<clinical_study>
  <!-- This xml conforms to an XML Schema at:
    https://clinicaltrials.gov/ct2/html/images/info/public.xsd -->
  <required_header>
    <download_date>ClinicalTrials.gov processed this data on September 20, 2023</download_date>
    <link_text>Link to the current ClinicalTrials.gov record.</link_text>
    <url>https://clinicaltrials.gov/ct2/show/NCT04404335</url>
  </required_header>
  <id_info>
    <org_study_id>LL-37PERIOURJC</org_study_id>
    <nct_id>NCT04404335</nct_id>
  </id_info>
  <brief_title>The Role of Anti-inflammatory Cytokines and Antimicrobial Peptide LL-37 Biomarkers in the Treatment of Periodontal Disease.</brief_title>
  <official_title>The Role of Anti-inflammatory Cytokines and Antimicrobial Peptide LL-37 Biomarkers in the Treatment of Periodontal Disease.</official_title>
  <sponsors>
    <lead_sponsor>
      <agency>Universidad Rey Juan Carlos</agency>
      <agency_class>Other</agency_class>
    </lead_sponsor>
  </sponsors>
  <source>Universidad Rey Juan Carlos</source>
  <oversight_info>
    <has_dmc>No</has_dmc>
    <is_fda_regulated_drug>No</is_fda_regulated_drug>
    <is_fda_regulated_device>No</is_fda_regulated_device>
  </oversight_info>
  <brief_summary>
    <textblock>
      Periodontal disease is an infectious pathology that consists of the destruction of the gums&#xD;
      and supporting structures around the teeth. There are several mechanisms involved in this&#xD;
      process. In healthy conditions there is a balance between the bacteria present in the gums&#xD;
      and the defence mechanisms of the host to fight the disease. One of the substances involved&#xD;
      in favouring this protective mechanism is a peptide called LL-37.&#xD;
&#xD;
      The aim of this study is to find out more about the rol of this peptide during this process&#xD;
      by using a non invasive method of analysis consisting of analysing some samples of the fluid&#xD;
      existing between the gums and the teeth.&#xD;
&#xD;
      This clinical study will take place at the University Clinic at the University Rey Juan&#xD;
      Carlos, Alcorcon (Madrid), Spain. Patients that attend the clinic suffering from gum disease&#xD;
      will be offered the possibility to participate in this study. A full questionnaire indicating&#xD;
      the risks and benefits of participating in the study as well as the management of&#xD;
      confidential personal data will be discussed with the participants in full detail. Full&#xD;
      consent will be sought before commencing the study.&#xD;
&#xD;
      Firstly, all participants will receive a through periodontal (gum) examination to assess if&#xD;
      they suffer from periodontal disease and to what extent (severity). Based on this preliminary&#xD;
      data, patients will be classified in different categories.&#xD;
&#xD;
      Patients will be divided in two groups:&#xD;
&#xD;
        -  30 Healthy patients (no signs of periodontitis)&#xD;
&#xD;
        -  30 Periodontal patients (subdivided in 2 groups of 30, depending of the severity of&#xD;
           their disease:&#xD;
&#xD;
             -  15 patients with Stage I-II periodontitis (mild/moderate)&#xD;
&#xD;
             -  15 patients with Stage III-IV periodontitis (severe).&#xD;
&#xD;
      The intervention will consist on taking samples of crevicular fluid (the fluid found in the&#xD;
      space between the gum and the root of the tooth) to quantify the presence of LL-37 as well as&#xD;
      IL-4, IL-6, IL-10, which seems to be associated with the defence mechanisms (immunity) of the&#xD;
      host against periodontal disease. These samples will be analysed in the laboratory by using&#xD;
      some specific procedure called ELISA.&#xD;
&#xD;
      All patients from the test group will receive a basic course of periodontal treatment (root&#xD;
      scale and polish), which is intended to remove the bacteria causing the disease from the&#xD;
      space between the gums and the roots (periodontal pocket). Samples of crevicular fluid will&#xD;
      be obtained before commencing the treatment. After 4-6 weeks, a new periodontal examination&#xD;
      will take place to assess if there is an improvement of the condition and also to quantify&#xD;
      the levels of LL-37, IL-4, IL-6, IL-10.&#xD;
&#xD;
      The findings of this study will help to understand the role of this peptide in the defence&#xD;
      mechanism of the host against periodontal disease. This could ultimately serve to develop new&#xD;
      therapies that could include the use of this peptide in addition to the routine periodontal&#xD;
      treatment prescribed to improve the healing conditions of the patient against periodontal&#xD;
      disease.&#xD;
    </textblock>
  </brief_summary>
  <detailed_description>
    <textblock>
      'The role of anti-inflammatory cytokines and antimicrobial peptide LL-37 biomarkers in the&#xD;
      treatment of periodontal disease.&#xD;
&#xD;
        1. Introduction&#xD;
&#xD;
           Periodontal disease is an infectious process that consists of the destruction of the&#xD;
           supporting anatomical structures of the tooth, as a consequence of the inflammatory&#xD;
           response to the periopathogenic activity of the subgingival bacterial plaque, which&#xD;
           leads to an innate, inflammatory and adaptative immune response.&#xD;
&#xD;
           The clinical manifestation of the disease depends of host and environmental factors as&#xD;
           well as the specific microbiological agent involved. The interrelation amongst bacteria&#xD;
           and the immunodefence mechanisms of the host constitute the base of the&#xD;
           immunopathological mechanisms that lead to the disease. In healthy conditions, there is&#xD;
           a balance between the activity of the periodontal microbiota and the host immune&#xD;
           response.&#xD;
&#xD;
           In the innate defence system of the oral cavity there are several antimicrobial peptides&#xD;
           involved, being one of them, the human cathelicidin LL-37.&#xD;
&#xD;
           The activity of antimicrobial peptides such as LL-37 in the gingival sulcus is&#xD;
           considered one of the greatest defence mechanisms of the periodontum. The gingival&#xD;
           epithelium not only constitutes a physical barrier to avoid bacterial invasion.&#xD;
           Neutrophils migrate to the junctional epithelium and create a barrier between the&#xD;
           bacterial plaque and the underlaying epithelium. They also produce antimicrobial&#xD;
           peptides, one of them the 18 kDa (hCAP18). Once the neutrophils are activated in&#xD;
           response to the bacterial aggression the release LL-37, with a similar activity to a&#xD;
           broad-spectrum antibiotic.&#xD;
&#xD;
           The total absence of this peptide (LL-37) has been associated to aggressive&#xD;
           periodontitis, in particular in patients with a neutrophil deficiency such as those who&#xD;
           suffer Morfan Kosmann syndrome&#xD;
&#xD;
           The nature of this research aims to elucidate the role of LL-37 in periodontal disease&#xD;
           by comparing the use of a none invasive technique such as crevicular fluid samples in&#xD;
           patients that suffer periodontal disease, before and after receiving a course of basic&#xD;
           periodontal treatment (root scaling and planing). In additionto the above, pro and&#xD;
           antiinflammatory cytokines (IL-6, IL-4, IL-10 in particular) will also be evaluated&#xD;
           before and after periodontal treatment. Samples obtained will be analysed by&#xD;
           immunohystochemical analysis (ELISA). The quantification of LL-37 before and after&#xD;
           periodontal treatment associated with the clinical findings observed may serve as&#xD;
           diagnostic biomarker of the immunopathological response inherent to the periodontal&#xD;
           disease.&#xD;
&#xD;
        2. Justification of research question. Current status&#xD;
&#xD;
           Over the last years, a great effort by scientific community has been made in order to&#xD;
           try to understand the complex immunopathological processes related to periodontal&#xD;
           disease and the relationship between the activity of the periodontal microbiota and the&#xD;
           host defence mechanisms.&#xD;
&#xD;
           The relevance of this research may serve to understand in more detail the role of the&#xD;
           antimicrobial peptide LL-37 in periodontal disease not only due to their bactericidal&#xD;
           effect but in the modulation of the immunological response of the host and its activity&#xD;
           before and after periodontal treatment. The lack of studies on this matter reflects the&#xD;
           need for further research on this topic.&#xD;
&#xD;
           Ultimately, the findings of this research may form the basis of a new therapeutic&#xD;
           approach based on the use of LL-37 to help modulate the immunological response of the&#xD;
           host in combination with conventional periodontal treatment. This may be particularly&#xD;
           relevant in patients susceptible to develop periodontal or periimplant diseases or in&#xD;
           particular cases that are resistant to conventional treatment alternatives.&#xD;
&#xD;
        3. Summary of intended research&#xD;
&#xD;
      The main aim of the study is to investigate the role of LL-37 in the immunopathological&#xD;
      response of the periodontum. Subsequent aims are to investigate the possibility of using this&#xD;
      microbial peptide to serve as diagnostic, prognostic as well as a therapeutic agent in the&#xD;
      treatment of periodontal disease.&#xD;
&#xD;
      Research hypothesis: There are significant quantitative differences in the levels of LL-37&#xD;
      before and after periodontal treatment, with a significant reduction of LL-37 at&#xD;
      re-evaluation (4-6 weeks after a course of basic periodontal treatment -root scaling and&#xD;
      planing).&#xD;
&#xD;
      Null hypothesis: There are no significant quantitative differences in the levels of LL-37&#xD;
      before and after periodontal treatment&#xD;
&#xD;
      The objectives of the present study are:&#xD;
&#xD;
        1. Determine if healthy patients and patients with periodontal disease have different&#xD;
           levels of LL-37, IL-6, IL-4 and IL-10 in crevicular fluid at baseline and related them&#xD;
           to the stage/severity of periodontal disease.&#xD;
&#xD;
        2. Evaluate if after a course of basic periodontal treatment these levels of LL-37, IL-6,&#xD;
           IL-4 and IL-10 at baseline are similar to the quantified levels on healthy patients at&#xD;
           baseline.&#xD;
&#xD;
        3. With respect to crevicular fluid samples, evaluate if levels of LL-37, IL-6, IL-4 and&#xD;
           IL-10 in teeth affected by periodontal disease differ from healthy sites on the same&#xD;
           individual or if there are no significant differences in the above before and after a&#xD;
           course of periodontal treatment.&#xD;
&#xD;
        4. Stablish a non-invasive protocol to evaluate the immunological state of the patient&#xD;
           before and after periodontal treatment as prognostic factor.&#xD;
&#xD;
      4. Material and methods&#xD;
&#xD;
      Ethical approval from the Ethical Commite of University Rey Juan Carlos of Madrid (URJC) has&#xD;
      been granted. A selection of patients of the &quot;Master in Advanced Implantology, Tissue&#xD;
      Regeneration and Implant supported prosthesis&quot; of the FCU-URJC that fulfil the inclusion&#xD;
      criteria and that consent to be part of the study will be made.&#xD;
&#xD;
      The criteria for entry were as follows: aged 18 or over, with no previous history of&#xD;
      periodontal treatment in the last six months, and no medical or dental history that could&#xD;
      contraindicate periodontal treatment. Exclusion criteria included current pregnancy or&#xD;
      lactation, immunological disorders, periodontal treatment within the last 6 months, oral&#xD;
      contraception and antibiotic usage within the last 3 months, infections (such as HIV,&#xD;
      hepatitis, and tuberculosis). In addition, third molar teeth were excluded from GCF sampling&#xD;
      for being often impacted or extracted&#xD;
&#xD;
      Smoker patients will not be excluded and will be classified as light smokers (less than 10&#xD;
      cigarettes/day) or heavy smokers (more than 10 cigarettes /day).&#xD;
&#xD;
      Diabetic patients will not be excluded either but will be identified and accounted for.&#xD;
&#xD;
      All patients included in the study will go through the following stages:&#xD;
&#xD;
        1. Complete periodontal examination (probing depth (PD), clinical attachment level (CAL),&#xD;
           plaque index (PI), blood on probing (BOP)).&#xD;
&#xD;
        2. Patients will be divided in two groups:&#xD;
&#xD;
             -  30 healthy patients (control)&#xD;
&#xD;
             -  30 periodontal patients (test- periodontitis stage I-II (15 patients),&#xD;
                periodontitis stage III- IV (15 patients).&#xD;
&#xD;
      Due to lack of in vivo studies, simple sample size calculation was made using a conservative&#xD;
      power analysis (G*Power 3.1.9.4 program, 2009). The simple size (27 subjects) was based on a&#xD;
      two-tailed test with an effective size of 0.05, an alphabetical error probability of 0.05,&#xD;
      difference of 50% in mean value, standard deviations to be maxium 80% of the mean values and&#xD;
      a power of 0.90.&#xD;
&#xD;
      Taking into account these parameters, a simple sample size between 21-30 patients per group&#xD;
      is estimated.&#xD;
&#xD;
      Crevicular fluid samples will also be obtained following the manufacturers protocol using&#xD;
      perio papers (Periopaper, Applied Biosystems, Inc, Foster City, CA). Teeth to be examined&#xD;
      will be isolated with cotton rolls and air dried. Paper strips will be introduced into the&#xD;
      gingival sulcus for approximately 30 seconds. Test group patients' samples will be taken from&#xD;
      periodontally pathogenic sites and healthy sites. Heavily contaminated samples with saliva or&#xD;
      blood will be discarded.&#xD;
&#xD;
      Samples will be stored at -80 C at the laboratory of Departamento de Ciencias Básicas de la&#xD;
      Salud, Edificio Departamental I, Fundación Clínica Universitaria, URJC (Alcorcon). Once all&#xD;
      samples are obtained, ELISA analysis of all samples will be performed by using a specific&#xD;
      ELISA kit commercially available for human LL-37 (HyCult Biotechnology, Uden, the&#xD;
      Netherlands) and commercially available kits for IL-4, IL-4, IL-6 and IL-10 ((Diaclone SAS;&#xD;
      Besançon, France; 850.890, 950.035 and 850.880, respectively).&#xD;
&#xD;
      Patients of the test group will receive a course of basic periodontal treatment (root scaling&#xD;
      and planing) and oral hygiene instructions after the first sample collection.&#xD;
&#xD;
      A re-evaluation appointment at 4-6 weeks will take place where new crevicular fluid samples&#xD;
      of both groups will be obtained (test and control), to evaluate any statistically significant&#xD;
      difference with respect to the quantity of LL-37 in relation to the baseline measurements&#xD;
      recorded. After that, samples will be scrutinised again by performing ELISA assays following&#xD;
      the protocol described above.&#xD;
&#xD;
      Once all results are obtained, statistical analysis of the samples will be carried out.&#xD;
    </textblock>
  </detailed_description>
  <overall_status>Completed</overall_status>
  <start_date type="Actual">May 17, 2021</start_date>
  <completion_date type="Actual">July 15, 2022</completion_date>
  <primary_completion_date type="Actual">June 30, 2022</primary_completion_date>
  <study_type>Observational [Patient Registry]</study_type>
  <has_expanded_access>No</has_expanded_access>
  <study_design_info>
    <observational_model>Cohort</observational_model>
    <time_perspective>Prospective</time_perspective>
  </study_design_info>
  <target_duration>2 Months</target_duration>
  <primary_outcome>
    <measure>LL-37 in crevicular fluid in healthy/periodontal patients.</measure>
    <time_frame>At baseline for healthy (control) and periodontal (test) groups and at 4-6 weeks after treatment for test group</time_frame>
    <description>Crevicular fluid samples will also be obtained following the manufacturers protocol using perio papers (Periopaper, Applied Biosystems, Inc, Foster City, CA). Samples will be stored at -80 C at the laboratory of Departamento de Epidemiología y Salud Pública, Edificio Departamental I, Fundación Clínica Universitaria, URJC (Alcorcon). Once all samples are obtained, appropriate transport will be arranged to the Department of Natural Sciences, Biomedical Sciences, Hendon Campus, Middlesex University of London. There ELISA analysis of all samples will be performed by using a specific ELISA kit commercially available for human LL-37 (HyCult Biotechnology, Uden, the Netherlands).</description>
  </primary_outcome>
  <secondary_outcome>
    <measure>Periodontal parameters</measure>
    <time_frame>At baseline for healthy (control) and periodontal (test) groups and at 4-6 weeks after treatment for test group</time_frame>
    <description>Probing depth (PD)</description>
  </secondary_outcome>
  <secondary_outcome>
    <measure>Probing depth (PD)</measure>
    <time_frame>At baseline for healthy (control) and periodontal (test) groups and at 4-6 weeks after treatment for test group</time_frame>
    <description>Assessment of the depth of the gingival sulcus by inserting a calibrated periodontal probe</description>
  </secondary_outcome>
  <secondary_outcome>
    <measure>Clinical attachment level (CAL)</measure>
    <time_frame>At baseline for healthy (control) and periodontal (test) groups and at 4-6 weeks after treatment for test group</time_frame>
    <description>Assessment of the soft tissue from the cemento enamel junction (CEJ) to the gingival margin</description>
  </secondary_outcome>
  <secondary_outcome>
    <measure>Plaque index (PI)</measure>
    <time_frame>At baseline for healthy (control) and periodontal (test) groups and at 4-6 weeks after treatment for test group</time_frame>
    <description>By using plaque diclosing tablets to assess levels of plaque/oral hygiene</description>
  </secondary_outcome>
  <secondary_outcome>
    <measure>Blood on probing (BOP)</measure>
    <time_frame>At baseline for healthy (control) and periodontal (test) groups and at 4-6 weeks after treatment for test group</time_frame>
    <description>Record of the presence of blood at the time of inserting the periodontal probe into the gingival sulcus</description>
  </secondary_outcome>
  <number_of_groups>2</number_of_groups>
  <enrollment type="Actual">60</enrollment>
  <condition>Periodontal Diseases</condition>
  <condition>Periodontitis</condition>
  <arm_group>
    <arm_group_label>Healthy (control group)</arm_group_label>
    <description>This group will include a total of 30 patients free of periodontitis Samples of crevicular fluid to quantity levels of antimicrobial peptide LL-37 will be taken at baseline&#xD;
ELISA analysis of all samples will be performed by using a specific ELISA kit commercially available for human LL-37 and also IL-4, IL-6 and IL-10</description>
  </arm_group>
  <arm_group>
    <arm_group_label>Periodontitis (test group)</arm_group_label>
    <description>30 Periodontal patients (subdivided in 2 groups of 15, depending of the severity of their disease):&#xD;
15 patients with Stage I-II periodontitis (mild/moderate)&#xD;
15 patients with Stage III-IV periodontitis (severe).&#xD;
Two sets of samples of crevicular fluid will be obtained before and after receiving a course of routine basic periodontal treatment (root scaling and planing).&#xD;
A reevaluation visit to re-assess clinical periodontal parameters will take place at 4-6 weeks after treatment. New samples of crevicular fluid will then be taken at this stage to compare the level of LL-37 before and after the treatment.&#xD;
ELISA analysis of all samples will be performed by using a specific ELISA kit commercially available for human LL-37 (HyCult Biotechnology, Uden, the Netherlands).</description>
  </arm_group>
  <intervention>
    <intervention_type>Diagnostic Test</intervention_type>
    <intervention_name>Crevicular fluid analysis by using ELISA testing.</intervention_name>
    <description>Samples of crevicular fluid (fluid existing in the space between the gums and the roots of the teeth) will be obtained from healthy and periodontal patients. In periodontal patients, samples will be taken before and after receiving a routine course of periodontal treatment (root scale and polish) to assess the levels of a peptide called LL-37 and inflammatory cytokines (IL-4, IL-6, IL-10): Samples will then be analysed using laboratory procedures (ELISA)</description>
    <arm_group_label>Healthy (control group)</arm_group_label>
    <arm_group_label>Periodontitis (test group)</arm_group_label>
  </intervention>
  <biospec_retention>Samples Without DNA</biospec_retention>
  <biospec_descr>
    <textblock>
      Crevicular fluid&#xD;
    </textblock>
  </biospec_descr>
  <eligibility>
    <study_pop>
      <textblock>
        This clinical study will take place at the University Clinic at the University Rey Juan&#xD;
        Carlos, Alcorcon (Madrid), Spain. Patients that attend the clinic suffering from gum&#xD;
        disease will be offered the possibility to participate in this study. A full questionnaire&#xD;
        indicating the risks and benefits of participating in the study as well as the management&#xD;
        of confidential personal data will be discussed with the participants in full detail. Full&#xD;
        consent will be sought before commencing the study.&#xD;
&#xD;
        Patients will be divided in two groups:&#xD;
&#xD;
          -  30 Healthy patients (no signs of periodontitis)&#xD;
&#xD;
          -  30 Periodontal patients (subdivided in 2 groups of 15, depending of the severity of&#xD;
             their disease:&#xD;
&#xD;
               -  15 patients with Stage I-II periodontitis (mild/moderate)&#xD;
&#xD;
               -  15 patients with Stage III-IV periodontitis (severe).&#xD;
      </textblock>
    </study_pop>
    <sampling_method>Non-Probability Sample</sampling_method>
    <criteria>
      <textblock>
        The criteria for entry were as follows: aged 18 or over, with no previous history of&#xD;
        periodontal treatment in the last six months, and no medical or dental history that could&#xD;
        contraindicate periodontal treatment. Exclusion criteria included current pregnancy or&#xD;
        lactation, immunological disorders, periodontal treatment within the last 6 months, oral&#xD;
        contraception and antibiotic usage within the last 3 months, infections (such as HIV,&#xD;
        hepatitis, and tuberculosis). In addition, third molar teeth were excluded from GCF&#xD;
        sampling for being often impacted or extracted&#xD;
      </textblock>
    </criteria>
    <gender>All</gender>
    <minimum_age>18 Years</minimum_age>
    <maximum_age>N/A</maximum_age>
    <healthy_volunteers>Accepts Healthy Volunteers</healthy_volunteers>
  </eligibility>
  <location>
    <facility>
      <name>Clinica Universitaria Universidad Rey Juan Carlos</name>
      <address>
        <city>Alcorcon</city>
        <state>Madrid</state>
        <zip>28923</zip>
        <country>Spain</country>
      </address>
    </facility>
  </location>
  <location_countries>
    <country>Spain</country>
  </location_countries>
  <reference>
    <citation>Davidopoulou S, Diza E, Sakellari D, Menexes G, Kalfas S. Salivary concentration of free LL-37 in edentulism, chronic periodontitis and healthy periodontium. Arch Oral Biol. 2013 Aug;58(8):930-4. doi: 10.1016/j.archoralbio.2013.01.003. Epub 2013 Feb 8.</citation>
    <PMID>23778112</PMID>
  </reference>
  <reference>
    <citation>Turkoglu O, Emingil G, Kutukculer N, Atilla G. Gingival crevicular fluid levels of cathelicidin LL-37 and interleukin-18 in patients with chronic periodontitis. J Periodontol. 2009 Jun;80(6):969-76. doi: 10.1902/jop.2009.080532.</citation>
    <PMID>19485828</PMID>
  </reference>
  <reference>
    <citation>Putsep K, Carlsson G, Boman HG, Andersson M. Deficiency of antibacterial peptides in patients with morbus Kostmann: an observation study. Lancet. 2002 Oct 12;360(9340):1144-9. doi: 10.1016/S0140-6736(02)11201-3.</citation>
    <PMID>12387964</PMID>
  </reference>
  <reference>
    <citation>de Haar SF, Hiemstra PS, van Steenbergen MT, Everts V, Beertsen W. Role of polymorphonuclear leukocyte-derived serine proteinases in defense against Actinobacillus actinomycetemcomitans. Infect Immun. 2006 Sep;74(9):5284-91. doi: 10.1128/IAI.02016-05.</citation>
    <PMID>16926422</PMID>
  </reference>
  <reference>
    <citation>Barros SP, Williams R, Offenbacher S, Morelli T. Gingival crevicular fluid as a source of biomarkers for periodontitis. Periodontol 2000. 2016 Feb;70(1):53-64. doi: 10.1111/prd.12107.</citation>
    <PMID>26662482</PMID>
  </reference>
  <reference>
    <citation>Kinney JS, Morelli T, Oh M, Braun TM, Ramseier CA, Sugai JV, Giannobile WV. Crevicular fluid biomarkers and periodontal disease progression. J Clin Periodontol. 2014 Feb;41(2):113-120. doi: 10.1111/jcpe.12194. Epub 2013 Dec 12.</citation>
    <PMID>24303954</PMID>
  </reference>
  <reference>
    <citation>McCrudden MT, Orr DF, Yu Y, Coulter WA, Manning G, Irwin CR, Lundy FT. LL-37 in periodontal health and disease and its susceptibility to degradation by proteinases present in gingival crevicular fluid. J Clin Periodontol. 2013 Oct;40(10):933-41. doi: 10.1111/jcpe.12141. Epub 2013 Aug 18.</citation>
    <PMID>23952216</PMID>
  </reference>
  <reference>
    <citation>Greer A, Zenobia C, Darveau RP. Defensins and LL-37: a review of function in the gingival epithelium. Periodontol 2000. 2013 Oct;63(1):67-79. doi: 10.1111/prd.12028.</citation>
    <PMID>23931055</PMID>
  </reference>
  <reference>
    <citation>Mallapragada S, Wadhwa A, Agrawal P. Antimicrobial peptides: The miraculous biological molecules. J Indian Soc Periodontol. 2017 Nov-Dec;21(6):434-438. doi: 10.4103/jisp.jisp_325_16.</citation>
    <PMID>29551860</PMID>
  </reference>
  <reference>
    <citation>Khurshid Z, Naseem M, Yahya I Asiri F, Mali M, Sannam Khan R, Sahibzada HA, Zafar MS, Faraz Moin S, Khan E. Significance and Diagnostic Role of Antimicrobial Cathelicidins (LL-37) Peptides in Oral Health. Biomolecules. 2017 Dec 5;7(4):80. doi: 10.3390/biom7040080.</citation>
    <PMID>29206168</PMID>
  </reference>
  <verification_date>September 2022</verification_date>
  <study_first_submitted>May 17, 2020</study_first_submitted>
  <study_first_submitted_qc>May 24, 2020</study_first_submitted_qc>
  <study_first_posted type="Actual">May 27, 2020</study_first_posted>
  <last_update_submitted>September 27, 2022</last_update_submitted>
  <last_update_submitted_qc>September 27, 2022</last_update_submitted_qc>
  <last_update_posted type="Actual">September 30, 2022</last_update_posted>
  <responsible_party>
    <responsible_party_type>Principal Investigator</responsible_party_type>
    <investigator_affiliation>Universidad Rey Juan Carlos</investigator_affiliation>
    <investigator_full_name>David Madruga-Gonzalez</investigator_full_name>
    <investigator_title>Associate Professor</investigator_title>
  </responsible_party>
  <keyword>Antimicrobial peptides</keyword>
  <keyword>Defensines</keyword>
  <keyword>LL-37</keyword>
  <keyword>Periodontal disease</keyword>
  <keyword>Crevicular fluid</keyword>
  <keyword>IL-4</keyword>
  <keyword>IL-6</keyword>
  <keyword>IL-10</keyword>
  <condition_browse>
    <!-- CAUTION:  The following MeSH terms are assigned with an imperfect algorithm            -->
    <mesh_term>Periodontitis</mesh_term>
    <mesh_term>Periodontal Diseases</mesh_term>
  </condition_browse>
  <patient_data>
    <sharing_ipd>No</sharing_ipd>
    <ipd_description>At present there is no consideration to share this data</ipd_description>
  </patient_data>
  <!-- Results have not yet been posted for this study                                          -->
</clinical_study>


download date: ClinicalTrials.gov processed this data on September 20, 2023
link text: Link to the current ClinicalTrials.gov record.
url: https://clinicaltrials.gov/ct2/show/NCT04404335
org study id: LL-37PERIOURJC
nct id: NCT04404335
lead sponsor: 
has dmc: No
is fda regulated drug: No
is fda regulated device: No
Periodontal disease is an infectious pathology that consists of the destruction of the gums
      and supporting structures around the teeth. There are several mechanisms involved in this
      process. In healthy conditions there is a balance between the bacteria present in the gums
      and the defence mechanisms of the host to fight the disease. One of the substances involved
      in favouring this protective mechanism is a peptide called LL-37.

      The aim of this study is to find out more about the rol of this peptide during this process
      by using a non invasive method of analysis consisting of analysing some samples of the fluid
      existing between the gums and the teeth.

      This clinical study will take place at the University Clinic at the University Rey Juan
      Carlos, Alcorcon (Madrid), Spain. Patients that attend the clinic suffering from gum disease
      will be offered the possibility to participate in this study. A full questionnaire indicating
      the risks and benefits of participating in the study as well as the management of
      confidential personal data will be discussed with the participants in full detail. Full
      consent will be sought before commencing the study.

      Firstly, all participants will receive a through periodontal (gum) examination to assess if
      they suffer from periodontal disease and to what extent (severity). Based on this preliminary
      data, patients will be classified in different categories.

      Patients will be divided in two groups:

        -  30 Healthy patients (no signs of periodontitis)

        -  30 Periodontal patients (subdivided in 2 groups of 30, depending of the severity of
           their disease:

             -  15 patients with Stage I-II periodontitis (mild/moderate)

             -  15 patients with Stage III-IV periodontitis (severe).

      The intervention will consist on taking samples of crevicular fluid (the fluid found in the
      space between the gum and the root of the tooth) to quantify the presence of LL-37 as well as
      IL-4, IL-6, IL-10, which seems to be associated with the defence mechanisms (immunity) of the
      host against periodontal disease. These samples will be analysed in the laboratory by using
      some specific procedure called ELISA.

      All patients from the test group will receive a basic course of periodontal treatment (root
      scale and polish), which is intended to remove the bacteria causing the disease from the
      space between the gums and the roots (periodontal pocket). Samples of crevicular fluid will
      be obtained before commencing the treatment. After 4-6 weeks, a new periodontal examination
      will take place to assess if there is an improvement of the condition and also to quantify
      the levels of LL-37, IL-4, IL-6, IL-10.

      The findings of this study will help to understand the role of this peptide in the defence
      mechanism of the host against periodontal disease. This could ultimately serve to develop new
      therapies that could include the use of this peptide in addition to the routine periodontal
      treatment prescribed to improve the healing conditions of the patient against periodontal
      disease.
'The role of anti-inflammatory cytokines and antimicrobial peptide LL-37 biomarkers in the
      treatment of periodontal disease.

        1. Introduction

           Periodontal disease is an infectious process that consists of the destruction of the
           supporting anatomical structures of the tooth, as a consequence of the inflammatory
           response to the periopathogenic activity of the subgingival bacterial plaque, which
           leads to an innate, inflammatory and adaptative immune response.

           The clinical manifestation of the disease depends of host and environmental factors as
           well as the specific microbiological agent involved. The interrelation amongst bacteria
           and the immunodefence mechanisms of the host constitute the base of the
           immunopathological mechanisms that lead to the disease. In healthy conditions, there is
           a balance between the activity of the periodontal microbiota and the host immune
           response.

           In the innate defence system of the oral cavity there are several antimicrobial peptides
           involved, being one of them, the human cathelicidin LL-37.

           The activity of antimicrobial peptides such as LL-37 in the gingival sulcus is
           considered one of the greatest defence mechanisms of the periodontum. The gingival
           epithelium not only constitutes a physical barrier to avoid bacterial invasion.
           Neutrophils migrate to the junctional epithelium and create a barrier between the
           bacterial plaque and the underlaying epithelium. They also produce antimicrobial
           peptides, one of them the 18 kDa (hCAP18). Once the neutrophils are activated in
           response to the bacterial aggression the release LL-37, with a similar activity to a
           broad-spectrum antibiotic.

           The total absence of this peptide (LL-37) has been associated to aggressive
           periodontitis, in particular in patients with a neutrophil deficiency such as those who
           suffer Morfan Kosmann syndrome

           The nature of this research aims to elucidate the role of LL-37 in periodontal disease
           by comparing the use of a none invasive technique such as crevicular fluid samples in
           patients that suffer periodontal disease, before and after receiving a course of basic
           periodontal treatment (root scaling and planing). In additionto the above, pro and
           antiinflammatory cytokines (IL-6, IL-4, IL-10 in particular) will also be evaluated
           before and after periodontal treatment. Samples obtained will be analysed by
           immunohystochemical analysis (ELISA). The quantification of LL-37 before and after
           periodontal treatment associated with the clinical findings observed may serve as
           diagnostic biomarker of the immunopathological response inherent to the periodontal
           disease.

        2. Justification of research question. Current status

           Over the last years, a great effort by scientific community has been made in order to
           try to understand the complex immunopathological processes related to periodontal
           disease and the relationship between the activity of the periodontal microbiota and the
           host defence mechanisms.

           The relevance of this research may serve to understand in more detail the role of the
           antimicrobial peptide LL-37 in periodontal disease not only due to their bactericidal
           effect but in the modulation of the immunological response of the host and its activity
           before and after periodontal treatment. The lack of studies on this matter reflects the
           need for further research on this topic.

           Ultimately, the findings of this research may form the basis of a new therapeutic
           approach based on the use of LL-37 to help modulate the immunological response of the
           host in combination with conventional periodontal treatment. This may be particularly
           relevant in patients susceptible to develop periodontal or periimplant diseases or in
           particular cases that are resistant to conventional treatment alternatives.

        3. Summary of intended research

      The main aim of the study is to investigate the role of LL-37 in the immunopathological
      response of the periodontum. Subsequent aims are to investigate the possibility of using this
      microbial peptide to serve as diagnostic, prognostic as well as a therapeutic agent in the
      treatment of periodontal disease.

      Research hypothesis: There are significant quantitative differences in the levels of LL-37
      before and after periodontal treatment, with a significant reduction of LL-37 at
      re-evaluation (4-6 weeks after a course of basic periodontal treatment -root scaling and
      planing).

      Null hypothesis: There are no significant quantitative differences in the levels of LL-37
      before and after periodontal treatment

      The objectives of the present study are:

        1. Determine if healthy patients and patients with periodontal disease have different
           levels of LL-37, IL-6, IL-4 and IL-10 in crevicular fluid at baseline and related them
           to the stage/severity of periodontal disease.

        2. Evaluate if after a course of basic periodontal treatment these levels of LL-37, IL-6,
           IL-4 and IL-10 at baseline are similar to the quantified levels on healthy patients at
           baseline.

        3. With respect to crevicular fluid samples, evaluate if levels of LL-37, IL-6, IL-4 and
           IL-10 in teeth affected by periodontal disease differ from healthy sites on the same
           individual or if there are no significant differences in the above before and after a
           course of periodontal treatment.

        4. Stablish a non-invasive protocol to evaluate the immunological state of the patient
           before and after periodontal treatment as prognostic factor.

      4. Material and methods

      Ethical approval from the Ethical Commite of University Rey Juan Carlos of Madrid (URJC) has
      been granted. A selection of patients of the "Master in Advanced Implantology, Tissue
      Regeneration and Implant supported prosthesis" of the FCU-URJC that fulfil the inclusion
      criteria and that consent to be part of the study will be made.

      The criteria for entry were as follows: aged 18 or over, with no previous history of
      periodontal treatment in the last six months, and no medical or dental history that could
      contraindicate periodontal treatment. Exclusion criteria included current pregnancy or
      lactation, immunological disorders, periodontal treatment within the last 6 months, oral
      contraception and antibiotic usage within the last 3 months, infections (such as HIV,
      hepatitis, and tuberculosis). In addition, third molar teeth were excluded from GCF sampling
      for being often impacted or extracted

      Smoker patients will not be excluded and will be classified as light smokers (less than 10
      cigarettes/day) or heavy smokers (more than 10 cigarettes /day).

      Diabetic patients will not be excluded either but will be identified and accounted for.

      All patients included in the study will go through the following stages:

        1. Complete periodontal examination (probing depth (PD), clinical attachment level (CAL),
           plaque index (PI), blood on probing (BOP)).

        2. Patients will be divided in two groups:

             -  30 healthy patients (control)

             -  30 periodontal patients (test- periodontitis stage I-II (15 patients),
                periodontitis stage III- IV (15 patients).

      Due to lack of in vivo studies, simple sample size calculation was made using a conservative
      power analysis (G*Power 3.1.9.4 program, 2009). The simple size (27 subjects) was based on a
      two-tailed test with an effective size of 0.05, an alphabetical error probability of 0.05,
      difference of 50% in mean value, standard deviations to be maxium 80% of the mean values and
      a power of 0.90.

      Taking into account these parameters, a simple sample size between 21-30 patients per group
      is estimated.

      Crevicular fluid samples will also be obtained following the manufacturers protocol using
      perio papers (Periopaper, Applied Biosystems, Inc, Foster City, CA). Teeth to be examined
      will be isolated with cotton rolls and air dried. Paper strips will be introduced into the
      gingival sulcus for approximately 30 seconds. Test group patients' samples will be taken from
      periodontally pathogenic sites and healthy sites. Heavily contaminated samples with saliva or
      blood will be discarded.

      Samples will be stored at -80 C at the laboratory of Departamento de Ciencias Básicas de la
      Salud, Edificio Departamental I, Fundación Clínica Universitaria, URJC (Alcorcon). Once all
      samples are obtained, ELISA analysis of all samples will be performed by using a specific
      ELISA kit commercially available for human LL-37 (HyCult Biotechnology, Uden, the
      Netherlands) and commercially available kits for IL-4, IL-4, IL-6 and IL-10 ((Diaclone SAS;
      Besançon, France; 850.890, 950.035 and 850.880, respectively).

      Patients of the test group will receive a course of basic periodontal treatment (root scaling
      and planing) and oral hygiene instructions after the first sample collection.

      A re-evaluation appointment at 4-6 weeks will take place where new crevicular fluid samples
      of both groups will be obtained (test and control), to evaluate any statistically significant
      difference with respect to the quantity of LL-37 in relation to the baseline measurements
      recorded. After that, samples will be scrutinised again by performing ELISA assays following
      the protocol described above.

      Once all results are obtained, statistical analysis of the samples will be carried out.
observational model: Cohort
time perspective: Prospective
measure: LL-37 in crevicular fluid in healthy/periodontal patients.
time frame: At baseline for healthy (control) and periodontal (test) groups and at 4-6 weeks after treatment for test group
description: Crevicular fluid samples will also be obtained following the manufacturers protocol using perio papers (Periopaper, Applied Biosystems, Inc, Foster City, CA). Samples will be stored at -80 C at the laboratory of Departamento de Epidemiología y Salud Pública, Edificio Departamental I, Fundación Clínica Universitaria, URJC (Alcorcon). Once all samples are obtained, appropriate transport will be arranged to the Department of Natural Sciences, Biomedical Sciences, Hendon Campus, Middlesex University of London. There ELISA analysis of all samples will be performed by using a specific ELISA kit commercially available for human LL-37 (HyCult Biotechnology, Uden, the Netherlands).
measure: Periodontal parameters
time frame: At baseline for healthy (control) and periodontal (test) groups and at 4-6 weeks after treatment for test group
description: Probing depth (PD)
measure: Probing depth (PD)
time frame: At baseline for healthy (control) and periodontal (test) groups and at 4-6 weeks after treatment for test group
description: Assessment of the depth of the gingival sulcus by inserting a calibrated periodontal probe
measure: Clinical attachment level (CAL)
time frame: At baseline for healthy (control) and periodontal (test) groups and at 4-6 weeks after treatment for test group
description: Assessment of the soft tissue from the cemento enamel junction (CEJ) to the gingival margin
measure: Plaque index (PI)
time frame: At baseline for healthy (control) and periodontal (test) groups and at 4-6 weeks after treatment for test group
description: By using plaque diclosing tablets to assess levels of plaque/oral hygiene
measure: Blood on probing (BOP)
time frame: At baseline for healthy (control) and periodontal (test) groups and at 4-6 weeks after treatment for test group
description: Record of the presence of blood at the time of inserting the periodontal probe into the gingival sulcus
arm group label: Healthy (control group)
description: This group will include a total of 30 patients free of periodontitis Samples of crevicular fluid to quantity levels of antimicrobial peptide LL-37 will be taken at baseline
ELISA analysis of all samples will be performed by using a specific ELISA kit commercially available for human LL-37 and also IL-4, IL-6 and IL-10
arm group label: Periodontitis (test group)
description: 30 Periodontal patients (subdivided in 2 groups of 15, depending of the severity of their disease):
15 patients with Stage I-II periodontitis (mild/moderate)
15 patients with Stage III-IV periodontitis (severe).
Two sets of samples of crevicular fluid will be obtained before and after receiving a course of routine basic periodontal treatment (root scaling and planing).
A reevaluation visit to re-assess clinical periodontal parameters will take place at 4-6 weeks after treatment. New samples of crevicular fluid will then be taken at this stage to compare the level of LL-37 before and after the treatment.
ELISA analysis of all samples will be performed by using a specific ELISA kit commercially available for human LL-37 (HyCult Biotechnology, Uden, the Netherlands).
intervention type: Diagnostic Test
intervention name: Crevicular fluid analysis by using ELISA testing.
description: Samples of crevicular fluid (fluid existing in the space between the gums and the roots of the teeth) will be obtained from healthy and periodontal patients. In periodontal patients, samples will be taken before and after receiving a routine course of periodontal treatment (root scale and polish) to assess the levels of a peptide called LL-37 and inflammatory cytokines (IL-4, IL-6, IL-10): Samples will then be analysed using laboratory procedures (ELISA)
arm group label: Healthy (control group)
arm group label: Periodontitis (test group)
Crevicular fluid
study pop: 
sampling method: Non-Probability Sample
criteria: 
gender: All
minimum age: 18 Years
maximum age: N/A
healthy volunteers: Accepts Healthy Volunteers
facility: 
country: Spain
citation: Davidopoulou S, Diza E, Sakellari D, Menexes G, Kalfas S. Salivary concentration of free LL-37 in edentulism, chronic periodontitis and healthy periodontium. Arch Oral Biol. 2013 Aug;58(8):930-4. doi: 10.1016/j.archoralbio.2013.01.003. Epub 2013 Feb 8.
PMID: 23778112
citation: Turkoglu O, Emingil G, Kutukculer N, Atilla G. Gingival crevicular fluid levels of cathelicidin LL-37 and interleukin-18 in patients with chronic periodontitis. J Periodontol. 2009 Jun;80(6):969-76. doi: 10.1902/jop.2009.080532.
PMID: 19485828
citation: Putsep K, Carlsson G, Boman HG, Andersson M. Deficiency of antibacterial peptides in patients with morbus Kostmann: an observation study. Lancet. 2002 Oct 12;360(9340):1144-9. doi: 10.1016/S0140-6736(02)11201-3.
PMID: 12387964
citation: de Haar SF, Hiemstra PS, van Steenbergen MT, Everts V, Beertsen W. Role of polymorphonuclear leukocyte-derived serine proteinases in defense against Actinobacillus actinomycetemcomitans. Infect Immun. 2006 Sep;74(9):5284-91. doi: 10.1128/IAI.02016-05.
PMID: 16926422
citation: Barros SP, Williams R, Offenbacher S, Morelli T. Gingival crevicular fluid as a source of biomarkers for periodontitis. Periodontol 2000. 2016 Feb;70(1):53-64. doi: 10.1111/prd.12107.
PMID: 26662482
citation: Kinney JS, Morelli T, Oh M, Braun TM, Ramseier CA, Sugai JV, Giannobile WV. Crevicular fluid biomarkers and periodontal disease progression. J Clin Periodontol. 2014 Feb;41(2):113-120. doi: 10.1111/jcpe.12194. Epub 2013 Dec 12.
PMID: 24303954
citation: McCrudden MT, Orr DF, Yu Y, Coulter WA, Manning G, Irwin CR, Lundy FT. LL-37 in periodontal health and disease and its susceptibility to degradation by proteinases present in gingival crevicular fluid. J Clin Periodontol. 2013 Oct;40(10):933-41. doi: 10.1111/jcpe.12141. Epub 2013 Aug 18.
PMID: 23952216
citation: Greer A, Zenobia C, Darveau RP. Defensins and LL-37: a review of function in the gingival epithelium. Periodontol 2000. 2013 Oct;63(1):67-79. doi: 10.1111/prd.12028.
PMID: 23931055
citation: Mallapragada S, Wadhwa A, Agrawal P. Antimicrobial peptides: The miraculous biological molecules. J Indian Soc Periodontol. 2017 Nov-Dec;21(6):434-438. doi: 10.4103/jisp.jisp_325_16.
PMID: 29551860
citation: Khurshid Z, Naseem M, Yahya I Asiri F, Mali M, Sannam Khan R, Sahibzada HA, Zafar MS, Faraz Moin S, Khan E. Significance and Diagnostic Role of Antimicrobial Cathelicidins (LL-37) Peptides in Oral Health. Biomolecules. 2017 Dec 5;7(4):80. doi: 10.3390/biom7040080.
PMID: 29206168
responsible party type: Principal Investigator
investigator affiliation: Universidad Rey Juan Carlos
investigator full name: David Madruga-Gonzalez
investigator title: Associate Professor
mesh term: Periodontitis
mesh term: Periodontal Diseases
sharing ipd: No
ipd description: At present there is no consideration to share this data