors, were computed using unconditional logistic regression with benign nonproliferative breast disease as the referent group. Atypical ductal hyperplasia (OR = 2.4; 95% CI, 1.3-4.5) or atypical lobular hyperplasia (OR = 5.3; 95% CI, 2.7-10.4) in a prior biopsy were associated with increased breast cancer risk. Atypical lobular hyperplasia was more strongly associated with the risk of premenopausal breast cancer (OR = 9.6; 95% CI, 3.3-27.8) than with the risk of postmenopausal breast cancer (OR = 3.7; 95% CI, 1.3-10.2). The association of atypical ductal hyperplasia and breast cancer risk varied little by menopausal status. The magnitude of breast cancer risk seems to vary according to the type of atypical hyperplasia present at biopsy.
Human erythrocyte glucose transporter (Glut1) is immunohistochemically detected as a late event during malignant progression in Barrett's metaplasia. We have previously demonstrated that the human erythrocyte glucose transporter (Glut1) is expressed in adenocarcinoma arising in Barrett's metaplasia (BM). We have also shown that Glut1 is expressed as a late event during colorectal carcinogenesis. The aim of this work was to determine the chronology of Glut1 expression during the neoplastic progression in Barrett's metaplasia. Formalin-fixed, paraffin-embedded tissue sections of 251 biopsies from 97 patients with BM were immunostained with the anti-Glut1 antibody MYM, after microwave-aided antigen retrieval, using the standard avidin-biotin complex immunoperoxidase technique. Dysplasia was graded as negative (ND), low grade (LGD)/indefinite or high grade (HGD). None of the 181 biopsies with ND (0%) or 51 biopsies with LGD (0%) showed Glut1 immunoreactivity. More importantly, although 0 of 6 biopsies with HGD (0%) expressed Glut1, 9 of 13 biopsies with adenocarcinoma (69%) were Glut1 positive (P = 0.0108, Fisher's exact test). Our results indicate that Glut1 is expressed as a late event during the neoplastic progression in BM. Prospective studies are needed to determine the clinical utility of Glut1 immunoreactivity as a marker of carcinoma in patients with BM.
Hierarchical modeling of gene-environment interactions: estimating NAT2 genotype-specific dietary effects on adenomatous polyps. Data sparseness currently limits gene-environment interaction estimation. To improve effect estimates of gene-environment interactions, we give an overview of one approach, hierarchical modeling, and propose a two-stage hierarchical model. The first stage is a logistic model for the joint effects of the genetic and environmental factors. The second stage regresses the joint effects on genotype-specific enzymatic activity of the environmentally derived substrate. The model is illustrated using a case-control study of adenomas of the large bowel, for which NAT2 genotype and dietary data were collected. The first-stage interactions of dietary components and genotype were regressed on initial conversion rates of dietary heterocyclic amines to aryl nitrenium ions. We fit the hierarchical model by penalized likelihood. Compared to effect estimates from maximum-likelihood logistic regression, hierarchical results are more reasonable and precise. These results lend further support to previous observations that hierarchical regression is preferable to ordinary logistic regression when multiple factors and their interactions are being studied. We propose that hierarchical modeling can act as a bridge between molecular epidemiology studies and laboratory data, combining both efficiently.