α-IRAK-4 Suppresses the Activation of RANK/RANKL Pathway on Macrophages Exposed to Endodontic Microorganisms.

Periapical lesions are common pathologies affecting the alveolar bone, often initiated by intraradicular lesions resulting from microbial exposure to dental pulp. These microorganisms trigger inflammatory and immune responses. When endodontic treatment fails to eliminate the infection, periapical lesions persist, leading to bone loss. The RANK/RANKL/OPG pathway plays a crucial role in both the formation and the destruction of the bone. In this study, the objective was to inhibit the RANK/RANKL pathway in vitro within exposed Thp-1 macrophages to endodontic microorganisms, specifically Enterococcus faecalis, which was isolated from root canals of 20 patients with endodontic secondary/persistent infection, symptomatic and asymptomatic, and utilizing an α-IRAK-4 inhibitor, we introduced endodontic microorganisms and/or lipoteichoic acid from Streptococcus spp. to cellular cultures in a culture plate, containing thp-1 cells and/or PBMC from patients with apical periodontitis. Subsequently, we assessed the percentages of RANK+, RANKL+, and OPG+ cells through flow cytometry and measured the levels of several inflammatory cytokines (IL-1ß, TNF-α, IL-6, IL-8, IL-10, and IL-12p70) in the cellular culture supernatant through a CBA kit and performed analysis by flow cytometry. A significant difference was observed in the percentages of RANK+RANKL+, OPG+ RANKL+ cells in thp-1 cells and PBMCs from patients with apical periodontitis. The findings revealed significant differences in the percentages of the evaluated cells, highlighting the novel role of the IRAK-4 inhibitor in addressing this oral pathology, apical periodontitis, where bone destruction is observed.

Differentiation of circulating monocytes into macrophages with metabolically activated phenotype regulates inflammation in dyslipidemia patients.

Macrophages are mediators of inflammation having an important role in the pathogenesis of cardiovascular diseases. Recently, a pro-inflammatory subpopulation, known as metabolically activated macrophages (MMe), has been described in conditions of obesity and metabolic syndrome where they are known to release cytokines that can promote insulin resistance. Dyslipidemia represents an important feature in metabolic syndrome and corresponds to one of the main modifiable risk factors for the development of cardiovascular diseases. Circulating monocytes can differentiate into macrophages under certain conditions. They correspond to a heterogeneous population, which include inflammatory and anti-inflammatory subsets; however, there is a wide spectrum of phenotypes. Therefore, we decided to investigate whether the metabolic activated monocyte (MoMe) subpopulation is already present under dyslipidemia conditions. Secondly, we assessed whether different levels of cholesterol and triglycerides play a role in the polarization towards the metabolic phenotype (MMe) of macrophages. Our results indicate that MoMe cells are found in both healthy and dyslipidemia patients, with cells displaying the following metabolic phenotype: CD14varCD36+ABCA1+PLIN2+. Furthermore, the percentages of CD14++CD68+CD80+ pro-inflammatory monocytes are higher in dyslipidemia than in healthy subjects. When analysing macrophage differentiation, we observed that MMe percentages were higher in the dyslipidemia group than in healthy subjects. These MMe have the ability to produce high levels of IL-6 and the anti-inflammatory cytokine IL-10. Furthermore, ABCA1 expression in MMe correlates with LDL serum levels. Our study highlights the dynamic contributions of metabolically activated macrophages in dyslipidemia, which may have a complex participation in low-grade inflammation due to their pro- and anti-inflammatory function.

Differential Expression of AhR in Peripheral Mononuclear Cells in Response to Exposure to Polycyclic Aromatic Hydrocarbons in Mexican Women.

The exposure to air pollutants causes significant damage to health, and inefficient cooking and heating practices produce high levels of household air pollution, including a wide range of health-damaging pollutants such as fine particles, carbon monoxide and PAHs. The exposure to PAHs has been associated with the development of neoplastic processes, asthma, genotoxicity, altered neurodevelopment and inflammation. The effects on the induction of proinflammatory cytokines are attributed to the activation of AhR. However, the molecular mechanisms by which the PAHs produce proinflammatory effects are unknown. This study was performed on a group of 41 Mexican women from two rural communities who had stoves inside their houses, used wood as biomass fuel, and, thus, were vulnerable. According to the urinary 1-OHP concentration, the samples were stratified into two groups for determination of the levels of TNF-α, AhR, CYP1B1, miR-125b and miR-155 expression. Our results showed that the CYP1B1, TNF-α, miR-125b and miR-155 expression levels were not statistically different between women with the lowest and highest levels of 1-OHP. Interestingly, high levels of PAHs promoted augmented expression of AhR, which is a protein involved in the modulation of inflammatory pathways in vivo, suggesting that cell signaling of AhR may be implicated in several pathogenesis processes.

Impact of type 2 diabetes on the capacity of human macrophages infected with Mycobacterium tuberculosis to modulate monocyte differentiation through a bystander effect.

Type 2 diabetes mellitus (T2D) is a risk factor for the development of tuberculosis (TB) through mechanisms poorly understood. Monocytes and macrophages are key effector cells to control TB, but they are also subverted by Mycobacterium tuberculosis (Mtb). Specifically, Mtb can induce a bystander effect that skews monocyte differentiation towards macrophages with a permissive phenotype to infection. Here, we evaluated whether T2D impacts this TB aspect. Our approach was to differentiate monocytes from healthy control (HC) subjects and T2D patients into macrophages (MDM), and then assess their response to Mtb infection, including their secretome content and bystander effect capacity. Through flow cytometric analyses, we found a lower level of activation markers in MDM from T2D patients than from HC in response to mock (HLA-DR, CD86 and CD163) or Mtb challenge (CD14 and CD80). In spite of high TGF-ß1 levels in mock-infected MDM from T2D patients, cytometric bead arrays indicated that there were no major differences in the secretome cytokine content in these cells relative to HC-MDM, even in response to Mtb. Mimicking a bystander effect, the secretome of Mtb-infected HC-MDM drove HC monocytes towards MDM with a permissive phenotype for Mtb intracellular growth. However, the secretome from Mtb-infected T2D-MDM did not exacerbate the Mtb load compared to secretome from Mtb-infected HC-MDM, possibly due to the high IL-1ß production relative to Mtb-infected HC-MDM. Collectively, despite T2D affecting the basal MDM activation, our approach revealed that it has no major consequence on their response to Mtb or capacity to generate a bystander effect influencing monocyte differentiation.

Both in vitro T cell proliferation and telomere length are decreased, but CD25 expression and IL-2 production are not affected in aged men.

INTRODUCTION: Aging is a natural process involving dysfunction of multiple organs and is characterized by increased susceptibility to infections, cancer and autoimmune diseases. The functionality of the immune system depends on the capacity of lymphocytes to proliferate in response to antigenic challenges, and telomere length has an important role regulating the number of cell divisions. The aim of this study was to determine the possible relationship between telomere length, interleukin 2 (IL-2) production, CD25 expression and proliferation of peripheral blood mononuclear cells (PBMCs) in aged men. MATERIAL AND METHODS: Telomere length was measured by RT-PCR in PBMCs from young and aged men. IL-2 production and CD25 expression were determined by ELISA and flow cytometry, respectively. Cell proliferation was measured by CFSE dilution assays upon in vitro stimulation with concanavalin A (Con A). RESULTS: PBMCs from aged men showed a shorter telomere length and a reduced capacity to proliferate in vitro, compared to young men. In contrast, no significant differences in the level of CD25 expression on T lymphocytes, and in vitro production of IL-2 were detected in both groups. In addition, no significant correlation was detected between levels of CD25 expression, IL-2 production, cell proliferation, and telomere length in aged men. CONCLUSIONS: In aged men the telomere length shortening and the reduced T cell proliferation are not related to the capacity of IL-2 production and CD25 expression on T lymphocytes.

Expression and activity of AIM2-inflammasome in rheumatoid arthritis patients.

INTRODUCTION: AIM2 inflammasome activation leads to the release of IL-ß, which plays an important role in rheumatoid arthritis pathogenesis. In this work, we evaluated AIM2 expression and activity in RA patients and healthy controls. METHODS: AIM2 and RANKL expression were evaluated by flow cytometry. Inflammasome activity was determined in monocyte cultures stimulated with synthetic DNA by measuring IL-1ß levels in supernatants using an ELISA assay. The caspase-1 expression in monocytes was measured by western blot, the POP3 expression was analysed by qPCR, and serum levels of IFN-γ were evaluated using ELISA assay. RESULTS: We observed a diminution of CD14+AIM2+ cells in RA patients, associated with disease activity and evolution. Likewise, the levels of IL-1ß were increased in monocyte cultures un-stimulated and stimulated with LPS from RA patients with DAS28 ≥ 4. The Caspase-1 activity and RANKL + monocytes in RA patients were slightly increased. Finally, augmented POP3 expression and diminished IFN-γ serum levels were detected in RA patients. CONCLUSION: Our results showed that the monocytes from RA patients were prone to release IL-1ß in the absence of the AIM2 inflammasome signal. The down-regulation of AIM2 to a systemic level in RA patients might be a consequence of augmented POP3 expression and might imply the survival of pro-inflammatory cells contributing to the inflammation process.

Células T reguladoras en lupus eritematoso generalizado / Regulatory T cells in systemic lupus erythematosus

Resumen El lupus eritematoso generalizado (LEG) es una enfermedad autoinmune crónica caracterizada por la pérdida de la tolerancia a los antígenos propios y la síntesis de diferentes autoanticuerpos con la formación y depósito de complejos inmunes y el daño de múltiples órganos. Las células T reguladoras (Treg) desempeñan un papel esencial en el mantenimiento de la tolerancia periférica, controlan el estado de activación del sistema inmune y limitan las respuestas autoinmunes. El estudio del número y la función de las diferentes subpoblaciones de células Treg en LEG ha sido objeto de una intensa investigación. Dependiendo del fenotipo de las células Treg analizado se ha reportado que la frecuencia de estas células en pacientes con LEG se encuentra disminuida, aumentada o sin alteraciones. Además, diferentes grupos han descrito que la función supresora de las células Treg de los pacientes con LEG se encuentra reducida o no se ve afectada. En conjunto, lo datos reportados sugieren que las células Treg desempeñan un papel relevante en la patogénesis del LEG y que estos linfocitos pueden ser considerados blancos potenciales para el diseño de nuevas estrategias terapéuticas para esta enfermedad.

Abstract Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by a loss of tolerance to self-antigens and synthesis of different autoantibodies, with the formation and deposition of immune complexes and damage to multiple organs. T regulatory cells (Tregs) play a crucial role in maintaining peripheral tolerance, controlling the state of activation of the immune system and limiting autoimmune responses. The study of the number and function of the different Treg cell subpopulations in SLE has been the subject of intense research. Depending on the analyzed Treg cell phenotype, the frequency of these cells has been reported to be reduced, increased or unaltered in patients with SLE. In addition, different groups have described that Treg cells suppressive function is reduced or unaffected in patients with SLE. Taken together, the reported data suggest that Treg cells play a relevant role in the pathogenesis of SLE and that these lymphocytes can be considered potential targets for the design of new therapeutic strategies for this condition.

Analysis of expression of the PD-1/PD-L1 immune checkpoint system and its prognostic impact in gastroenteropancreatic neuroendocrine tumors.

The immune checkpoint based therapy targeting the programmed death-1 (PD-1) receptor and its PD-L1 ligand has recently been approved for the therapy of different malignant conditions, but not yet for gastroenteropancreatic neuroendocrine tumors (GEP-NETs). In this context, we evaluated the expression of PD-1 and PD-L1 in GEP-NETs and its potential correlations with clinical outcomes. Expression of PD-1/PD-L1 was analyzed by immunohistochemistry in 116 GEP-NETs and 48 samples of peritumoral tissue. In addition, the expression of these molecules was assessed by flow cytometry in peripheral blood mononuclear cells (PBMC) from patients with GEP-NETs (n = 32) and healthy controls (n = 32) and in intratumoral mononuclear cells (TMCs) (n = 3). Expression of PD-L1 and PD-1 was detected by immunohistochemistry in 6% and 1% of tumor tissue samples, respectively, and in 8% of peritumoral tissue samples, for both markers. We also observed that PD-1 expression by TMCs was associated with metastatic disease at diagnosis, and the levels of circulating PD-1+ PBMCs were associated with progressive disease upon follow-ups. In addition, circulating PD-1+ PBMCs were significantly correlated with PD-L1 expression by tumor cells. Our data suggest that PD-1/PD-L1 is expressed in 1 to 8% of GEP-NETs, and that this feature is significantly associated with disease evolution (p < 0.01).

Patients With Autoimmune Thyroiditis Show Diminished Levels and Defective Suppressive Function of Tr1 Regulatory Lymphocytes.

Context: T regulatory type 1 (Tr1) cells are a subpopulation of T lymphocytes (CD4+CD49+LAG-3+IL-10+) that exert a considerable immunosuppressive effect. However, their possible role in autoimmune thyroid disease (AITD) has not been explored so far. Purpose: To analyze the levels and function of Tr1 cells in peripheral blood and thyroid tissue of patients with AITD. Design: Cases and controls, observational study. Setting: Department of Endocrinology, Hospital Universitario de la Princesa, Madrid, Spain. Patients: Thirty-eight patients with AITD (23 with Graves disease and 15 with Hashimoto thyroiditis) and 26 controls. Intervention: Multiparametric flow cytometry and immunofluorescence techniques were used to analyze the levels in peripheral blood (n = 38) and thyroid mononuclear cells (n = 5). An in vitro assay of suppression of cellular activation and cytokine release was performed to study the function of Tr1 cells. Main Outcome Measure: Levels and function of Tr1 cells in patients with AITD and controls. Results: Levels of Tr1 cells were significantly diminished in peripheral blood from patients with AITD. Functional studies showed that Tr1 cells from patients with AITD exhibit a diminished suppressive function compared with healthy controls. Tr1 levels were associated with disease severity, including longer duration of the disease and ophthalmopathy activity, and with autoantibody titers. Conclusions: The low levels of Tr1 cells and their diminished function may have a relevant role in the defective immune-regulatory function characteristic of patients with AITD.

Patients with Systemic Lupus Erythematosus Show Increased Levels and Defective Function of CD69+ T Regulatory Cells.

T regulatory (Treg) cells have a key role in the pathogenesis of chronic inflammatory and autoimmune diseases. A CD4+CD69+ T cell subset has been described that behaves as Treg lymphocytes, exerting an important immune suppressive effect. In this study, we analyzed the levels and function of CD4+CD69+ Treg cells in patients with systemic lupus erythematosus (SLE). Blood samples were obtained from 22 patients with SLE and 25 healthy subjects. Levels of CD4+CD69+ Treg cells were analyzed by multiparametric flow cytometry, and their function was measured by an assay of suppression of lymphocyte activation and through the inhibition of cytokine synthesis. We found an increased percent of CD4+CD25varCD69+TGF-ß+IL-10+Foxp3- lymphocytes in patients with SLE compared to controls. In addition, a significant diminution in the suppressive effect of these cells on the activation of autologous T lymphocytes was observed in most patients with SLE. Accordingly, CD69+ Treg cells from SLE patients showed a defective capability to inhibit the release of IL-2, IL-6, IL-10, and IL-17A by autologous lymphocytes. Our findings suggest that while CD4+CD69+ Treg lymphocyte levels are increased in SLE patients, these cells are apparently unable to contribute to the downmodulation of the autoimmune response and the tissue damage seen in this condition.

FICZ generates human tDCs that induce CD4+ CD25high Foxp3+ Treg-like cell differentiation.

Dendritic cells (DCs) play a central role in the maintenance of immune homeostasis, their participation as professional antigen presenting cells is essential to the initiation of the adaptive immune response as well as to the induction of tolerance. The recently described role of the aryl hydrocarbon receptor (AhR) in the immune system, particularly in the modulation of the adaptive immune response has attracted the attention as a potential player in the induction of immune tolerance. However, the effects of AhR activation through endogenous ligands on human DCs have been poorly evaluated. In this study, we investigated the effect of FICZ, a natural AhR ligand, on monocyte-derived dendritic cells (Mo-DCs) from healthy subjects. We found that the activation of AhR through FICZ during DCs differentiation and maturation processes resulted in a decreased expression of CD83, an increased expression of the enzyme IDO and a reduced production of the pro-inflammatory cytokines IL-6 and TNF-α. More importantly, FICZ-treated DCs were able to induce the differentiation of naive T lymphocytes into CD4+ CD25high Foxp3+ T reg-like cells. Our results show that the activation of the AhR on human DCs induces a tolerogenic phenotype with potential implications in immunotherapy.

Levels of regulatory T cells CD69(+)NKG2D(+)IL-10(+) are increased in patients with autoimmune thyroid disorders.

Regulatory T (Treg) cells play an important role in the pathogenesis of autoimmune thyroid disorders (AITD). New subsets of CD4(+)CD69(+) and CD4(+)NKG2D(+) T lymphocytes that behave as regulatory cells have been recently reported. The role of these immunoregulatory lymphocytes has not been previously explored in AITD. We analyzed by multi-parametric flow cytometry different Treg cell subsets in peripheral blood from 32 patients with AITD and 19 controls, and in thyroid tissue from seven patients. The suppressive activity was measured by an assay of inhibition of lymphocyte activation. We found a significant increased percentage of CD4(+)CD69(+)IL-10(+), CD4(+)CD69(+)NKG2D(+), and CD4(+)CD69(+)IL-10(+)NKG2D(+) cells, in peripheral blood from GD patients compared to controls. The increase in CD4(+)CD69(+)IL-10(+) and CD4(+)CD69(+)IL-10(+)NKG2D(+) T cells was especially remarkable in patients with active Graves' ophthalmopathy (GO), and a significant positive correlation between GO activity and CD4(+)CD69(+)IL-10(+) or CD4(+)CD69(+)IL-10(+)NKG2D(+) cells was also found. In addition, these cells were increased in patients with a more severe and/or prolonged disease. Thyroid from AITD patients showed an increased proportion of CD69(+) regulatory T cells subpopulations compared to autologous peripheral blood. The presence of CD69(+), NKG2D(+), and IL-10(+) cells was confirmed by immunofluorescence microscopy. In vitro functional assays showed that CD69(+) Treg cells exerted an important suppressive effect on the activation of T effector cells in controls, but not in AITD patients. Our findings suggest that the levels of CD69(+) regulatory lymphocytes are increased in AITD patients, but they are apparently unable to down-modulate the autoimmune response and tissue damage.

T regulatory (Treg) and T helper 17 (Th17) lymphocytes in thyroid autoimmunity.

Different immune cell subsets have a relevant role in the pathogenesis of and tissue damage seen in autoimmune thyroid diseases (AITD), including T regulatory (Treg) lymphocytes and T helper (Th) 17 cells. There are several types of CD4+ Treg cells (Foxp3+, CD69+, Tr1), which are able to prevent the appearance of autoimmune diseases, down regulating the immune response and the inflammatory phenomenon. However, despite their presence in peripheral blood and thyroid tissue from patients with AITD, these cells are apparently unable to put down the autoimmune process. Moreover, many reports indicate the involvement of Th17 cells in chronic inflammatory diseases, including AITD. Nevertheless, it is now evident that these lymphocytes show a remarkable plasticity, giving rise to anti-inflammatory (including Treg lymphocytes) and pro-inflammatory cell subtypes. Nowadays, both Treg and Th17 cells must be considered as key elements in the pathogenesis of AITD as well as plausible potential targets for the next generation of therapeutic options of this condition.

Circulating Microvesicles Regulate Treg and Th17 Differentiation in Human Autoimmune Thyroid Disorders.

BACKGROUND: Microvesicles (MVs) are emerging as important contributors to the development of inflammatory and autoimmune diseases. MVs can mediate immune modulation carrying genetic information, including microRNAs that can be transferred between cells. DESIGN: We determined the plasma levels of annexin-V+ MVs derived from different immune cells and platelets in patients with autoimmune thyroid diseases (AITDs) and in healthy controls. T lymphocyte polarization assays were performed in the presence of MVs to evaluate their effect in T regulatory and T helper 17 cells differentiation. microRNA content into plasma MVs and their corresponding mRNA targets were evaluated by RT-PCR. RESULTS: The percentage of platelet-derived MVs (CD41a+) was significantly increased in plasma samples from AITD patients compared with healthy controls. In contrast, patients with AITD showed a lower percentage of leukocyte and endothelial cell-derived MVs compared with controls. In addition, functional assays showed that MVs from AITD patients inhibited the in vitro differentiation of Foxp3+ T regulatory cells (11.35% vs 4.40%, P = .01) and induced the expression of interferon-γ by CD4+ lymphocytes (10.91% vs 13.99%, P = .01) as well as the differentiation of T helper 17 pathogenic (IL-17+interferon-γ+) cells (1.98% vs 5.13%, P = .03). Furthermore, in AITD patients, whereas miR-146a and miR-155 were increased in circulating MVs, their targets IL-8 and SMAD4 were decreased in peripheral blood mononuclear cells. CONCLUSIONS: Our data indicate that circulating MVs seem to have a relevant role in the modulation of the inflammatory response observed in AITD.

CD39 expression on Treg and Th17 cells is associated with metabolic factors in patients with type 2 diabetes.

Th17 cells are involved in the pathogenesis of multiple inflammatory diseases such as type two diabetes (T2D). CD39(+) Treg cells have been implicated as responsible for suppressing Th17 cells. The aim of this study was to evaluate the number and function of CD4(+)CD25(high)CD39(+) Treg and Th17 cells in peripheral blood mononuclear cells (PBMC) from T2D patients and healthy control subjects. The Th17 cells were detected in PBMC under culture with human anti-CD3/CD28 and PMA/ionomycin and the levels of IL-17 were assessed by ELISA and qPCR. The T2D patients with obesity showed significantly lower percentages of CD39(+) Treg cells. A negative correlation between CD39(+) Treg cells and weight, and body mass index was detected. In contrast, the low levels of CD4(+)IL-17(+) cells in overweight and obese T2D patients showed a positive correlation with glucose and HbA1c. Additionally, we found a subpopulation of Th17 cells that express CD39 and were correlated with glucose and HbA1c. Our findings suggest that the expression of CD39 on Treg cells and also in CD4(+)IL-17(+) cells from T2D patients is related to hyperglycemia as well as to overweight and obesity and therefore may participate as a modulator of the effector capacity of Th17 cells.

Graves' disease is associated with a defective expression of the immune regulatory molecule galectin-9 in antigen-presenting dendritic cells.

INTRODUCTION: Patients with autoimmune thyroid disease (AITD) show defects in their immune-regulatory mechanisms. Herein we assessed the expression and function of galectin-1 and galectin-9 (Gal-1, Gal-9) in dendritic cells (DCs) from patients with AITD. MATERIALS AND METHODS: Peripheral blood samples from 25 patients with Graves' disease (GD), 11 Hashimoto's thyroiditis (HT), and 24 healthy subjects were studied. Thyroid tissue samples from 44 patients with AITD and 22 patients with goiter were also analyzed. Expression and function of Gal-1 and Gal-9 was assessed by quantitative RT-PCR, immunofluorescence and flow cytometry. RESULTS: A diminished expression of Gal-9, but not of Gal-1, by peripheral blood DCs was observed in GD patients, mainly in those with Graves´ ophthalmopathy, and a significant negative association between disease severity and Gal-9 expression was detected. In addition, the mRNA levels of Gal-9 and its ligand TIM-3 were increased in thyroid tissue from AITD patients and its expression was associated with the levels of Th1/Th12/Th17 cytokines. Immunofluorescence studies proved that intrathyroidal Gal-9 expression was confined to DCs and macrophages. Finally, in vitro functional assays showed that exogenous Gal-9 had a suppressive effect on the release of Th1/Th2/Th17 cytokines by DC/lymphocyte autologous co-cultures from both AITD patients and healthy controls. CONCLUSIONS: The altered pattern of expression of Gal-9 in peripheral blood DCs from GD patients, its correlation with disease severity as well as its ability to suppress cytokine release suggest that Gal-9 could be involved in the pathogenesis of AITD.

DDE and PCB 153 independently induce aryl hydrocarbon receptor (AhR) expression in peripheral blood mononuclear cells.

Recent studies have demonstrated that compounds inducing pro-inflammatory cytokines enhance AhR expression. The aim of this study was 2-fold: (1) to determine if two pro-inflammatory compounds, dichlorodiphenyldichloroethylene (DDE) and 2,2',4,4',5,5'-hexa-chlorobiphenyl (PCB 153), independently affect AhR gene expression in peripheral blood mononuclear cells (PBMC); and (2) if affected, to determine whether the mechanism involved was due to AhR activation or to a pro-inflammatory effect of the chemicals. PBMC isolated from healthy individuals were incubated in the presence of DDE (10 µg/ml) and PCB 153 (20 ng/ml) over time and AhR and CYP1A1 expression was assessed with a real-time PCR technique. The results indicated there was over-expression of the AhR mRNA in PBMC when the cells were treated with DDE and PCB 153. No changes in expression levels of CYP1A1 mRNA were found. Importantly, when the cells were exposed to DDE and PCB 153 in the presence of an antagonist of tumor necrosis factor (TNF)-α, the over-expression of AhR was abolished; as expected, the expression of CYP1A1 was unaffected. In conclusion, these studies demonstrated for the first time an increment of AhR expression "in vitro" in PBMC treated with two pro-inflammatory environmental pollutants, DDE and PCB153. Moreover, the over-expression of AhR was dependent of TNFα induced by DDE and PCB 153 and was independent of AhR activation.

Laboratory biomarkers for guiding therapy with methotrexate in rheumatoid arthritis.

Methotrexate (MTX) is a first-line drug for the treatment of several rheumatic diseases. However, it is difficult to predict the response to this drug based on clinical manifestations. Although different mechanisms of action have been proposed for the antiinflammatory and immunosuppressive effects of MTX, the best characterized are blockade of the de novo synthesis of purines and pyrimidines, which inhibits DNA synthesis, and induction of adenosine release, which downregulates the effector functions of different immune cells. Thus, variants of the enzymes and other molecules involved in these metabolic pathways are expected to play a relevant role in the therapeutic effect or toxicity of MTX in patients with rheumatoid arthritis (RA). Accordingly, polymorphisms of the genes encoding these proteins have been widely associated with the response to or discontinuation of MTX. In addition, variants of the genes involved in the transportation of MTX inside and outside cells and in its metabolism have also been associated with the efficacy or toxicity of this drug in patients with RA. However, published results are contradictory, and no consensus regarding the best laboratory markers of MTX efficacy has been reached. Therefore, additional prospective studies with a large number of patients are necessary to identify the combination of genetic and nongenetic factors that can predict, with a reasonable level of confidence, the efficacy and toxicity of MTX in patients with RA.

Specific therapy to regulate inflammation in rheumatoid arthritis: molecular aspects.

Rheumatoid arthritis (RA) is a chronic inflammatory disease in which persistent inflammation of synovial tissue results in a progressive functional decline of the joint and premature mortality. TNF inhibitors were the first biological disease-modifying antirheumatic drugs (DMARDs) used to treat RA. Since then, new biological drugs have emerged, such as inhibitors of IL-1, IL-6 and others, with different mechanisms of action that include the depletion of B cells and the inhibition of T-cell costimulation. Recently, RA treatments have incorporated the use of synthetic DMARDs. This review describes the molecular aspects of the mechanisms of action of biological and synthetic DMARDs, discusses the adverse effects and limitations of established therapies and analyses the alternative approaches to RA treatment.