Clinical, immunological, and microbiological analysis of the association between periodontitis and COVID-19: a case-control study.

PURPOSE: Periodontitis and coronavirus disease (COVID-19) share risk factors and activate similar immunopathological pathways, intensifying systemic inflammation. This study investigated the clinical, immunological and microbiological parameters in individuals with COVID-19 and controls, exploring whether periodontitis-driven inflammation contributes to worsening COVID-19 endpoints. METHODS: Case (positive RT-PCR for SARS-CoV-2) and control (negative RT-PCR) individuals underwent clinical and periodontal assessments. Salivary levels of TNF-α, IL-6, IL-1ß, IL-10, OPG, RANKL, neutrophil extracellular traps, and subgingival biofilm were analyzed at two timepoints. Data on COVID-19-related outcomes and comorbidity information were evaluated from medical records. RESULTS: Ninety-nine cases of COVID-19 and 182 controls were included for analysis. Periodontitis was associated with more hospitalization (p = 0.009), more days in the intensive care unit (ICU) (p = 0.042), admission to the semi-ICU (p = 0.047), and greater need for oxygen therapy (p = 0.042). After adjustment for confounders, periodontitis resulted in a 1.13-fold increase in the chance of hospitalization. Salivary IL-6 levels (p = 0.010) were increased in individuals with COVID-19 and periodontitis. Periodontitis was associated with increased RANKL and IL-1ß after COVID-19. No significant changes were observed in the bacterial loads of the periodontopathogens Porphyromona gingivalis, Aggregatibacter actinomycetemcomitans, Tanerella forsythia, and Treponema denticola. CONCLUSIONS: Periodontitis was associated with worse COVID-19 outcomes, suggesting the relevance of periodontal care to reduce the burden of overall inflammation. Understanding the crosstalk between SARS-CoV-2 infection and chronic conditions such as periodontitis that can influence disease outcome is important to potentially prevent complications of COVID-19.

Microtomographic, histomorphometric, and molecular features show a normal alveolar bone healing process in iNOS-deficient mice along a compensatory upregulation of eNOS and nNOS isoforms.

METHODOLOGY: Inducible nitric oxide synthase (iNOS) is one of the enzymes responsible for the synthesis of nitric oxide (NO), which is an important signaling molecule with effects on blood vessels, leukocytes, and bone cells. However, the role of iNOS in alveolar bone healing remains unclear. This study investigated the role of iNOS in alveolar bone healing after tooth extraction in mice. C57Bl/6 wild type (WT) and iNOS genetically deficient (iNOS-KO) mice were subjected to upper incision tooth extraction, and alveolar bone healing was evaluated by micro-computed tomography (µCT) and histological/histomorphometric, birefringence, and molecular methods. RESULTS: The expression of iNOS had very low control conditions, whereas a significant increase is observed in healing sites of WT mice, where iNOS mRNA levels peak at 7d time point, followed by a relative decrease at 14d and 21d. Regarding bone healing, both WT and iNOS-KO groups showed the usual phases characterized by the presence of clots, granulation tissue development along the inflammatory cell infiltration, angiogenesis, proliferation of fibroblasts and extracellular matrix synthesis, bone neoformation, and remodeling. The overall micro-computed tomography and histomorphometric and birefringence analyses showed similar bone healing readouts when WT and iNOS-KO strains are compared. Likewise, Real-Time PCR array analysis shows an overall similar gene expression pattern (including bone formation, bone resorption, and inflammatory and immunological markers) in healing sites of WT and iNOS-KO mice. Moreover, molecular analysis shows that nNOS and eNOS were significantly upregulated in the iNOS-KO group, suggesting that other NOS isoforms could compensate the absence of iNOS. CONCLUSION: The absence of iNOS does not result in a significant modulation of bone healing readouts in iNOS-KO mice. The upregulation of nNOS and eNOS may compensate iNOS absence, explaining the similar bone healing outcome in WT and iNOS-KO strains.

Microtomographic, histomorphometric, and molecular features show a normal alveolar bone healing process in iNOS-deficient mice along a compensatory upregulation of eNOS and nNOS isoforms

Abstract Inducible nitric oxide synthase (iNOS) is one of the enzymes responsible for the synthesis of nitric oxide (NO), which is an important signaling molecule with effects on blood vessels, leukocytes, and bone cells. However, the role of iNOS in alveolar bone healing remains unclear. This study investigated the role of iNOS in alveolar bone healing after tooth extraction in mice. Methodology C57Bl/6 wild type (WT) and iNOS genetically deficient (iNOS-KO) mice were subjected to upper incision tooth extraction, and alveolar bone healing was evaluated by micro-computed tomography (μCT) and histological/histomorphometric, birefringence, and molecular methods. Results The expression of iNOS had very low control conditions, whereas a significant increase is observed in healing sites of WT mice, where iNOS mRNA levels peak at 7d time point, followed by a relative decrease at 14d and 21d. Regarding bone healing, both WT and iNOS-KO groups showed the usual phases characterized by the presence of clots, granulation tissue development along the inflammatory cell infiltration, angiogenesis, proliferation of fibroblasts and extracellular matrix synthesis, bone neoformation, and remodeling. The overall micro-computed tomography and histomorphometric and birefringence analyses showed similar bone healing readouts when WT and iNOS-KO strains are compared. Likewise, Real-Time PCR array analysis shows an overall similar gene expression pattern (including bone formation, bone resorption, and inflammatory and immunological markers) in healing sites of WT and iNOS-KO mice. Moreover, molecular analysis shows that nNOS and eNOS were significantly upregulated in the iNOS-KO group, suggesting that other NOS isoforms could compensate the absence of iNOS. Conclusion The absence of iNOS does not result in a significant modulation of bone healing readouts in iNOS-KO mice. The upregulation of nNOS and eNOS may compensate iNOS absence, explaining the similar bone healing outcome in WT and iNOS-KO strains.

Macrophage Polarization and Alveolar Bone Healing Outcome: Despite a Significant M2 Polarizing Effect, VIP and PACAP Treatments Present a Minor Impact in Alveolar Bone Healing in Homeostatic Conditions.

Host inflammatory immune response comprises an essential element of the bone healing process, where M2 polarization allegedly contributes to a favorable healing outcome. In this context, immunoregulatory molecules that modulate host response, including macrophage polarization, are considered potential targets for improving bone healing. This study aims to evaluate the role of the immunoregulatory molecules VIP (Vasoactive intestinal peptide) and PACAP (Pituitary adenylate cyclase activating polypeptide), which was previously described to favor the development of the M2 phenotype, in the process of alveolar bone healing in C57Bl/6 (WT) mice. Experimental groups were submitted to tooth extraction and maintained under control conditions or treated with VIP or PACAP were evaluated by microtomographic (µCT), histomorphometric, immunohistochemical, and molecular analysis at 0, 3, 7, and 14 days to quantify tissue healing and host response indicators at the healing site. Gene expression analysis demonstrates the effectiveness of VIP or PACAP in modulating host response, evidenced by the early dominance of an M2-type response, which was paralleled by a significant increase in M2 (CD206+) in treated groups. However, despite the marked effect of M1/M2 balance in the healing sites, the histomorphometric analysis does not reveal an equivalent/corresponding modulation of the healing process. µCT reveals a slight increase in bone matrix volume and the trabecular thickness number in the PACAP group, while histomorphometric analyzes reveal a slight increase in the VIP group, both at a 14-d time-point; despite the increased expression of osteogenic factors, osteoblastic differentiation, activity, and maturation markers in both VIP and PACAP groups. Interestingly, a lower number of VIP and PACAP immunolabeled cells were observed in the treated groups, suggesting a reduction in endogenous production. In conclusion, while both VIP and PACAP treatments presented a significant immunomodulatory effect with potential for increased healing, no major changes were observed in bone healing outcome, suggesting that the signals required for bone healing under homeostatic conditions are already optimal, and additional signals do not improve an already optimal process. Further studies are required to elucidate the role of macrophage polarization in the bone healing process.

TBX21-1993T/C polymorphism association with Th1 and Th17 response at periapex and with periapical lesions development risk.

TBX21-1993T/C (rs4794067) polymorphism increases the transcriptional activity of the Tbx21, essential for interferon gamma (IFNg) transcription, but its functional impact on development Th1- response in vivo remains unclear, as well its potential influence over inflammatory osteolytic conditions, such as periapical lesions. Therefore, this study comprises a case-control and functional investigation of Tbx21 genetic variations impact on Th1 response in vivo and in vitro, and its impact on periapical lesions risk and outcome, performed with a population of healthy controls (H; N = 283) and patients presenting periapical lesions (L; N = 188) or deep caries (DC; N = 152). TBX21-1993T/C genotyping demonstrated that the polymorphic allele C, as well TC/TC+CC genotypes, was significantly less frequent in the L patients compared to H and DC groups. Additionally, gene expression analysis demonstrates that T-cell-specific T-box transcription factor (Tbet) and IFNg transcripts levels were downregulated whereas IL-17 levels were upregulated in the TBX21-1993 C carriers (TC/TC+CC) in comparison with the TT group. Also, while TT and TC+CC genotypes are equally prevalent in the lesions presenting low IFN/IL17 ratio, a significant decrease in polymorphic TC+CC genotypes was observed in lesions presenting intermediate and high IFN/IL17 ratio. In vitro experiments confirmed the predisposition to Th1 polarization associated with TBX21-1993, since PBMC CD4 T cells from T allele carriers produce higher IFNg levels upon CD3/CD28 stimulation than the C group, in both standard/neutral and Th1-polarizing culture conditions. In conclusion, the TBX21-1993 T allele and TC/CC genotypes predispose to Th1-type immune response development in vitro, influence immune response polarization in vivo, and consequently account for the risk for apical periodontitis development.

Genetic Association with Subgingival Bacterial Colonization in Chronic Periodontitis.

Chronic periodontitis is the most prevalent form of inflammatory destructive bone disease and has been affecting humans since antiquity. Evidence suggest that genetic factors can highly influence periodontitis risk, modulating disease elements such as the susceptibility to microbial colonization and the nature of subsequent host-microbe interaction. Several single-nucleotide polymorphisms (SNPs) have been associated with the occurrence of periodontitis, but the full range of genetic influence in periodontitis outcomes remains to be determined. In this context, this study comprises an analysis of possible correlation between periodontitis-related genetic variants with changes in the subgingival microbiological pattern performed in a Brazilian population (n = 167, comprising 76 chronic periodontitis patients and 91 healthy subjects). For the genetic characterization, 19 candidate SNPs were selected based on the top hits of previous large genome wide association studies (GWAS), while the subgingival microbiota was characterized for the presence and relative quantity of 40 bacterial species by DNA-DNA checkerboard. The case/control association test did not demonstrate a significant effect of the target SNPs with the disease phenotype. The polymorphism rs2521634 proved significantly associated with Tannerella. forsythia, Actinomyces gerencseriae, Fusobacterium periodonticum, and Prevotella nigrescens; rs10010758 and rs6667202 were associated with increased counts of Porphyromonas gingivalis; and rs10043775 proved significantly associated with decreased counts of Prevotella intermedia. In conclusion, we present strong evidence supporting a direct connection between the host's genetic profile, specifically rs2521634, rs10010758, rs6667202, and rs10043775 polymorphisms, and the occurrence of chronic periodontitis-associated bacteria.

Simultaneous analysis of multiple T helper subsets in leprosy reveals distinct patterns of Th1, Th2, Th17 and Tregs markers expression in clinical forms and reactional events.

Leprosy is a chronic infectious disease caused by Mycobacterium leprae. Previous studies have demonstrated that the difference among clinical forms of leprosy can be associated with the immune response of patients, mainly by T helper (Th) and regulatory T cells (Tregs). Then, aiming at clarifying the immune response, the expression of cytokines related to Th1, Th2, Th17 and Tregs profiles were evaluated by qPCR in 87 skin biopsies from leprosy patients. Additionally, cytokines and anti-PGL-1 antibodies were determined in serum by ELISA. The results showed that the expression of various targets (mRNA) related to Th1, Th2, Th17 and Tregs were significantly modulated in leprosy when compared with healthy individuals, suggesting the presence of a mixed profile. In addition, the targets related to Th1 predominated in the tuberculoid pole and side and Th2 and Tregs predominated in the lepromatous pole and side; however, Th17 targets showed a mixed profile. Concerning reactional events, Tregs markers were decreased and IL-15 was increased in reversal reaction and IL-17F, CCL20 and IL-8 in erythema nodosum leprosum, when compared with the respective non-reactional leprosy patients. Additionally, ELISA analysis demonstrated that IL-22, IL-6, IL-10 and anti-PGL-1 antibody levels were significantly higher in the serum of patients when compared with healthy individuals, and IL-10 and anti-PGL-1 antibodies were also increased in the lepromatous pole and side. Together, these results indicate that Th1, Th2 and Th17 are involved in the determination of clinical forms of leprosy and suggest that decreased Tregs activity may be involved in the pathogenesis of reactional events.

qPCR detection of Mycobacterium leprae in biopsies and slit skin smear of different leprosy clinical forms

Abstract Leprosy, whose etiological agent is Mycobacterium leprae, is a chronic infectious disease that mainly affects the skin and peripheral nervous system. The diagnosis of leprosy is based on clinical evaluation, whereas histopathological analysis and bacilloscopy are complementary diagnostic tools. Quantitative PCR (qPCR), a current useful tool for diagnosis of infectious diseases, has been used to detect several pathogens including Mycobacterium leprae. The validation of this technique in a robust set of samples comprising the different clinical forms of leprosy is still necessary. Thus, in this study samples from 126 skin biopsies (collected from patients on all clinical forms and reactional states of leprosy) and 25 slit skin smear of leprosy patients were comparatively analyzed by qPCR (performed with primers for the RLEP region of M. leprae DNA) and routine bacilloscopy performed in histological sections or in slit skin smear. Considering clinical diagnostic as the gold standard, 84.9% of the leprosy patients were qPCR positive in skin biopsies, resulting in 84.92% sensitivity, with 84.92 and 61.22% positive (PPV) and negative (NPV) predictive values, respectively. Concerning bacilloscopy of histological sections (BI/H), the sensitivity was 80.15% and the PPV and NPV were 80.15 and 44.44%, respectively. The concordance between qPCR and BI/H was 87.30%. Regarding the slit skin smear, 84% of the samples tested positive in the qPCR. Additionally, qPCR showed 100% specificity, since all samples from different mycobacteria, from healthy individuals, and from other granulomatous diseases presented negative results. In conclusion, the qPCR technique for detection of M. leprae using RLEP primers proved to be specific and sensitive, and qPCR can be used as a complementary test to diagnose leprosy irrespective of the clinical form of disease.

qPCR detection of Mycobacterium leprae in biopsies and slit skin smear of different leprosy clinical forms.

Leprosy, whose etiological agent is Mycobacterium leprae, is a chronic infectious disease that mainly affects the skin and peripheral nervous system. The diagnosis of leprosy is based on clinical evaluation, whereas histopathological analysis and bacilloscopy are complementary diagnostic tools. Quantitative PCR (qPCR), a current useful tool for diagnosis of infectious diseases, has been used to detect several pathogens including Mycobacterium leprae. The validation of this technique in a robust set of samples comprising the different clinical forms of leprosy is still necessary. Thus, in this study samples from 126 skin biopsies (collected from patients on all clinical forms and reactional states of leprosy) and 25 slit skin smear of leprosy patients were comparatively analyzed by qPCR (performed with primers for the RLEP region of M. leprae DNA) and routine bacilloscopy performed in histological sections or in slit skin smear. Considering clinical diagnostic as the gold standard, 84.9% of the leprosy patients were qPCR positive in skin biopsies, resulting in 84.92% sensitivity, with 84.92 and 61.22% positive (PPV) and negative (NPV) predictive values, respectively. Concerning bacilloscopy of histological sections (BI/H), the sensitivity was 80.15% and the PPV and NPV were 80.15 and 44.44%, respectively. The concordance between qPCR and BI/H was 87.30%. Regarding the slit skin smear, 84% of the samples tested positive in the qPCR. Additionally, qPCR showed 100% specificity, since all samples from different mycobacteria, from healthy individuals, and from other granulomatous diseases presented negative results. In conclusion, the qPCR technique for detection of M. leprae using RLEP primers proved to be specific and sensitive, and qPCR can be used as a complementary test to diagnose leprosy irrespective of the clinical form of disease.

qPCR detection of Mycobacterium leprae in biopsies and slit skin smear of different leprosy clinical forms

Leprosy, whose etiological agent is Mycobacterium leprae, is a chronic infectious disease that mainly affects the skin and peripheral nervous system. The diagnosis of leprosy is based on clinical evaluation, whereas histopathological analysis and bacilloscopy are complementary diagnostic tools. Quantitative PCR (qPCR), a current useful tool for diagnosis of infectious diseases, has been used to detect several pathogens including Mycobacterium leprae. The validation of this technique in a robust set of samples comprising the different clinical forms of leprosy is still necessary. Thus, in this study samples from 126 skin biopsies (collected from patients on all clinical forms and reactional states of leprosy) and 25 slit skin smear of leprosy patients were comparatively analyzed by qPCR (performed with primers for the RLEP region of M. leprae DNA) and routine bacilloscopy performed in histological sections or in slit skin smear. Considering clinical diagnostic as the gold standard, 84.9% of the leprosy patients were qPCR positive in skin biopsies, resulting in 84.92% sensitivity, with 84.92 and 61.22% positive (PPV) and negative (NPV) predictive values, respectively. Concerning bacilloscopy of histological sections (BI/H), the sensitivity was 80.15% and the PPV and NPV were 80.15 and 44.44%, respectively. The concordance between qPCR and BI/H was 87.30%. Regarding the slit skin smear, 84% of the samples tested positive in the qPCR. Additionally, qPCR showed 100% specificity, since all samples from different mycobacteria, from healthy individuals, and from other granulomatous diseases presented negative results. In conclusion, the qPCR technique for detection of M. leprae using RLEP primers proved to be specific and sensitive, and qPCR can be used as a complementary test to diagnose leprosy irrespective of the clinical form of disease.

Simultaneous analysis of multiple T helper subsets in leprosy reveals distinct patterns of Th1, Th2, Th7 and tregs markers expression in clinical forms and reactional events

Leprosy is a chronic infectious disease caused by Mycobacterium leprae. Previous studies have demonstrated that the difference among clinical forms of leprosy can be associated with the immune response of patients, mainly by T helper (Th) and regulatory T cells (Tregs). Then, aiming at clarifying the immune response, the expression of cytokines related to Th1, Th2, Th17 and Tregs profiles were evaluated by qPCR in 87 skin biopsies from leprosy patients. Additionally, cytokines and anti-PGL-1 antibodies were determined in serum by ELISA. The results showed that the expression of various targets (mRNA) related to Th1, Th2, Th17 and Tregs were significantly modulated in leprosy when compared with healthy individuals, suggesting the presence of a mixed profile. In addition, the targets related to Th1 predominated in the tuberculoid pole and side and Th2 and Tregs predominated in the lepromatous pole and side; however, Th17 targets showed a mixed profile. Concerning reactional events, Tregs markers were decreased and IL-15 was increased in reversal reaction and IL-17F, CCL20 and IL-8 in erythema nodosum leprosum, when compared with the respective non-reactional leprosy patients. Additionally, ELISA analysis demonstrated that IL-22, IL-6, IL-10 and anti-PGL-1 antibody levels were significantly higher in the serum of patients when compared with healthy individuals, and IL-10 and anti-PGL-1 antibodies were also increased in the lepromatous pole and side. Together, these results indicate that Th1, Th2 and Th17 are involved in the determination of clinical forms of leprosy and suggest that decreased Tregs activity may be involved in the pathogenesis of reactional events.