Ignite Creation Date:
2024-05-06 @ 8:07 PM
Last Modification Date:
2024-10-26 @ 3:21 PM
Study NCT ID:
NCT06261723
Status:
RECRUITING
Last Update Posted:
2024-04-16
First Post:
2024-01-30
Brief Title:
Effect of Non-chirurgical Periodontal Treatment on the Immune System From a Gender Perspective
Sponsor:
Milagros Rocha Barajas
Organization:
Fundación para el Fomento de la Investigación Sanitaria y Biomédica de la Comunitat Valenciana
Study Overview
Official Title:
Effect of Non-chirurgical Periodontal Treatment on the Innate and Adaptive Immunity From a Gender Perspective Potential Therapeutic Implications of microRNAs
Status:
RECRUITING
Status Verified Date:
2024-04
Last Known Status:
None
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
None
Brief Summary:
The goal of this observational study is to evaluate non-surgical periodontal treatment in women and men with periodontitis with and without obesity The main questions it aims to answer are
If non-surgical periodontal treatment of patients with chronic periodontitis can modulate the innate and adaptive immune response taking into account patient gender and the coexistence of obesity
If there are specific miRNAs that can regulate this immune response and can be considered as suitable biomarkers and therapeutic targets
Obese or non-obese participants with periodontitis will receive non-surgical periodontal treatment consisting of oral health guidance and mechanical periodontal debridement throughout the mouth using an ultrasonic device and manual curettes Researchers will compare four groups obese women non-obese women obese men and non-obese men to clarify the involment of immune response after treatment considering the coexistence of obesity and potential gender differences
If non-surgical periodontal treatment of patients with chronic periodontitis can modulate the innate and adaptive immune response taking into account patient gender and the coexistence of obesity
If there are specific miRNAs that can regulate this immune response and can be considered as suitable biomarkers and therapeutic targets
Obese or non-obese participants with periodontitis will receive non-surgical periodontal treatment consisting of oral health guidance and mechanical periodontal debridement throughout the mouth using an ultrasonic device and manual curettes Researchers will compare four groups obese women non-obese women obese men and non-obese men to clarify the involment of immune response after treatment considering the coexistence of obesity and potential gender differences
Detailed Description:
Blood gingival crevicular fluid GCF and saliva samples will be collected from patients at baseline and 12 weeks after non-surgical periodontal treatment From fasting blood samples 12h peripheral blood mononuclear cells PBMCs and neutrophil fractions will be isolated using an immunomagnetic method following the manufacturers protocol and plasma and serum will be stored at -80C until analysis LUNA-FL will be used to determine cell count and viability acridine orange and propidium iodide double stain in cell samples GCF samples will be collected in duplicate by carefully inserting sterile paper points into the periodontal pocket 1 mm for 30 s The volume will be determined using the Periotron 8000 and stored at -80C for future determinations Unstimulated saliva will be collected in RNase-free tubes by expectoration and after centrifugation the supernatant will be stored at -80C for microRNA miRNA analysis
Clinical periodontal parameters of probing pockets depth PD millimetres of clinical attachment level CAL bleeding on probing BOP simplified plaque index of Silness and Löe and simplified calculus index of Greene and Vermillion will be determined using a conventional manual periodontal probe UNC-15 PCP A periodontal examination will be performed to measure PD CAL and BOP at six sites per tooth for all teeth excluding third molars as previously described Patients will be interviewed about their medical history lifestyle habits smoking frequency of tooth brushing physical activity and sociodemographic characteristics Weight height and blood pressure will be measured using standardized methods Biochemical parameters of carbohydrate metabolism - glucose insulin glycated hemoglobin A1c - lipid profile - total cholesterol LDL HDL triglycerides TG apolipoproteins AI and B - emerging inflammatory and cardiovascular risk markers - C-Reactive Protein CRP C3c and retinol-binding protein 4 RBP4 - and complete blood count will be determined at the hospitals Clinical Analysis Service
For detection of differences in protein expression cells will be incubated in lysis buffer with protease and phosphatase inhibitors RIPA Buffer for 15 minutes at 4 degrees celsius C The supernatant will be collected after centrifugation for 15 minutes at 16000g The total protein concentration will be quantified using a bicinchoninic acid BCA protein assay Aliquots of 25 µg of protein will be resolved on 8-16 gradient SDS-polyacrylamide gels and transferred to nitrocellulose membranes Target proteins will be detected by incubating the membranes with anti-actin JNK NFkB MCP1 GPX-1 NLRP3 ASC procaspase 1 caspase 1 NADPH oxidase catalase GPX1 SOD1 Beclin ATG5-ATG12 p62 LC3 I LC3 II Pink1 GRP78 eIF2alpha IRE1 alpha ATF6 CHOP PGC1 alpha mTFA VDAC Complex I II III IV and V The protein signal will be detected by chemiluminescence and analyzed by densitometry
Inflammasome complex assembly first stage of activation through co-localization studies of NLRP3-ASC in PBMCs will be conducted using the confocal andor fluorescence microscopy Briefly PBMCs will be seeded on coverslips coated with Poly-D-Lysine fixed with paraformaldehyde PFA 4 for 20 minutes permeabilized with Triton X-100 for 20 minutes and blocking with Phosphate-buffered saline buffer-Bovine serum albumn PBS-BSA 3 for 1 hour at room temperature Hybridization with specific primary antibodies diluted in PBS-BSA 1 will be carry out overnight at 4 ºC and then secondary antibodies conjugated with AlexaFluor fluorophores will be incubated for 1 hour in the dark at room temperature Stained samples will be transferred the coverslip onto microscope slide and conserved in anti-fade fluorescence mounting medium
Circulating levels of cytokines adhesion molecules and serum oxidative stress markers will be measured in serum samples but for secretome studies 1x106 PBMCs will be previously incubated in 1 mL RPMI with 10 fetal bovine serum FBS for 4 hours 37C 5 CO2 to obtain the supernatant Both serums and supernatants will be analyzed with a Luminex 200 analyzer system following the Milliplex MAP Kit manufacturers procedure These same determinations will be carried out on GCF and serumplasma samples from patients
The quantification of functional autophagosomes in PBMCs will be performed using flow cytometry on our BD Accuri equipment with the CYTO-ID Autophagy detection kit following manufacturers procedure In parallel leucocyte from whole blood samples will be incubated with redox status detecting fluorescent probes for 15 minutes and then they will be analysed using flow cytometry Additionally a multicolor panels for flow cytometry based on cluster of differentiation CD antigens detection CD3CD4CD8CD45RACCR7CD38 and CD14CD16 will be designed to analyze the percentage of lymphocyte T subpopulations naive T cells TN central memory TCM effector memory TEM and terminally differentiated TEMRA cells and monocytes subpopulations classical non-classical and intermediate respectively in blood samples In both procedures 10000 events will be acquired and single staining and FMO controls for all fluorochrome-conjugated antibodies in the panels will be performed to establish adequate compensation and define positive signals
A parallel plate flow chamber connected to an inverted microscope will enable the researchers to measure neutrophil-endothelial cell interactions in vitro Through this system the leukocyte suspension obtained from patients will be perfused over a monolayer of immortalized endothelial cells HUVECTERT 2 under conditions simulating blood flow Videos will be analyzed afterward to determine flow rolling velocity and firm adhesion of leukocytes to endothelial cells as previously described Antioxidants 2020 Aug 1198734
Changes in gene expression levels will be evaluated using Nanostring technology for nCounter To sum up total RNA will be extracted from PBMCs using the GeneAllR RibospinTM total kit and starting from 100-200 ng of total RNA Diagnostica Longwood SL will analyze the differential gene expression response thus obtaining the multiplex metabolism panel
For differential gene expression analysis of miRNAs DEGs RNA will be extracted using the miRNeasy SerumPlasma kit Qiagen After short-chain RNA extraction libraries will be prepared for sequencing using the TruSeq Small RNA library preparation kit Illumina Inc Libraries will be pooled equimolarly and quantified using the KAPA SYBR FAST Universal qPCR kit with Illumina Primer Premix and group size will be measured using a Bioanalyzer Agilent Finally 2 nanomoles of the group will be sequenced on the Illumina NovaSeq6000 platform with a 1 PhiX control in the FISABIO facilities Libraries will be sequenced using 2x100 bp chemistry in an SP flow cell Illumina Inc After multiplexing raw data will be processed using the COMPSRA a COMprehensive Platform for Small RNA-Seq data Analysis pipeline
DNA will also be extracted from plasma according to the blood and body fluid protocol of the QIAamp blood reagent set QIAgen Hilden Germany 400 μL of plasma will be applied to each column DNA will be eluted in 200 μL of supplied buffer and will be stored at -20C until use mtDNA will be quantified with reverse transcription-quantitative polymerase chain reaction RT-qPCR using specific primers for obtaining circulating mtDNA levels
Data analysis will be performed with SPSS 170 Groups will be compared using unpaired Students t-tests or Mann-Whitney U tests for parametric and non-parametric data respectively Changes after intervention will be evaluated using paired Students t-tests or Wilcoxon tests depending on the variable distribution Pearson or Spearman correlation coefficients will be used to measure the strength of association between variables In multivariable regression models the relationship between two or more explanatory variables independent variables and a response variable dependent variable will be evaluated by fitting a linear equation to the obtained data Qualitative data will be expressed in percentages and proportions will be compared using a Chi-square test All tests will use a 95 confidence interval and differences will be considered statistically significant when p 005
Clinical periodontal parameters of probing pockets depth PD millimetres of clinical attachment level CAL bleeding on probing BOP simplified plaque index of Silness and Löe and simplified calculus index of Greene and Vermillion will be determined using a conventional manual periodontal probe UNC-15 PCP A periodontal examination will be performed to measure PD CAL and BOP at six sites per tooth for all teeth excluding third molars as previously described Patients will be interviewed about their medical history lifestyle habits smoking frequency of tooth brushing physical activity and sociodemographic characteristics Weight height and blood pressure will be measured using standardized methods Biochemical parameters of carbohydrate metabolism - glucose insulin glycated hemoglobin A1c - lipid profile - total cholesterol LDL HDL triglycerides TG apolipoproteins AI and B - emerging inflammatory and cardiovascular risk markers - C-Reactive Protein CRP C3c and retinol-binding protein 4 RBP4 - and complete blood count will be determined at the hospitals Clinical Analysis Service
For detection of differences in protein expression cells will be incubated in lysis buffer with protease and phosphatase inhibitors RIPA Buffer for 15 minutes at 4 degrees celsius C The supernatant will be collected after centrifugation for 15 minutes at 16000g The total protein concentration will be quantified using a bicinchoninic acid BCA protein assay Aliquots of 25 µg of protein will be resolved on 8-16 gradient SDS-polyacrylamide gels and transferred to nitrocellulose membranes Target proteins will be detected by incubating the membranes with anti-actin JNK NFkB MCP1 GPX-1 NLRP3 ASC procaspase 1 caspase 1 NADPH oxidase catalase GPX1 SOD1 Beclin ATG5-ATG12 p62 LC3 I LC3 II Pink1 GRP78 eIF2alpha IRE1 alpha ATF6 CHOP PGC1 alpha mTFA VDAC Complex I II III IV and V The protein signal will be detected by chemiluminescence and analyzed by densitometry
Inflammasome complex assembly first stage of activation through co-localization studies of NLRP3-ASC in PBMCs will be conducted using the confocal andor fluorescence microscopy Briefly PBMCs will be seeded on coverslips coated with Poly-D-Lysine fixed with paraformaldehyde PFA 4 for 20 minutes permeabilized with Triton X-100 for 20 minutes and blocking with Phosphate-buffered saline buffer-Bovine serum albumn PBS-BSA 3 for 1 hour at room temperature Hybridization with specific primary antibodies diluted in PBS-BSA 1 will be carry out overnight at 4 ºC and then secondary antibodies conjugated with AlexaFluor fluorophores will be incubated for 1 hour in the dark at room temperature Stained samples will be transferred the coverslip onto microscope slide and conserved in anti-fade fluorescence mounting medium
Circulating levels of cytokines adhesion molecules and serum oxidative stress markers will be measured in serum samples but for secretome studies 1x106 PBMCs will be previously incubated in 1 mL RPMI with 10 fetal bovine serum FBS for 4 hours 37C 5 CO2 to obtain the supernatant Both serums and supernatants will be analyzed with a Luminex 200 analyzer system following the Milliplex MAP Kit manufacturers procedure These same determinations will be carried out on GCF and serumplasma samples from patients
The quantification of functional autophagosomes in PBMCs will be performed using flow cytometry on our BD Accuri equipment with the CYTO-ID Autophagy detection kit following manufacturers procedure In parallel leucocyte from whole blood samples will be incubated with redox status detecting fluorescent probes for 15 minutes and then they will be analysed using flow cytometry Additionally a multicolor panels for flow cytometry based on cluster of differentiation CD antigens detection CD3CD4CD8CD45RACCR7CD38 and CD14CD16 will be designed to analyze the percentage of lymphocyte T subpopulations naive T cells TN central memory TCM effector memory TEM and terminally differentiated TEMRA cells and monocytes subpopulations classical non-classical and intermediate respectively in blood samples In both procedures 10000 events will be acquired and single staining and FMO controls for all fluorochrome-conjugated antibodies in the panels will be performed to establish adequate compensation and define positive signals
A parallel plate flow chamber connected to an inverted microscope will enable the researchers to measure neutrophil-endothelial cell interactions in vitro Through this system the leukocyte suspension obtained from patients will be perfused over a monolayer of immortalized endothelial cells HUVECTERT 2 under conditions simulating blood flow Videos will be analyzed afterward to determine flow rolling velocity and firm adhesion of leukocytes to endothelial cells as previously described Antioxidants 2020 Aug 1198734
Changes in gene expression levels will be evaluated using Nanostring technology for nCounter To sum up total RNA will be extracted from PBMCs using the GeneAllR RibospinTM total kit and starting from 100-200 ng of total RNA Diagnostica Longwood SL will analyze the differential gene expression response thus obtaining the multiplex metabolism panel
For differential gene expression analysis of miRNAs DEGs RNA will be extracted using the miRNeasy SerumPlasma kit Qiagen After short-chain RNA extraction libraries will be prepared for sequencing using the TruSeq Small RNA library preparation kit Illumina Inc Libraries will be pooled equimolarly and quantified using the KAPA SYBR FAST Universal qPCR kit with Illumina Primer Premix and group size will be measured using a Bioanalyzer Agilent Finally 2 nanomoles of the group will be sequenced on the Illumina NovaSeq6000 platform with a 1 PhiX control in the FISABIO facilities Libraries will be sequenced using 2x100 bp chemistry in an SP flow cell Illumina Inc After multiplexing raw data will be processed using the COMPSRA a COMprehensive Platform for Small RNA-Seq data Analysis pipeline
DNA will also be extracted from plasma according to the blood and body fluid protocol of the QIAamp blood reagent set QIAgen Hilden Germany 400 μL of plasma will be applied to each column DNA will be eluted in 200 μL of supplied buffer and will be stored at -20C until use mtDNA will be quantified with reverse transcription-quantitative polymerase chain reaction RT-qPCR using specific primers for obtaining circulating mtDNA levels
Data analysis will be performed with SPSS 170 Groups will be compared using unpaired Students t-tests or Mann-Whitney U tests for parametric and non-parametric data respectively Changes after intervention will be evaluated using paired Students t-tests or Wilcoxon tests depending on the variable distribution Pearson or Spearman correlation coefficients will be used to measure the strength of association between variables In multivariable regression models the relationship between two or more explanatory variables independent variables and a response variable dependent variable will be evaluated by fitting a linear equation to the obtained data Qualitative data will be expressed in percentages and proportions will be compared using a Chi-square test All tests will use a 95 confidence interval and differences will be considered statistically significant when p 005
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
False
Is a FDA Regulated Device?:
False
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None