SARS Antibody Testing in Children: Development of Oral Fluid Assays for IgG Measurements.

Seroepidemiological studies to monitor antibody kinetics are important for assessing the extent and spread of SARS-CoV-2 in a population. Noninvasive sampling methods are advantageous for reducing the need for venipuncture, which may be a barrier to investigations, particularly in pediatric populations. Oral fluids are obtained by gingiva-crevicular sampling from children and adults and are very well accepted. Enzyme immunoassays (EIAs) based on these samples have acceptable sensitivity and specificity compared to conventional serum-based antibody EIAs and are suitable for population-based surveillance. We describe the development and evaluation of SARS-CoV-2 IgG EIAs using SARS-CoV-2 viral nucleoprotein (NP) and spike (S) proteins in IgG isotype capture format and an indirect receptor-binding-domain (RBD) IgG EIA, intended for use in children as a primary endpoint. All three assays were assessed using a panel of 1,999 paired serum and oral fluids from children and adults participating in school SARS-CoV-2 surveillance studies during and after the first and second pandemic wave in the United Kingdom. The anti-NP IgG capture assay was the best candidate, with an overall sensitivity of 75% (95% confidence interval [CI]: 71 to 79%) and specificity of 99% (95% CI: 78 to 99%) compared with paired serum antibodies. Sensitivity observed in children (80%, 95% CI: 71 to 88%) was higher than that in adults (67%, CI: 60% to 74%). Oral fluid assays (OF) using spike protein and RBD antigens were also 99% specific and achieved reasonable but lower sensitivity in the target population (78%, 95% CI [68% to 86%] and 53%, 95% CI [43% to 64%], respectively). IMPORTANCE We report on the first large-scale assessment of the suitability of oral fluids for detection of SARS-CoV-2 antibody obtained from healthy children attending school. The sample type (gingiva-crevicular fluid, which is a transudate of blood but is not saliva) can be self collected. Although detection of antibodies in oral fluids is less sensitive than that in blood, our study suggests an optimal format for operational use. The laboratory methods we have developed can reliably measure antibodies in children, who are able to take their own samples. Our findings are of immediate practical relevance for use in large-scale seroprevalence studies designed to measure exposure to infection, as they typically require venipuncture. Overall, our data indicate that OF assays based on the detection of SARS-CoV-2 antibodies are a tool suitable for population-based seroepidemiology studies in children and highly acceptable in children and adults, as venipuncture is no longer necessary.

Association between rheumatoid factors and proinflammatory biomarkers with implant health in rheumatoid arthritis patients with dental implants.

OBJECTIVE: To evaluate the levels of crevicular fluid rheumatoid factors (RFs), and other proinflammatory cytokines including interleukin (IL)-6, and tumor necrosis factor-alpha (TNF-ɑ) and correlate these biomarkers with the clinical peri-implant parameters among rheumatoid arthritis (RA) patients with or without concomitant connective tissue disorder (CTD). PATIENTS AND METHODS: Three groups of 14 participants each [Group-I: healthy, Group-II: RA without CTD, and Group-III: RA with CTD] were selected. The clinical parameters observed were bleeding on probing (BOP), plaque scores (PS), pocket depth (PD) and alveolar bone loss (ABL). RFs, IL-6 and TNF-α were Enzyme-linked immunosorbent assay (ELISA) was incorporated to quantify RFs, IL-6 and TNF-α. RESULTS: BOP was significantly higher in Groups II and III as compared with Group I. Alveolar bone loss was significantly higher in Group III followed by Group II and least in Group I. Patients with RA with CTD presented with statistically higher levels of RF, IL-6 and TNF-α followed by Group II compared with Group I (p<0.05). A positive correlation existed between BOP and all the three biomarkers RF (r=0.0562; p=0.0039), IL-6 and TNF-α for Group-II patients. Similarly, a significant positive correlation existed between BOP and all the three biomarkers RF, IL-6 and TNF-α for Group-III patients. In addition, a positive correlation was also seen between ABL and RF, PD, and IL-6 in Group-III patients. CONCLUSIONS: RF might influence peri-implant inflammation in RA patients with CTD. Moreover, the increased RF levels are indicative of diagnostic marker for peri-implant complications in RA.

NLRP3 Inflammasome Expression in Gingival Crevicular Fluid of Patients with Periodontitis and Chronic Hepatitis C.

The study is aimed at assessing the impact that periodontal disease and chronic hepatitis C could have on gingival crevicular fluid levels of the NLRP3 inflammasome, caspase-1 (CASP-1), and interleukin-18 (IL-18) and at evaluating whether the increased local inflammatory reaction with clinical periodontal consequences is correlated to their upregulation. Patients were divided into four groups, according to their periodontal status and previously diagnosed hepatitis C, as follows: (i) CHC group, chronic hepatitis C patients; (ii) P group, periodontal disease patients, systemically healthy; (iii) CHC + P group, patients suffering from both conditions; and (iv) H group, systemically and periodontally healthy controls. Gingival crevicular samples were collected for quantitative analysis of the NLRP3 inflammasome, CASP-1, and IL-18. CHC + P patients expressed the worse periodontal status and the highest NLRP3, CASP-1, and IL-18 levels, the difference being statistically significant (p < 0.05). The P group patients also expressed significantly more elevated NLRP3, CASP-1, and IL-18 levels, as compared to nonperiodontal patients (CHC and H groups). Chronic hepatitis C and periodontal disease could have a significant influence on the upregulation of NLRP3 inflammasome and its components, possibly contributing to an increased local inflammatory reaction and clinical periodontal consequences.

Profiles of inflammation factors and inflammatory pathways around the peri-miniscrew implant.

BACKGROUND: Peri-miniscrew implant is a temporary assistant armamentarium for the treatment of severe malocclusion and complex tooth movement, the inflammation around it is the main reason for the failure of orthodontic treatment due to the implant loosening and falling out. Inflammation around the peri-miniscrew implant is associated with the release of pro-inflammatory cytokines. These pro-inflammatory cytokines, in turn, recruit immune cells (such as macrophages, dendritic cells, T cells, and B cells), which can produce and release inflammatory biomarkers, regulate the interaction between immune cells, periodontal ligament cells, osteoblasts, and so on. However, there is currently no effective clinical treatment plan to prevent inflammation around implants. PURPOSE: To investigate the potentially essential factors in the inflammatory response around the peri-miniscrew implant and explore the signaling pathways involved. METHODS: Here, we review the studies focused on inflammatory biomarkers (Interleukins, tumor necrosis factor-α (TNF-α), receptor activator of NF-κB ligand (RANKL), matrix metalloproteinases (MMPs), and cellular adhesion molecules (CAMs)) in peri-miniscrew implant crevicular fluid (PMICF), as well as inflammatory signaling pathways (Wnt5a, JNK, Erk1/2, NF-κBp65 and TAB/TAK) in periodontal cells from 1998 to 2020. RESULTS: A literature search revealed TLR-2, TLR-4, LOX-1, and BMPs are involved in regulating ILs (IL-1ß, IL-6, IL-8, and IL-17), TNF-α, RANKL, MMP-2, MMP-9 expression via JNK, Erk1/2, Wnt5a, NF-κBp65, OPN, and TAB/TAK signaling pathways. Among them, IL-1ß and IL-6 are the critical inflammation factors in the signaling pathways inducing the inflammatory reaction surrounding implants. Besides, CAM-1 was also regulated by MMP-9 and IL-17. CONCLUSION: There are considerable potential factors involving regulating inflammatory biomarkers on downstream signaling pathways in peri-minisrew implant crevicular fluid. CLINICAL SIGNIFICANCE: This review provides the substantiation of these cell factors and signaling pathways around peri-miniscrew implants, proposes more practical clinical therapeutic ideas and schemes for improving the stability and clinical efficacy of peri-miniscrew implants.

Cytomorphometric Analysis of Inflammation Dynamics in the Periodontium Following the Use of Fixed Dental Prostheses.

Cytomorphometry is used in the sampling of biological materials and diagnostic procedures. The use of cytological studies in periodontal diseases is not well described in the literature. Our study aimed to quantitatively assess the inflammation dynamics using cytomorphometric analysis of the periodontium before and after the use of fixed dental prostheses. Following ethics approval, a total of 105 subjects were divided in 3 groups as gingivitis (n = 23), periodontitis (n = 58), and healthy periodontium (control) (n = 24). The fixed dental prostheses (crowns and fixed partial dentures) were fabricated from cobalt-chrome metal-ceramic prostheses using the conventional method (C/M-CoCr), cobalt-chrome metal-ceramic prostheses by the computer-aided design and computer-aided manufacturing (CAD/CAM) technique (C/C-CoCr), and zirconia-based ceramic prostheses by the CAD/CAM technique (C/C-Zr) among subjects with gingivitis and periodontitis. The gingival crevicular fluid (GCF) was obtained from subjects before and after the use of the prostheses. The total count of epithelial cells and the connective tissue cells or polymorphonuclear neutrophils (PMNs) in GCF were studied using cytomorphometric analysis. The Statistical Package Tor the Social Sciences (SPSS), Version 20 (IBM Company, Chicago, IL, USA) was used to analyze the results and the significance level was set at p = 0.05. The data for before and after the use of the prostheses were compared using independent t-Tests. Similarly, the results after the use of prostheses in gingivitis, periodontitis, and control in each type of prostheses were compared using One-way ANOVA with post hoc using Scheffe. The total epithelial cells and the PMNs were determined along with the epithelium/leukocyte index. Regardless of the prostheses type used, no significant change in the parameters was identified among patients with a healthy periodontium, before and after prosthetic treatment. In all study groups, a statistically increase (p value < 0.05) was observed in the oral epithelial cell counts and a statistically decrease (p < 0.05) in the PMNs count following the use of the fixed prostheses. Data on cytomorphometric analysis could enable the selection of the most appropriate prostheses for use in patients with periodontal pathologies. When choosing prostheses, changes in the composition of GCF could be considered as a useful criterion for their use.

IL-10 Dampens an IL-17-Mediated Periodontitis-Associated Inflammatory Network.

Emerging evidence suggests comprehensive immune profiling represents a highly promising, yet insufficiently tapped approach to identify potentially prognostic signatures for periodontitis. In this report, we agnostically identified a periodontitis-associated inflammatory expression network with multiple biomarkers identified within gingival crevicular fluid samples from study participants by applying principal component analysis. We identified an IL-17-dominated trait that is associated with periodontal disease and is inversely modified by the level of IL-10. IL-10 mitigated chemokine CXCL5 and CXCL1 expressions in IL-17-stimulated peripheral blood monocytic cells and peripheral blood monocytic cell-derived macrophages. Il10-deficient mice presented more bone loss, which was associated with more Il17 and IL-17-mediated chemokine and cytokine expression at the transcriptional levels in comparison with control wild-type mice in both the Porphyromonas gingivalis-induced experimental murine periodontitis and ligature-induced alveolar bone-loss models. The dampening effect of IL-10 on the excessive signaling of IL-17 appeared to be mediated by innate immune cells populations rather than by gingival epithelial cells, which are the major cell target for IL-17 signaling. Additionally, elevated IL-17 response in Il10-deficient mice specifically elicited an M1-skewing macrophage phenotype in the gingiva that was associated with the advanced bone loss in the ligature model. In summary, IL-17 dominated an inflammatory network characteristic of periodontitis, and IL-10 dampens this excessive IL-17-mediated periodontitis trait.

Effect of dental caries on periodontal inflammatory status: A split-mouth study.

OBJECTIVE: This controlled split-mouth study aimed to estimate the effect of caries and related treatment on concentrations of interleukin (IL)-2, interferon (IFN)-γ, IL-12, IL-17A, IL-13, IL-10, IL-6, IL-5, IL-4, IL-22, tumor necrosis factor (TNF)-α, and IL1-ß in gingival crevicular fluid (GCF) of caries affected teeth before (B), 7 (7D) and 30 (30D) days post-treatment and to compare them with concentrations from healthy teeth. DESIGN: Study population included 81 systemically and periodontally healthy non-smokers exhibiting at least one shallow occlusal/ inter-proximal caries and one healthy tooth from the same morphologic group at the contralateral position. Following clinical exam, the GCF samples were collected baseline as well as 7D and 30D, while the biomarker measurement was performed using multiplex flowcytometry. RESULTS: Caries affected teeth exhibited significantly higher levels of IFN-γ, IL-1ß, IL-2, IL-4 and IL-6 when compared to healthy teeth. Post-treatment cytokines levels showed general trend of increase when compared to baseline, that was significant for IL-22 and IL-17 at 7D, while IFN-γ was significantly increased at 7D compared to the healthy teeth. At 30D, IFN-γ, TNF-α, IL-17 and IL-4 levels were significantly increased when compared to healthy teeth, while IL-2 levels were significantly higher than baseline levels. CONCLUSION: Considering significantly increased periodontal levels of inflammatory markers in caries affected teeth and in response to performed treatment, it seems that dental caries and related restorative treatment might contribute to periodontal inflammation via additive effects already in early-stage caries.

Clinical, microbiological and immunological outcomes with photodynamic therapy as an adjunct to full-mouth scaling in patients undergoing fixed orthodontic treatment.

AIM: The purpose of this clinical study was to evaluate the adjunctive role of photodynamic therapy (PDT) in the improvement of clinical, microbiological and immunological parameters among adolescent patients undergoing fixed orthodontic treatment (FOT) during the period of 6 weeks. MATERIALS AND METHODS: Fifty adolescent patients undergoing FOT were divided into following two groups; Group-A: PDT as an adjunct to full-mouth scaling (FMS) and, Group-B: FMS alone. Full-mouth plaque scores (PS), bleeding on probing (BOP) and probing depth (PD) were estimated using full-mouth six point periodontal charting. Plaque samples were collected for percentage bacterial counts of P. intermedia and P. gingivalis. Gingival crevicular fluid (GCF) sampling technique was employed for estimation of interleukin (IL)-6 and tumor necrosis factor-alpha (TNF-α) using enzyme-linked immunosorbent assay. All measurements were performed at baseline, 3 weeks and 6 weeks. RESULTS: The mean age of patients in Group-A and Group-B was 15.4 years and 14.2 years, respectively. The mean duration for fixed orthodontic therapy in patients for Group A and B were 1.25 and 1.3 years, respectively. At 3 weeks follow up, both groups showed significant reduction in PS and BOP (p < 0.001). There was a statistically significant difference in BOP and PI at 6 weeks (p < 0.05). There was a statistically significant difference in IL-6 at 3 weeks and TNF-α at 6 weeks between both groups (p < 0.05). The percentage levels for both Pi and Pg significantly reduced at 3 weeks follow up in both the groups, however, these levels were again raised at 6 weeks follow up. There was a statistically significant difference between Group-A and Group-B (p < 0.05) at 3 weeks and 6 weeks follow up. CONCLUSIONS: Photodynamic therapy as an adjunct to FMS helps to improve clinical gingival parameters, microbial and inflammatory load. PDT showed additional benefits in reducing bleeding scores in adolescent patients undergoing FOT.

Cytokines in gingivocrevicular fluid of rheumatoid arthritis patients: A review of the literature.

OBJECTIVE: Rheumatoid arthritis (RA) and periodontitis share several pathological features including bone and soft tissue destruction and high levels of circulating inflammatory proteins. Studies related to cytokines in the periodontal inflammatory exudate (gingivocrevicular fluid, GCF) of RA patients might provide insight into the association between periodontitis and RA. The aim of our study was to review the literature on cytokines in GCF of RA patients including the effect of anti-rheumatic treatment with biological disease-modifying anti-rheumatic drugs (DMARDs) and periodontal treatment on these cytokines. MATERIALS AND METHODS: MedLine/PubMed searches with different combinations of keywords "rheumatoid arthritis or RA" and "crevicular fluid or GCF" until June 2019 revealed 64 articles. Ten cross-sectional observational studies and nine treatment studies fulfilled the inclusion criteria. RESULTS: Rheumatoid arthritis patients have increased circulating and GCF levels of pro-inflammatory cytokines and proteins, despite anti-rheumatic treatment with biological DMARDs. Presence of periodontitis was accompanied by higher cytokine and protein levels. Treatment of periodontitis resulted in a decrease of these levels. CONCLUSION: Analysis of GCF of RA patients reveals that the relationship between periodontitis and RA is bidirectional, probably caused by a non-specific inflammatory burden. Data for a specific relationship are barely present in GCF.

[Effect of photodynamic therapy on interleukin in gingival crevicular fluid from patients receiving oral implantation].

PURPOSE: Photodynamic therapy (PDT) has been applied in treatment of peri-implantitis for its antimicrobial effects. The aim of the study was to evaluate the effects of a Denfotex PAD Light System's photodynamic therapy on prevention of peri-implantitis. METHODS: Twenty patients with implants in molar area were treated with photoactivated disinfection right after completion of suprastructures. The irradiation time was 30 seconds for mesial, distal, buccal and lingual/palatal sites respectively at a power output of 100mW. Gingival crevicular fluid (GCF) samples from these patients were obtained before photodynamic treatment, 2 weeks and 3 months after treatment. GCF was collected using paper points, and enzyme-linked immunosorbent assay (ELISA) was performed to determine the cytokine (IL-1ß, ΤΝF-α, IL-6 and ΙL-17) levels. The data were analyzed with SPSS 23.0 software package. RESULTS: ELISA showed IL-1ß and ΤΝF-α levels of all patients were decreased 2 weeks and 3 months after treatment. Reduction of IL-1ß, ΤΝF-α, IL-6 and IL-17 from 2 weeks to 3 months and from baseline to 3 months all showed a significant difference (P＜0.05). CONCLUSIONS: Denfotex PAD light system is effective in reducing peri-implant inflammatory mediators and can work as an effective treatment to prevent peri-implantitis.

Evaluation of photodynamic therapy in pericoronitis: Protocol of randomized, controlled, double-blind study.

INTRODUCTION: Pericoronitis is a common disease in the eruption phase of third molars, sometimes debilitating, with an impact on the quality of life. The most indicated treatment in the initial phase is the irrigation for cleanliness of the region. In order to reduce the chances of systemic dissemination of the infection and antibiotics use, it is mandatory to test effective treatments in the initial phase of pericoronitis avoiding the evolution of the infectious disease. Photodynamic therapy (PDT) is an interesting alternative because it is an effective antimicrobial treatment that is easy to perform and does not select bacterial resistance. The methylene blue (MB) used in PDT has been studied in an oral formulation, which optimizes the formation of monomers increasing its antimicrobial action. OBJECTIVE: The aim of this study is to evaluate the effectiveness of PDT with MB in an astringent vehicle in pericoronitis on the initial phase in healthy patients through microbiological, clinical, and immune response. The impact of pericoronitis on oral health-related quality of life (OHRQoL) of these patients will also be evaluated. METHOD: In this randomized, controlled, double-blind clinical bioequivalence protocol, 64 healthy patients with pericoronitis will be evaluated. Patients will be randomized into the positive control group (G1) (n = 32): irrigation with sterile saline and PDT (conventional MB at 0.005% concentration and irradiation with low intensity laser λ = 660 nm, 9J per point and radiant exposure of 318 J/cm), and the experimental group (G2) (n = 32): treatment identical to G1, however, MB will be delivered in a new formulation for oral use. Microbiological analysis will be performed by RT-PCR for the bacterium Tannerella forsythia. Gingival crevicular fluid and saliva will be collected to evaluate cytokines by Luminex assay (Luminex Corporation, Austin, TX). The pain (visual analogue scale), swelling and buccal opening (digital caliper), and OHRQoL will also be evaluated through the OHIP-14 questionnaire. The variables will be evaluated in T1 (baseline), T2 (immediately after PDT), and T3 (4th day after PDT). Registration: clinicaltrials.gov NCT03576105. Registered in July 2018.

Cytokine levels in gingival crevicular fluid during orthodontic treatment with aligners compared to conventional labial fixed appliances: a 3-week clinical study.

Objective: To test the hypothesis that the levels of IL-1ß and TNF-α increased more and IL-1α, IL-2, IL-6, IL-8 increased less, after 3 weeks of treatment with conventional labial fixed appliance and with aligners. Material and methods: Forty patients who were treated either with labial brackets (n = 20) or aligners (n = 20). Gingival crevicular fluid (GCF) samples were collected at baseline and after 21 days. Cytokine levels were evaluated by enzyme-linked immune sorbent assay (ELISA). Plaque index (PI), gingival index (GI), and bleeding on probing (POB) were also examined. Results: The levels of IL-1α, IL-1ß, IL-2, IL-6, IL-8 and TNF-α in the GCF were significantly increased in both groups. The levels of IL-2, IL-6, IL-8 increased more in patients treated with aligners compared to those treated by labial fixed appliances. There was a statistically significant difference in change of the mean cytokine levels of IL-1α, IL-2, IL-6, IL-8 and TNF-α compared to labial fixed appliances and aligners. Conclusions: The levels of the six studied cytokines in GCF (IL-1α, IL-1ß, IL-2, IL-6, IL-8 and TNF-α) increased after 3 weeks both after treatment with conventional labial fixed appliance and with aligners. IL-1ß and TNF-α showed a prominent increase compared to the other cytokines in the GCF of teeth by both the labial fixed appliance and aligners. However, there were only minor differences in the changes of the cytokine levels from baseline to 3 weeks between the two groups. There were no differences between the groups regarding PI, GI or POB.

Macrophage Migration Inhibitory Factor Levels in Gingival Crevicular Fluid, Saliva, and Serum of Chronic Periodontitis Patients.

Chronic periodontitis (CP) is an infection that affects the teeth supporting structure. Macrophage migration inhibitory factor (MIF) is an important effector cytokine of the innate immune system. Due to its functional characteristics, MIF may be involved in the immunopathology of CP. The aim of the present study was to evaluate MIF levels in gingival crevicular fluid (GCF), saliva, and serum of CP patients. A cross-sectional study was conducted on 60 subjects divided into two groups: subjects with CP (n= 30) and periodontally healthy subjects without CP (n=30). MIF was quantified in GCF, saliva, and serum of all participants by enzyme-linked immunosorbent assay. MIF concentrations were higher in GCF, saliva, and serum in the group with CP compared with the group without CP and a higher MIF concentration was observed in GCF (p=0.001) and saliva (p=0.009) in the group with CP. MIF intragroup comparisons between fluids demonstrated significant high levels of MIF in saliva compared with GCF and serum in both study groups (p<0.05). A positive correlation was found between clinical signs and MIF concentration in GCF (p<0.05). There is an association between the MIF and the clinical signs of the disease. Therefore, MIF could have an important role in the pathology and progression of CP.

The relationship between T-helper cell polarization and the RANKL/OPG ratio in gingival tissues from chronic periodontitis patients.

This study aimed to investigate the relationship between inflammation-related T-helper cell polarization and the receptor activator for nuclear factor-κB ligand (RANKL)/osteoprotegerin (OPG) ratio, which is associated with bone resorption or remodeling of chronic periodontitis patients. Gingival crevicular fluid (GCF) and gingival tissues were obtained from periodontally healthy individuals (PH group) and chronic periodontitis patients (CP group). The GCF levels of IFN-γ, IL-4, IL-17, and IL-10 linked to T-helper cell polarization toward the Th1, Th2, Th17, and Treg phenotypes, respectively, were determined by ELISA. The expression levels of these cytokines and the polarized T-helper cells in gingival tissues were assessed through immunohistochemical and immunofluorescence assays. In addition, the RANKL and OPG expression levels in gingival tissues were detected by immunohistochemical assays, and linear regression analysis was used to identify the potential relationship between T-helper cell polarization and the RANKL/OPG ratio. In total, 22 individuals and 35 patients were enrolled in the present study. In both GCF and gingival tissues, increased levels of IL-17 and the decreased levels of IL-4 and IL-10 were observed in the CP group. When polarized T-helper cells were identified in gingival tissues, more Th1 and Th17 cells were found in the CP group, whereas more Th2 and Treg cells were found in the PH group. Although there was no significant difference in OPG expression between the two groups, the RANKL/OPG ratio in the CP group was higher than that in the PH group. The linear regression analysis showed that the presence of more Th1 and Th17 cells correlated with a higher RANKL/OPG ratio, whereas the presence of more Th2 cells correlated with a lower RANKL/OPG ratio. Th1 and Th17 cells are positively correlated and Th2 cells are negatively correlated with the RANKL/OPG ratio. Our data suggest that T-helper cell polarization is closely linked to the RANKL/OPG ratio in gingival tissues from chronic periodontitis patients.

Plaque Accumulation and Inflammation Adjacent to Restorations of Amorphous Calcium Phosphate-containing Composite in Early Childhood Caries.

PURPOSE: To evaluate the clinical, biochemical, and microbiological reactions to nanocomposite containing amorphous calcium phosphate (ACP) in comparison to a traditional composite restorative material in early childhood caries. MATERIALS AND METHODS: Eighteen teeth were restored with the test material (ACP-containing resin) and 18 teeth were restored with the control material (traditional composite, TC) in fourteen paediatric patients using a split-mouth design. One caries- and restoration-free intact tooth in each patient was selected as the healthy control. Gingival crevicular fluid (GCF) and supragingival plaque samples were collected at baseline before the treatment and also on days 1, 7, 14 and 30 after treatment. Unstimulated whole saliva samples were obtained from each patient at baseline, and 1 and 6 months after restoration. GCF and saliva samples were assayed for IL-17A, IL-17F IL-17A/F, IL-17E, OPG and RANKL levels by ELISA, and plaque composition was assessed using RT-PCR. RESULTS: Clinical evaluation indicated no statistically significant differences between the two restorative materials according to the FDI criteria surface lustre, material retention and marginal adaptation properties. Pro-inflammatory IL-17 levels decreased statistically significantly at 6 months compared to baseline and 1-month values (p < 0.05). The baseline pro-inflammatory IL-17 cytokine levels in GCF samples around the carious teeth were higher than those obtained around the healthy teeth (p < 0.05), but similar in GCF from the ACP-test and TC teeth. Microbiological findings were similar in the ACP and T groups. CONCLUSION: It may be suggested that both ACP-containing and traditional resin composites show similar antimicrobial and biochemical effects in early childhood caries.

Interleukin-6 and Interferon-α Levels in Gingival Crevicular Fluid in HIV-1 Patients with Chronic Periodontitis / Niveles de Interleukin-6 e Interferon-α de Fluido Crevicular Gingival en Pacientes VIH-1 con Periodontitis Crónica

ABSTRACT: The influence of cytokine on the progression of chronic periodontitis in human immunodeficiency virus (HIV) patients is still controversial and poorly investigated. This study aimed to analyze and compare IL-6 and IFN-α levels in the gingival crevicular fluid of HIV-1-positive and HIV-1-negative patients with chronic periodontitis and different grades of tissue destruction and inflammation. Samples from the gingival crevicular sulcus were obtained from 35 HIV-1-positive individuals with chronic periodontitis and 35 seronegative patients with chronic periodontitis. Probing depth and clinical attachment level, as well as the results of the Enzyme-Linked Immunosorbent Assay for confirmation of patient diagnostics, were evaluated. Statistical analyses were performed using Student t, Mann-Whitney and Spearman tests. IL-6 levels were significantly lower, while IFN-α levels were significantly higher in HIV-1 patients. Clinical attachment level was directly associated with IFN-α levels in HIV-1 carriers, connected to probing depth in these patients. Clinical data in association with gingival crevicular fluid cytokine levels may reveal a localized immunological response pattern, which may contribute to the understanding of periodontitis pathogenesis in HIV-1 carriers.

RESUMEN: La influencia de la citocina en la progresión de la periodontitis crónica en pacientes con el virus de la inmunodeficiencia humana (VIH) sigue siendo controvertida y poco investigada. Este estudio tuvo como objetivo analizar y comparar los niveles de interleuquina-6 (IL6) e interferón-α (IFN-α) en el líquido crevicular gingival de pacientes VIH-1-positivos y VIH-1-negativos con periodontitis crónica y diferentes grados de destrucción e inflamación tisular. Se obtuvieron muestras del surco crevicular gingival de 35 individuos VIH-1 positivos con periodontitis crónica y 35 pacientes seronegativos con periodontitis crónica. Se evaluaron la profundidad de sondeo y el nivel de inserción clínica, así como los resultados del Ensayo Inmunoabsorbente Ligado a Enzimas para la confirmación del diagnóstico del paciente. Los análisis estadísticos se realizaron utilizando pruebas t de Student, Mann-Whitney y Spearman. Los niveles de IL-6 fueron significativamente más bajos, mientras que los niveles de IFN-a fueron significativamente más altos en los pacientes con VIH-1. El nivel de inserción clínica se asoció directamente con los niveles de IFN-α en los portadores del VIH1, conectados a la profundidad del sondaje en estos pacientes. Los datos clínicos en asociación con los niveles de citoquinas de los fluidos creviculares gingivales pueden revelar un patrón de respuesta inmunológica localizado, que puede contribuir a la comprensión de la patogénesis de la periodontitis en los portadores del VIH-1.

Gingival Exudatome Dynamics Implicate Inhibition of the Alternative Complement Pathway in the Protective Action of the C3 Inhibitor Cp40 in Nonhuman Primate Periodontitis.

Periodontitis is a prevalent chronic inflammatory disease associated with dysbiosis. Although complement inhibition has been successfully used to treat periodontitis in animal models, studies globally analyzing inflamed tissue proteins to glean insight into possible mechanisms of action are missing. Using quantitative shotgun proteomics, we aimed to investigate differences in composition of inflammatory gingival tissue exudate ("gingival crevicular fluid"; GCF), before and after local administration of an inhibitor of the central complement component, C3, in nonhuman primates. The C3 inhibitor, Cp40 (also known as AMY-101) was administered locally in the maxillary gingival tissue of cynomolgus monkeys with established periodontitis, either once a week (1×-treatment; n = 5 animals) or three times per week (3×-treatment; n = 10 animals), for 6 weeks followed by another 6 weeks of observation in the absence of treatment. 45 GCF samples were processed for FASP digestion and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Data were processed using the ProgenesisQI software. The statistical significance of differences between the groups was determined by RM-ANOVA, and a protein expression change was considered as a true regulation at >2-fold and p < 0.05. The human orthologues were subjected to Gene Ontology analyses using PANTHER. Data are available via ProteomeXchange with identifier PXD009502. 573 proteins with >2 peptides were longitudinally quantified. Both 3× and 1× administration of Cp40 resulted in significant down-regulation of dozens of proteins during the 6-week course of treatment as compared to baseline. Following drug withdrawal at 6 weeks, more than 50% of the down-regulated proteins showed increased levels at week 12. The top scored pathway was "complement activation, alternative pathway", and several proteins involved in this pathway were down-regulated at 6 weeks. We mapped the proteomic fingerprint changes in local tissue exudate of cynomolgus monkey periodontitis in response to C3 inhibition and identified the alternative pathway of complement activation and leukocyte degranulation as main targets, which are thus likely to play significant roles in periodontal disease pathogenesis. Label-free quantitative proteomics strategies utilizing GCF are powerful tools for the identification of treatment targets and providing insights into disease mechanisms.

Interleukin-34 Levels in Gingival Crevicular Fluid and Plasma in Healthy and Diseased Periodontal Tissue in Presence or Absence of Obesity: A Clinico-biochemical Study.

Interleukin (IL)-34 has recently been identified as an alternative ligand for colonystimulating factor-1 receptor and plays an important role in osteoclastogenesis. The aim of this study was to assess and compare IL-34 levels in gingival crevicular fluid (GCF) and plasma in obese individuals in the presence or absence of periodontal disease and to determine whether they showed a correlation with disease severity. Forty patients aged between 25 and 40 yr were enrolled and categorized into 4 groups: 10 non-obese patients with healthy periodontium (Group I); 10 obese patients with healthy periodontium (Group II); 10 non-obese patients with chronic periodontitis (Group III); and 10 obese patients with chronic periodontitis (Group IV). Demographic variables such as age and body mass index score were recorded and assessed, together with clinical periodontal parameters such as the gingival index, probing pocket depth, and clinical attachment level scores in all groups. The GCF and plasma levels of IL-34 were quantified using enzyme-linked immunosorbent assay. The results showed that the mean IL-34 concentrations in GCF or plasma were highest in Group IV, followed by Group III, Group II, and Group I, with the difference among them being statistically significant (p<0.05). These results suggest that obese individuals with periodontitis have higher GCF and plasma IL-34 levels than non-obese individuals with healthy periodontium. This suggests IL-34 as a potential inflammatory marker of periodontal disease and obesity.