Blocking activation of CD4-CD8- T cells modulates their cytotoxic potential and decreases the expression of inflammatory and chemotactic receptors.

CD4-CD8- (double negative - DN) T cells represent a small fraction of circulating T lymphocytes but are a major source of pro-inflammatory cytokines in patients with infectious diseases, including chronic Chagas cardiomyopathy (CCC), one of the deadliest cardiopathies known. Chagas disease is caused by an infection with the protozoan parasite Trypanosoma cruzi and can lead to either an asymptomatic form or a high-mortality cardiac disease. While circulating DN T cells represent a major inflammatory cytokine-expressing cell population in Chagas disease, their potential to be recruited to the heart and to perform cytotoxicity has not been determined. Our previous studies showed that blocking DN T cell activation decreases the expression of IFN-gamma, a cytokine involved in the severity of CCC. Here, studying a well-characterized cohort of Chagas patients with CCC or the asymptomatic form of Chagas disease (indeterminate form, IND), we evaluated the expression of cytotoxic molecules, cytokine and chemokine receptors in γδ+ and αß+ DN T cells by multiparameter flow cytometry, and investigated whether blocking the activation of DN T cells influences the expression of these molecules. We observed that DN T cells from CCC display a higher expression of granzyme A, perforin, inflammatory molecules, and inflammatory chemokine receptors than cells from IND. Messenger RNA coding for these molecules is also upregulated in the heart of CCC patients. Importantly, blocking the activation of DN T cells from CCC modulates their cytotoxic potential and the expression of inflammatory and of chemokine receptors, suggesting that targeting DN T cell activation may be a valid strategy to reduce recruitment to the heart, inflammation, cytotoxicity and, thereby diminish CCC progression and severity.

Distinct CD4-CD8- (Double-Negative) Memory T-Cell Subpopulations Are Associated With Indeterminate and Cardiac Clinical Forms of Chagas Disease.

CD4-CD8- (double-negative, DN) T cells are critical orchestrators of the cytokine network associated with the pathogenic inflammatory response in one of the deadliest cardiomyopathies known, Chagas heart disease, which is caused by Trypanosoma cruzi infection. Here, studying the distribution, activation status, and cytokine expression of memory DN T-cell subpopulations in Chagas disease patients without cardiac involvement (indeterminate form-IND) or with Chagas cardiomyopathy (CARD), we report that while IND patients displayed a higher frequency of central memory, CARD had a high frequency of effector memory DN T cells. In addition, central memory DN T cells from IND displayed a balanced cytokine profile, characterized by the concomitant expression of IFN-γ and IL-10, which was not observed in effector memory DN T cells from CARD. Supporting potential clinical relevance, we found that the frequency of central memory DN T cells was associated with indicators of better ventricular function, while the frequency of effector memory DN T cells was not. Importantly, decreasing CD1d-mediated activation of DN T cells led to an increase in IL-10 expression by effector memory DN T cells from CARD, restoring a balanced profile similar to that observed in the protective central memory DN T cells. Targeting the activation of effector memory DN T cells may emerge as a strategy to control inflammation in Chagas cardiomyopathy and potentially in other inflammatory diseases where these cells play a key role.

Systemic cytokines, chemokines and growth factors reveal specific and shared immunological characteristics in infectious cardiomyopathies.

Heart disease is a major cause of death worldwide. Chronic Chagas cardiomyopathy (CCC) caused by infection with Trypanosoma cruzi leading to high mortality in adults, and rheumatic heart disease (RHD), resulting from infection by Streptococcus pyogenes affecting mainly children and young adults, are amongst the deadliest heart diseases in low-middle income countries. Despite distinct etiology, the pathology associated with both diseases is a consequence of inflammation. Here we compare systemic immune profile in patients with these cardiopathies, to identify particular and common characteristics in these infectious heart diseases. We evaluated the expression of 27 soluble factors, employing single and multivariate analysis combined with machine-learning approaches. We observed that, while RHD and CCC display higher levels of circulating mediators than healthy individuals, CCC is associated with stronger immune activation as compared to RHD. Despite distinct etiologies, univariate analysis showed that expression of TNF, IL-17, IFN-gamma, IL-4, CCL4, CCL3, CXCL8, CCL11, CCL2, PDGF-BB were similar between CCC and RHD, consistent with their inflammatory nature. Network analysis revealed common inflammatory pathways between CCC and RHD, while highlighting the broader reach of the inflammatory response in CCC. The final multivariate model showed a 100% discrimination power for the combination of the cytokines IL-12p70, IL-1Ra, IL-4, and IL-7 between CCC and RHD groups. Thus, while clear immunological distinctions were identified between CCC and RHD, similarities indicate shared inflammatory pathways in these infectious heart diseases. These results contribute to understanding the pathogenesis of CCC and RHD and may impact the design of immune-based therapies for these and other inflammatory cardiopathies that may also share immunological characteristics.

Mutational Signatures Driven by Epigenetic Determinants Enable the Stratification of Patients with Gastric Cancer for Therapeutic Intervention.

DNA mismatch repair deficiency (dMMR) is associated with the microsatellite instability (MSI) phenotype and leads to increased mutation load, which in turn may impact anti-tumor immune responses and treatment effectiveness. Various mutational signatures directly linked to dMMR have been described for primary cancers. To investigate which mutational signatures are associated with prognosis in gastric cancer, we performed a de novo extraction of mutational signatures in a cohort of 787 patients. We detected three dMMR-related signatures, one of which clearly discriminates tumors with MLH1 gene silencing caused by promoter hypermethylation (area under the curve = 98%). We then demonstrated that samples with the highest exposure of this signature share features related to better prognosis, encompassing clinical and molecular aspects and altered immune infiltrate composition. Overall, the assessment of the prognostic value and of the impact of modifications in MMR-related genes on shaping specific dMMR mutational signatures provides evidence that classification based on mutational signature exposure enables prognosis stratification.

CD14 genotype and functional dichotomy of CD14+ and CD14- cells are associated with activated immune response and development of Chagas dilated cardiomyopathy.

We aimed to investigate the association of CD14 -260C/T (rs2569190) polymorphism and Chagas cardiomyopathy and the functional characteristics of CD14+ and CD14- monocytes upon infection with Trypanosoma cruzi. We observed an association between the T- genotype (absence of allele -260T) related to low CD14 expression and the dilated cardiomyopathy type of Chagas disease. Furthermore, we observed that CD14- monocytes showed a more activated profile upon in vitro infection with T. cruzi than CD14+ monocytes. Our findings suggest that T- genotype is associated with susceptibility to develop Chagas dilated cardiomyopathy, likely linked to the T. cruzi-induced inflammatory profile of CD14- monocytes.

Resinous adhesive systems differentially affect the expression of cytokines by human monocytes stimulated or not with Streptococcus mutans in vitro.

OBJECTIVES: The polymerization of adhesive systems is incomplete and the residual monomers that have been released have a cytotoxic capacity. Some teeth develop into pulp necrosis after composite resin restorations. Considering frequent pulpal inflammation in response to cariogenic bacteria, substances released from the patches could affect the cells of the inflammatory infiltrate and interfere with the mechanisms of defense against microorganisms and protection of pulpal tissue. The aim of this study was to evaluate the effect of substances released by different resinous adhesive systems on cell viability and cytokine expression by human monocytes stimulated in vitro with Streptococcus mutans. DESIGN: Peripheral blood mononuclear cells from 10 healthy subjects were stimulated with S. mutans and then incubated with supernatants obtained from the Single Bond Universal (SBU) or Clearfil SE Bond (CSEB) adhesive systems for eight hours. Staining with Annexin V and 7AAD for analysis of apoptosis were performed and detection of monocytes expressing cytokines IL-1α, IL-6, IL-8, IL-10, IL-12 and TNF-α were performed by flow cytometry. RESULTS: No treatment significantly affected apoptosis in monocytes. SBU supernatant increased the frequency of monocytes expressing IL-8 and decreased the monocytes expressing IL-10. Considering S. mutans-stimulated cells, while SBU increased the frequency of IL-8+ monocytes, CSEB reduced the frequency of IL-6 and TNF-α positive monocytes. CONCLUSIONS: Products released from SBU seem to induce proinflammatory effects on monocytes while those from CSEB show an anti-inflammatory outcome. These effects may interfere in the control of cytokine-mediated immunoinflammatory pulp reactions, both in the presence and absence of stimulation by cariogenic bacteria.

Increased Tumor Immune Microenvironment CD3+ and CD20+ Lymphocytes Predict a Better Prognosis in Oral Tongue Squamous Cell Carcinoma.

Background: Oral tongue squamous cell carcinoma (OTSCC) causes over 350,000 cases annually and particularly impacts populations in developing countries. Smoking and alcohol consumption are major risk factors. Determining the role of the tumor immune microenvironment (TIME) in OTSCC outcomes can elucidate immune mechanisms behind disease progression, and can potentially identify prognostic biomarkers. Methods: We performed a retrospective study of 48 OTSCC surgical specimens from patients with tobacco and alcohol exposures. A panel of immunoregulatory cell subpopulations including T (CD3, CD4, CD8) and B (CD20) lymphocytes, dendritic cells (CD1a, CD83), macrophages (CD68), and immune checkpoint molecules programmed cell death protein 1 (PD-1) and ligand 1 (PD-L1) were analyzed using immunohistochemistry. The levels of immune effector cell subpopulations and markers were analyzed in relation to overall survival. Results: Pathological characteristics of the tumor microenvironment included inflammatory infiltrates (83.3%), desmoplasia (41.6%), and perineural invasion (50.0%). The TIME contained high levels of T cells (CD3+, CD4+, and CD8+) and B cells (CD20+), as well as immature (CD1a) and mature (CD83) dendritic cells, PD-1, and PD-L1. Higher numbers of TIME infiltrating CD3+ T cells and CD20+ B cells were predictive of better survival, while higher levels of CD83+ mature dendritic cells predicted better survival. CD3+ T cells were identified as an independent prognostic marker for OTSCC. Lastly, CD3+ T cells were strongly correlated with the number of CD8+ cells and PD-L1 expression. Conclusion: Our findings provide evidence that the TIME profile of OTSSC impacted prognosis. The high expression of CD3+ T cells and B cells are predictive of better overall survival and indicative of an immunologically active, inflammatory TIME in patients with better survival. The number of CD3+ T cells was an independent prognostic marker.

CD14 genotype and functional dichotomy of CD14+ and CD14- cells are associated with activated immune response and development of Chagas dilated cardiomyopathy

We aimed to investigate the association of CD14 -260C/T (rs2569190) polymorphism and Chagas cardiomyopathy and the functional characteristics of CD14+ and CD14- monocytes upon infection with Trypanosoma cruzi. We observed an association between the T- genotype (absence of allele -260T) related to low CD14 expression and the dilated cardiomyopathy type of Chagas disease. Furthermore, we observed that CD14- monocytes showed a more activated profile upon in vitro infection with T. cruzi than CD14+ monocytes. Our findings suggest that T- genotype is associated with susceptibility to develop Chagas dilated cardiomyopathy, likely linked to the T. cruzi-induced inflammatory profile of CD14- monocytes.

Effect of porous tantalum on the biological response of human peripheral mononuclear cells exposed to Porphyromonas gingivalis.

AIM: To evaluate biological behavior of human peripheral mononuclear cells (PBMC) in contact with porous tantalum (PT) and Porphyromonas gingivalis (Pg). METHODS: Pg was incubated for 8 hours. The groups formed were: PBMC (control), PBMC + PT, PBMC + Pg and PBMC + PT + Pg. Cell viability was evaluated using MTT assay. The morphology and adhesion of PBMC to PT was evaluated using scanning electron microscopy. Expression of interleukin (IL)-10, transforming growth factor (TGF)-ß, matrix metallopeptidase (MMP)-9 and receptor activator of nuclear factor-κΒ ligand (RANKL) was determined by enzyme-linked immunosorbent assay. RESULTS: MTT assay revealed that PT did not interfere in the mitochondrial activity of PBMC (P > .05). Scanning electron microscopy showed the adherence of PBMC to PT. IL-10 levels in PBMC + PT were similar to PBMC and lower than PBMC + Pg. TGF-ß levels in PBMC + PT were higher than PBMC and PBMC + Pg. MMP-9 levels in PBMC + PT were similar to PBMC and lower than PBMC + Pg and PBMC + PT + Pg. RANKL levels in PBMC + PT were lower than in PBMC. CONCLUSION: PT did not affect PBMC viability, allowed cell adhesion, reduced expression of RANKL and enhanced TGF-ß in comparison with the control group.

Composite-derived monomers affect cell viability and cytokine expression in human leukocytes stimulated with Porphyromonas gingivalis.

OBJECTIVES: Dental composites release unreacted resin monomers into the oral environment, even after polymerization. Periodontal cells are, therefore, exposed to substances that potentially elicit the immune inflammatory response. The underlying molecular mechanisms associated with the interaction between resin monomers and human immune cells found in the gingival crevicular fluid are not fully understood yet. This study investigated the ability of bisphenol A-glycidyl methacrylate (BISGMA), urethane dimethacrylate (UDMA) and triethylene glycol dimethacrylate (TEGDMA) to induce apoptosis and cytokine release by human leukocytes stimulated with a periodontal pathogen. METHODOLOGY: Peripheral blood mononuclear cells (PBMC) from 16 healthy individuals were included in this study. To determine the toxicity, the PBMC were incubated for 20 hours, with monomers, for the analysis of cell viability using MTT assay. To evaluate cell death in the populations of monocytes and lymphocytes, they were exposed to sub-lethal doses of each monomer and of heat-inactivated Porphyromonas gingivalis (P. gingivalis) for 5 hours. Secretions of IL-1ß, IL-6, IL-10 and TNF-α were determined by ELISA after 20 hours. RESULTS: UDMA and TEGDMA induced apoptosis after a short-time exposure. Bacterial challenge induced significant production of IL-1ß and TNF-α (p<0.05). TEGDMA reduced the bacterial-induced release of IL-1ß and TNF-α, whereas UDMA reduced IL-1ß release (p<0.05). These monomers did not affect IL-10 and IL-6 secretion. BISGMA did not significantly interfere in cytokine release. CONCLUSIONS: These results show that resin monomers are toxic to PBMC in a dose-dependent manner, and may influence the local immune inflammatory response and tissue damage mechanisms via regulation of bacterial-induced IL-1ß and TNF-α secretion by PBMC.

Composite-derived monomers affect cell viability and cytokine expression in human leukocytes stimulated with Porphyromonas gingivalis

Abstract Objectives: Dental composites release unreacted resin monomers into the oral environment, even after polymerization. Periodontal cells are, therefore, exposed to substances that potentially elicit the immune inflammatory response. The underlying molecular mechanisms associated with the interaction between resin monomers and human immune cells found in the gingival crevicular fluid are not fully understood yet. This study investigated the ability of bisphenol A-glycidyl methacrylate (BISGMA), urethane dimethacrylate (UDMA) and triethylene glycol dimethacrylate (TEGDMA) to induce apoptosis and cytokine release by human leukocytes stimulated with a periodontal pathogen. Methodology: Peripheral blood mononuclear cells (PBMC) from 16 healthy individuals were included in this study. To determine the toxicity, the PBMC were incubated for 20 hours, with monomers, for the analysis of cell viability using MTT assay. To evaluate cell death in the populations of monocytes and lymphocytes, they were exposed to sub-lethal doses of each monomer and of heat-inactivated Porphyromonas gingivalis (P. gingivalis) for 5 hours. Secretions of IL-1β, IL-6, IL-10 and TNF-α were determined by ELISA after 20 hours. Results: UDMA and TEGDMA induced apoptosis after a short-time exposure. Bacterial challenge induced significant production of IL-1β and TNF-α (p<0.05). TEGDMA reduced the bacterial-induced release of IL-1β and TNF-α, whereas UDMA reduced IL-1β release (p<0.05). These monomers did not affect IL-10 and IL-6 secretion. BISGMA did not significantly interfere in cytokine release. Conclusions: These results show that resin monomers are toxic to PBMC in a dose-dependent manner, and may influence the local immune inflammatory response and tissue damage mechanisms via regulation of bacterial-induced IL-1β and TNF-α secretion by PBMC.

Loss of STI1-mediated neuronal survival and differentiation in disease-associated mutations of prion protein.

Cellular prion protein (PrPC ) is widely expressed and displays a variety of well-described functions in the central nervous system (CNS). Mutations of the PRNP gene are known to promote genetic human spongiform encephalopathies, but the components of gain- or loss-of-function mutations to PrPC remain a matter for debate. Among the proteins described to interact with PrPC is Stress-inducible protein 1 (STI1), a co-chaperonin that is secreted from astrocytes and triggers neuroprotection and neuritogenesis through its interaction with PrPC . In this work, we evaluated the impact of different PrPC pathogenic point mutations on signaling pathways induced by the STI1-PrPC interaction. We found that some of the pathogenic mutations evaluated herein induce partial or total disruption of neuritogenesis and neuroprotection mediated by mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases 1 and 2 (ERK1/2) and protein kinase A (PKA) signaling triggered by STI1-PrPC engagement. A pathogenic mutant PrPC that lacked both neuroprotection and neuritogenesis activities fail to promote negative dominance upon wild-type PrPC . Also, a STI1-α7-nicotinic acetylcholine receptor-dependent cellular signaling was present in a PrPC mutant that maintained both neuroprotection and neuritogenesis activities similar to what has been previously observed by wild-type PrPC . These results point to a loss-of-function mechanism underlying the pathogenicity of PrPC mutations.

Effects of Bio-Oss® and Cerasorb® dental M on the expression of bone-remodeling mediators in human monocytes.

In contribution to diverse techniques of bone reconstruction involving biomaterials in contemporary dentistry, this study aimed to evaluate the effect of the bone-grafting materials Bio-Oss® and Cerasorb® Dental M on the expression of cytokines associated with bone remodeling by human monocytes in vitro. Bio-Oss® and Cerasorb® Dental M were incubated in separate culture media, and their supernatants were added to mononuclear cells of human peripheral blood, some of which had been stimulated with Porphyromonas gingivalis. The frequency of total monocytes and CD14+ monocytes producing cytokines interleukin 6 (IL-6), IL-8, IL-10, IL-12, and tumor necrosis factor alpha (TNF-α) were determined by flow cytometry. One-way analysis of variance with repeated measures, followed by Tukey's post hoc test, revealed that stimulation with P. gingivalis increased the expression of IL-6 and IL-8 and reduced the expression of TNF-α compared to effects demonstrated in the control group (p < 0.05). Adding biomaterial supernatants did not significantly affect the expression of any cytokine evaluated, however, either in the absence or in the presence of bacterial stimulation. Our data suggest that Bio-Oss® and Cerasorb® Dental M neither stimulate cytokine production in human monocytes nor interfere with mechanisms of cell communication mediated by cytokines evaluated during stimulation with P. gingivalis. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2066-2073, 2017.

Persistent Sydenham’s chorea is not associated with sustained lymphocyte dysfunction / A coreia de Sydenham persistente não está associada à disfunção sustentada de linfócitos

The mechanisms involved in the symptoms of Sydenham’s chorea (SC) remain obscure. Taking into account the autoreactive antibody-mediated hypothesis of SC pathogenesis, the persistence of chorea may be associated with increased levels of B1 lymphocytes and other lymphocyte subsets. We evaluated lymphocyte subsets, including B1 and T cells, in patients with remitted (RSC) and persistent (PSC) SC by flow cytometry. Our results showed neither difference in the frequency of T and B lymphocytes subpopulations nor in their activation and functional states. These findings undermine the view of PSC as a sustained cytotoxic cellular-mediated condition. Alternative mechanisms may explain the pathogenesis of PSC.

Os mecanismos subjacentes aos sintomas da coreia de Sydenham (CS) permanecem desconhecidos. Considerando-se a hipótese de que a patogênese da CS é mediada por anticorpos autorreativos, a persistência da coreia está provavelmente associada a níveis aumentados de linfócitos B1 e outros subtipos de linfócitos. No presente trabalho, foram avaliados subtipos de linfócitos B e T em pacientes com CS em remissão (CSR) e persistente (CSP), por citometria de fluxo. Nossos resultados demonstraram que não há diferença na frequência das subpopulações de linfócitos T e B circulantes e no perfil de ativação e estado funcional dessas células. Esses resultados enfraquecem a hipótese de que a CSP seja uma condição imune sustentada mediada por células citotóxicas. São necessários estudos que investiguem mecanismos alternativos que expliquem a patogênese da CSP.

Persistent Sydenham's chorea is not associated with sustained lymphocyte dysfunction.

The mechanisms involved in the symptoms of Sydenham's chorea (SC) remain obscure. Taking into account the autoreactive antibody-mediated hypothesis of SC pathogenesis, the persistence of chorea may be associated with increased levels of B1 lymphocytes and other lymphocyte subsets. We evaluated lymphocyte subsets, including B1 and T cells, in patients with remitted (RSC) and persistent (PSC) SC by flow cytometry. Our results showed neither difference in the frequency of T and B lymphocytes subpopulations nor in their activation and functional states. These findings undermine the view of PSC as a sustained cytotoxic cellular-mediated condition. Alternative mechanisms may explain the pathogenesis of PSC.

A side-by-side comparison of T cell reactivity to fifty-nine Mycobacterium tuberculosis antigens in diverse populations from five continents.

We compared T cell recognition of 59 prevalently recognized Mycobacterium tuberculosis (MTB) antigens in individuals latently infected with MTB (LTBI), and uninfected individuals with previous BCG vaccination, from nine locations and populations with different HLA distribution, MTB exposure rates, and standards of TB care. This comparison revealed similar response magnitudes in diverse LTBI and BCG-vaccinated cohorts and significant correlation between responses in LTBIs from the USA and other locations. Many antigens were uniformly recognized, suggesting suitability for inclusion in vaccines targeting diverse populations. Several antigens were similarly immunodominant in LTBI and BCG cohorts, suggesting applicability for vaccines aimed at boosting BCG responses. The panel of MTB antigens will be valuable for characterizing MTB-specific CD4 T cell responses irrespective of ethnicity, infecting MTB strains and BCG vaccination status. Our results illustrate how a comparative analysis can provide insight into the relative immunogenicity of existing and novel vaccine candidates in LTBIs.

Effects of terlipressin as early treatment for protection of brain in a model of haemorrhagic shock.

INTRODUCTION: We investigated whether treatment with terlipressin during recovery from hypotension due to haemorrhagic shock (HS) is effective in restoring cerebral perfusion pressure (CPP) and brain tissue markers of water balance, oxidative stress and apoptosis. METHODS: In this randomised controlled study, animals undergoing HS (target mean arterial pressure (MAP) 40 mmHg for 30 minutes) were randomised to receive lactated Ringer's solution (LR group; n =14; volume equal to three times the volume bled), terlipressin (TERLI group; n =14; 2-mg bolus), no treatment (HAEMO group; n =12) or sham (n =6). CPP, systemic haemodynamics (thermodilution technique) and blood gas analyses were registered at baseline, shock and 5, 30, 60 (T60), 90 and 120 minutes after treatment (T120). After the animals were killed, brain tissue samples were obtained to measure markers of water balance (aquaporin-4 (AQP4)), Na(+)-K(+)-2Cl(-) co-transporter (NKCC1)), oxidative stress (thiobarbituric acid reactive substances (TBARS) and manganese superoxide dismutase (MnSOD)) and apoptotic damage (Bcl-x and Bax). RESULTS: Despite the HS-induced decrease in cardiac output (CO) and hyperlactataemia, resuscitation with terlipressin recovered MAP and resulted in restoration of CPP and in cerebral protection expressed by normalisation of AQP4, NKCC1, TBARS and MnSOD expression and Bcl-x/Bax ratio at T60 and T120 compared with sham animals. In the LR group, CO and blood lactate levels were recovered, but the CPP and MAP were significantly decreased and TBARS levels and AQP4, NKCC1 and MnSOD expression and Bcl-x/Bax ratio were significantly increased at T60 and T120 compared with the sham group. CONCLUSIONS: During recovery from HS-induced hypotension, terlipressin was effective in normalising CPP and cerebral markers of water balance, oxidative damage and apoptosis. The role of this pressor agent on brain perfusion in HS requires further investigation.

Evaluation of IL17A expression and of IL17A, IL17F and IL23R gene polymorphisms in Brazilian individuals with periodontitis.

The IL23/Th17 axis plays an important role in the pathogenesis of cell-mediated tissue damage caused either by autoimmunity or immune responses against bacterial infection. Single nucleotide polymorphisms in the IL17A, IL17F and IL23R genes have been associated with several inflammatory diseases. However, these polymorphisms have not yet been studied in periodontitis. The aim of present study was to evaluate the expression of IL17A and occurrence of the IL17A (rs2275913), IL17F (rs763780) and IL23R (rs11209026) gene polymorphisms in different clinical forms or severity of periodontitis in a sample of Brazilian individuals. Peripheral blood was obtained from 30 non-smoker individuals and analyzed by flow cytometry to determine IL-17 expression. Genomic DNA was obtained from oral swabs in 180 individuals and analyzed by Real-time PCR. The study group was composed by individuals without periodontitis (control), with aggressive periodontitis (AP) and with chronic periodontitis (CP). Higher frequency of IL17A+CD4+ T cells was observed in control group. The A+ genotype from IL17A (rs2275913) was associated with lack of disease. No association was found considering the IL17F and IL23R polymorphisms. Our data suggest that IL17A and the presence of IL17A (rs2275913) A allele are associated with the absence of periodontal disease.

Unequivocal identification of subpopulations in putative multiclonal Trypanosoma cruzi strains by FACs single cell sorting and genotyping.

Trypanosoma cruzi, the etiological agent of Chagas disease, is a polymorphic species. Evidence suggests that the majority of the T. cruzi populations isolated from afflicted humans, reservoir animals, or vectors are multiclonal. However, the extent and the complexity of multiclonality remain to be established, since aneuploidy cannot be excluded and current conventional cloning methods cannot identify all the representative clones in an infection. To answer this question, we adapted a methodology originally described for analyzing single spermatozoids, to isolate and study single T. cruzi parasites. Accordingly, the cloning apparatus of a Fluorescence-Activated Cell Sorter (FACS) was used to sort single T. cruzi cells directly into 96-wells microplates. Cells were then genotyped using two polymorphic genomic markers and four microsatellite loci. We validated this methodology by testing four T. cruzi populations: one control artificial mixture composed of two monoclonal populations--Silvio X10 cl1 (TcI) and Esmeraldo cl3 (TcII)--and three naturally occurring strains, one isolated from a vector (A316A R7) and two others derived from the first reported human case of Chagas disease. Using this innovative approach, we were able to successfully describe the whole complexity of these natural strains, revealing their multiclonal status. In addition, our results demonstrate that these T. cruzi populations are formed of more clones than originally expected. The method also permitted estimating of the proportion of each subpopulation of the tested strains. The single-cell genotyping approach allowed analysis of intrapopulation diversity at a level of detail not achieved previously, and may thus improve our comprehension of population structure and dynamics of T. cruzi. Finally, this methodology is capable to settle once and for all controversies on the issue of multiclonality.

Toll receptors type-2 and CR3 expression of canine monocytes and its correlation with immunohistochemistry and xenodiagnosis in visceral leishmaniasis.

The aim of the present study was to investigate TLR2 expression in peripheral blood monocytes from dogs naturally infected with Leishmania (Leishmania) infantum to determine whether it correlates with CD11b/CD18 (CR3) expression, and to evaluate the potential of dogs as sources of infection using phlebotomine xenodiagnosis. Forty eight dogs were serologically diagnosed with L. infantum infection by indirect immunofluorescence antibody test (IFAT) and enzyme linked immunosorbent assay (ELISA). Parasitological exams from bone-marrow aspirates were positive by PCR analysis. All dogs were clinical defined as symptomatic. Ear skin tissue samples were obtained for immunohistochemistry (IHQ) analysis. The potential of these dogs as a source of infection using phlebotomine xenodiagnosis (XENO) was evaluated. Flow cytometry was carried out on peripheral blood mononuclear cells using superficial receptors including CD14, CD11b, TLR2 and MHCII. IHQ ear skin tissue parasite load and XENO where done where we found a strict correlation (r = 0.5373). Dogs with higher expression of MFI of CD11b inside CD14 monocytes were represented by dogs without parasite ear tissue load that were unable to infect phlebotomines (IHQ⁻/XENO⁻). Dogs with lower expression of MFI of CD11b inside CD14 monocytes were represented by dogs with parasite ear tissue load and able to infect phlebotomines (IHQ⁺/XENO⁺) (p = 0,0032). Comparable results were obtained for MFI of MHCII (p = 0.0054). In addition, considering the population frequency of CD11b⁺TLR2⁺ and CD11b⁺MHCII⁺, higher values were obtained from dogs with IHQ⁻/XENO⁻ than dogs with IHQ⁺/XENO⁺ (p = 0.01; p = 0.0048, respectively). These data, together with the TLR2 and NO assays results (CD11b⁺TLR2⁺ and NO with higher values for dogs with IHQ⁻/XENO⁻ than dogs with IHQ⁺/XENO⁺, led to the conclusion that IHQ⁻/XENO⁻ dogs are more resistant or could modulate the cellular immune response essential for Leishmania tissue clearance.