Bacteria associated with apical periodontitis promotes in vitro the differentiation of macrophages to osteoclasts.

OBJECTIVE: To analyze the possible in vitro effect of the cytokine RANKL and bacteria involved in apical periodontitis on the differentiation of macrophages into osteoclasts. MATERIAL AND METHODS: Bacteria were isolated (mainly E. faecium and E. faecalis) from the root canal of fifty patients with apical periodontitis, the possible effect of these bacteria on the phagocytic activity of the monocyte cell line THP-1 was analyzed by flow cytometry. Furthermore, the effect of these bacteria (alone or in combination with the cytokine RANKL) on the differentiation of THP-1 macrophages into osteoclasts was analyzed through the expression of the receptor RANK and the tartrate-resistant acid phosphatase TRAP. Finally, the release of different cytokines (IL-1ß, TNF-α, IL-6, IL-8, IL-10, and IL-12p70) by THP-1 cells induced to differentiate into osteoclasts was also analyzed. RESULTS: We observed a significant proportion of THP-1 cells were able to internalize E. faecium and E. faecalis. Furthermore, these bacteria were able to induce (alone or in combination with RANKL) a significant expression of RANK by THP-1 macrophages; accordingly, E. faecium and E. faecalis induced very significant levels of TRAP in these cells. Finally, during the differentiation of THP-1 macrophages induced by RANKL or bacteria, a significant release of the pro-inflammatory cytokines IL-6 and TNF-α was observed. CONCLUSIONS AND CLINICAL RELEVANCE: Our data suggest that the causative agents of apical periodontitis can induce the differentiation of osteoclasts as well as the release of pro-inflammatory cytokines, phenomena that may have an important role in the bone damage observed in this condition.

Induction of transcription factors, miRNAs and cytokines involved in T lymphocyte differentiation in BCG-vaccinated subjects.

The BCG vaccine induces a Th1 phenotype, which is essential for protection against Mycobacterium tuberculosis. However, the effects of BCG vaccination over time on the T helper subpopulation and the microRNAs involved in adulthood have not been studied. In the present study, we explored the involvement of microRNAs, transcription factors and multifunctional cytokines in BCG vaccination by examining their levels both before and after vaccination of healthy adults. Peripheral blood mononuclear cells were obtained at 0, 2 and 6 months after vaccination. Cells were cultured in the presence or absence of ESAT-6 and CFP-10 or M. tuberculosis filtrate. The expression levels of miRNAs and transcription factors were evaluated using qRT-PCR. Cytokine production in supernatants and serum samples was evaluated using ELISA. Multifunctional CD4+ T cells were analyzed using multiparametric flow cytometry. We observed a decrease in the expression levels of T-BET, GATA3 and FOXP3 at 2 months and miR-146a, miR-326 and miR-155 at 6 months after receiving the vaccine. In the supernatant, the production of IL-17 was increased after 6 months, with both stimuli. In contrast, IL-10, TNF-α and IFN-γ increased at 2 months. In the serum, high levels of IL-10 were found after 2 months compared to time 0 and 6 months. The production of multifunctional cells that expressed the cytokine profiles CD4+TNF-α+IFN-γ-IL-10-, CD4+TNF-α+IL-1IFN-γ-, CD4+IL-10+IFN-γ-TNF-α- and CD4+IL-17+IFN-γ- predominantly increased after 2 months with and without the stimulus. Correlation analysis revealed a negative association between FOXP3 and miR-155 (r=-0.5120, p=0.0176) and between IL-17 and miR-326 (r=-0.5832, p=0.0364). This study is the first to demonstrate roles for microRNAs, transcription factors and cytokines in the T helper differentiation lineage and to describe the possible mechanism by which their expression is modulated by the presence of the BCG vaccine in adulthood. In conclusion, our results suggest that the BCG vaccine induces a modulation in transcription factors and miRNAs with high production of multifunctional cells CD4+TNF-α+IL-10+IFN-γ-.

Analysis of expression and function of the co-stimulatory receptor SLAMF1 in immune cells from patients with systemic lupus erythematosus (SLE).

The signaling lymphocytic activation molecule SLAMF1 (CD150) is a co-stimulatory molecule that is expressed by most immune cells, including T regulatory (Treg) lymphocytes. Since different abnormalities have been reported regarding the number and function of Foxp3+ Treg cells in patients with systemic lupus erythematosus (SLE), we decided to analyze the expression and function of CD150 in these regulatory lymphocytes in this condition. We isolated peripheral blood mononuclear cells from 20 patients with SLE, and 20 healthy controls. The expression of SLAMF1 was determined by multi-parametric flow cytometry and the suppressive function of CD4+CD25+ lymphocytes, upon engagement or not of CD150 with an agonistic monoclonal antibody, was analyzed by an assay of inhibition of cell proliferation. We observed a significantly increased expression of SLAMF1 by CD3+CD4+ helper T cells and CD19+ B cells in patients with SLE and active disease. However, similar levels of SLAMF1 expression were detected in Foxp3+ Treg cells from patients and controls. In contrast, a higher proportion of SLE patients increased their suppressive function of Treg cells upon CD150 engagement compared to healthy controls. Our data suggest that SLAMF1 is another significant piece in the intricate defective immune-regulatory function of patients with SLE.

Decreased blood levels of B lymphocytes and NK cells in patients with systemic lupus erythematosus (SLE) infected with papillomavirus (HPV).

Patients with systemic lupus erythematosus (SLE) show an enhanced risk to develop human papillomavirus (HPV) infection, and aggressive forms of this condition are seen in these patients. The aim of this study was to assess the possible relationship among HPV infection, immunosuppressive therapy and levels of leukocyte subsets in patients with SLE. The following individuals were included in the study: (1) SLE patients under immunosuppressive therapy and with lesions caused by HPV (n = 16); (2) SLE patients under immunosuppressive therapy and no evidence of HPV infection (n = 20); (3) untreated SLE patients with no evidence of HPV infection (n = 7), and; (4) healthy female subjects without evidence of HPV infection (n = 10). Peripheral blood was obtained and the percentages of different lymphocyte subsets were determined by flow cytometry, with the use of the following monoclonal antibodies: CD3, CD4, CD8, CD16, CD19, CD20, CD22, CD56, and CD335 (NKp46). We found that SLE patients under immunosuppressive therapy and with lesions caused by HPV showed significantly lower levels of B lymphocytes and NK cells compared to other groups. In contrast, SLE patients receiving immunosuppressive drugs and with no evidence of HPV infection showed similar levels of B and NK cells than healthy controls. Those patients receiving mycophenolate mofetil (MMF) had a diminished number of B cells, and a positive correlation was detected between the dose of MMF and the number of HPV skin lesions. Our data suggest that therapy of SLE patients with MMF is associated with diminished levels of B and NK cells and an enhanced risk for HPV infection.

Expression and function of Cbl-b in T cells from patients with systemic lupus erythematosus, and detection of the 2126 A/G Cblb gene polymorphism in the Mexican mestizo population.

Systemic lupus erythematosus (SLE) is characterized by abnormalities in the function of T and B lymphocytes and in the signaling pathways induced through their receptors. Cbl-b is an intracellular adaptor protein that plays a key role in the negative regulation of lymphocyte activity. We explored the expression and function of Cbl-b in T lymphocytes from SLE patients. In addition, the possible association of SLE and a single nucleotide polymorphism (SNP) of the Cblb gene was determined. We studied 150 SLE patients, 163 healthy individuals, and 14 patients with rheumatoid arthritis (RA). The expression of Cbl-b was analyzed in the peripheral blood mononuclear cells, and the negative regulatory function of Cbl-b was assessed by analyzing actin polymerization and the phosphorylation of JNK and c-Jun induced through CD3. Furthermore, the 2126(A/G) SNP of the Cblb gene was detected by real-time polymerase chain reaction. We found a significant small reduction in the expression of Cbl-b as well as increased levels of activation of c-Jun and actin polymerization in T lymphocytes from patients with SLE compared with healthy controls or RA patients. In addition, a significant association between the 2126(A/G) SNP and SLE was detected. Our data suggest that Cbl-b may contribute to the deregulated activation of T lymphocytes observed in SLE.

Effect of arsenic on regulatory T cells.

INTRODUCTION: Arsenic (As) affects the function and survival of lymphocytes, and some arsenic compounds exert a relevant antineoplastic effect. We have explored the effect of As on T regulatory cells. RESULTS AND DISCUSSION: In vitro experiments with peripheral blood mononuclear cells from healthy subjects showed that low concentrations of As tended to increase the number of natural T regulatory (nTreg) lymphocytes, whereas concentrations >5.0 muM had an opposite effect. Furthermore, rats exposed to As showed redistribution of nTreg cells, and As administration to rats with experimental allergic encephalomyelitis increased the levels of nTreg cells in spleen and diminished the severity of this condition. On the other hand, in 47 apparently healthy subjects chronically exposed to As, we found significant inverse correlation between urinary As levels and the number and function of nTreg lymphocytes. Although most of these individuals showed enhanced levels of apoptotic lymphocytes in peripheral blood, with a diminution of mitochondrial membrane potential, no significant correlation between these parameters and urinary As was detected. CONCLUSION: Our data indicate that As seems to have a relevant and complex effect on nTreg cells.

P2X7 and NRAMP1/SLC11 A1 gene polymorphisms in Mexican mestizo patients with pulmonary tuberculosis.

Tuberculosis remains one of the most important infectious diseases worldwide. Several studies have suggested that genetic factors may affect susceptibility to tuberculosis, but the specific genes involved have not yet been fully characterized. NRAMP1/SLC11 A1 and P2X(7) genes have been linked to increased risk for tuberculosis in some African and Asiatic populations. To explore the potential role of these genes in the susceptibility to pulmonary tuberculosis in a Mexican mestizo population, we evaluated the association of D543N and 3'-UTR polymorphisms in NRAMP1/SLC11 A1 and - 762 and A1513C polymorphisms in P2X(7) genes with the risk for tuberculosis. Polymerase chain reaction (PCR) amplification of genomic DNA followed by restriction fragment length polymorphism analysis, and allelic-specific PCR was employed. We found no significant differences in allelic frequency in NRAMP1/SLC11 A1 gene polymorphisms in 94 patients with tuberculosis compared to 100 healthy contacts. Similarly, no significant association of the P2X(7)-762 gene polymorphism with tuberculosis was detected. In contrast, the P2X(7) A1513C polymorphism was associated significantly with tuberculosis (P = 0.02, odds ratio = 5.28, 95% CI, 0.99-37.69), an association that had not been reported previously. However, when the function of P2X(7) was assessed by an L-selectin loss assay, we did not find significant differences in patients compared to healthy contacts or between PPD(+) and PPD(-) control individuals. This study further supports the complex role of P2X(7) gene in host regulation of Mycobacterium tuberculosis infection, and demonstrates that different associations of gene polymorphisms and tuberculosis are found in distinct racial populations.