Differential gene expression of peripheral blood mononuclear cells from rheumatoid arthritis patients may discriminate immunogenetic, pathogenic and treatment features

This study aimed to evaluate the association between the differential gene expression profiling of peripheral blood mononuclear cells of rheumatoid arthritis patients with their immunogenetic (human leucocyte antigen shared-epitope, HLA-SE), autoimmune response [anti-cyclic citrullinated peptide (CCP) antibodies], disease activity score (DAS-28) and treatment (disease-modifying antirheumatic drugs and tumour necrosis factor blocker) features. Total RNA samples were copied into Cy3-labelled complementary DNA probes, hybridized onto a glass slide microarray containing 4500 human IMAGE complementary DNA target sequences. The Cy3-monocolour microarray images from patients were quantified and normalized. Analysis of the data using the significance analysis of microarrays algorithm together with a Venn diagram allowed the identification of shared and of exclusively modulated genes, according to patient features. Thirteen genes were exclusively associated with the presence of HLA-SE alleles, whose major biological function was related to signal transduction, phosphorylation and apoptosis. Ninety-one genes were associated with disease activity, being involved in signal transduction, apoptosis, response to stress and DNA damage. One hundred and one genes were associated with the presence of anti-CCP antibodies, being involved in signal transduction, cell proliferation and apoptosis. Twenty-eight genes were associated with tumour necrosis factor blocker treatment, being involved in intracellular signalling cascade, phosphorylation and protein transport. Some of these genes had been previously associated with rheumatoid arthritis pathogenesis, whereas others were unveiled for future research.

Genetic Susceptibility Loci in Rheumatoid Arthritis Establish Transcriptional Regulatory Networks with Other Genes

Linkage studies have identified the human leukocyte antigen (HLA)-DRB1 as a putative rheumatoid arthritis (RA) susceptibility locus (SL). Nevertheless, it was estimated that its contribution was partial, suggesting that other non-HLA genes may play a role in RA susceptibility. To test this hypothesis, we conducted microarray transcription profiling of peripheral blood mononuclear cells in 15 RA patients and analyzed the data, using bioinformatics programs (significance analysis of microarrays method and GeneNetwork), which allowed us to determine the differentially expressed genes and to reconstruct transcriptional networks. The patients were grouped according to disease features or treatment with tumor necrosis factor blocker. Transcriptional networks that were reconstructed allowed us to identify the interactions occurring between RA SL and other genes, for example, HLA-DRB1 interacting with FNDC3A (fibronectin type III domain containing 3A). Given that fibronectin fragments can stimulate mediators of matrix and cartilage destruction in RA, this interaction is of special interest and may contribute to a clearer understanding of the functional role of HLA-DRB1 in RA pathogenesis.

Gene Expression Profiles Stratified according to Type 1 Diabetes Mellitus Susceptibility Regions

The MHC region (6p21) aggregates the major genes that contribute to susceptibility to type 1 diabetes (T1D). Three additional relevant susceptibility regions mapped on chromosomes 1p13 (PTPN22), 2q33 (CTLA-4), and 11p15 (insulin) have also been described by linkage studies. To evaluate the contribution of these susceptibility regions and the chromosomes that house these regions, we performed a large-scale differential gene expression on lymphomononuclear cells of recently diagnosed T1D patients, pinpointing relevant modulated genes clustered in these regions and their respective chromosomes. A total of 4608 cDNAs from the IMAGE library were spotted onto glass slides using robotic technology. Statistical analysis was carried out using the SAM program, and data regarding gene location and biological function were obtained at the SOURCE, NCBI, and FATIGO programs. Three induced genes were observed spanning around the MHC region (6p21-6p23), and seven modulated genes (5 repressed and 2 repressed) were seen spanning around the 6q21-24 region. Additional modulated genes were observed in and around the 1p13, 2q33, and 11p15 regions. Overall, modulated genes in these regions were primarily associated with cellular metabolism, transcription factors and signaling transduction. The differential gene expression characterization may identify new genes potentially involved with diabetes pathogenesis.

Correlation between beta-2-glycoprotein I gene polymorphism and anti-beta-2 glycoprotein I antibodies in patients with multibacillary leprosy

Antiphospholipid antibodies, such as anti-beta 2-glycoprotein I (beta 2GPI), are present in multibacillary leprosy (MB) patients; however, MB patients do not usually present with antiphospholipid antibody syndrome (APS), which is characterized by thromboembolic phenomena (TEP). Rare cases of TEP occur in leprosy patients, but the physiopathology of this condition remains unclear. In this case-control study, we examined whether single-nucleotide polymorphisms (SNPs) of the beta 2GPI gene contributed to the risk of leprosy and APS co-morbidity. SNPs Ser88Asn, Leu247Val, Cys306Gly and Trp316Ser were identified in 113 Brazilian leprosy patients. Additionally, anti-beta 2GPI antibodies and plasma concentrations of beta 2GPI were quantified. The Ser88Asn, Cys306Gly and Trp316Ser SNPs were not risk factors for APS in leprosy. A higher frequency of Val/Val homozygosity was observed in leprosy patients compared to controls (36 vs. 5%; P < 0.001). Forty-two percent of MB and 17% of paucibacillary leprosy patients were positive for anti-beta 2GPI IgM (P = 0.014). There was no correlation between SNP Ser88Asn or Cys306Gly and anti-beta 2GPI antibody levels. In MB patients with positive anti-beta 2GPI IgM, the frequency of Val/Val homozygosity was higher than in controls (32 vs. 15%; P = 0.042). The frequency of the mutant allele Ser316 was higher in MB patients with positive rather than negative anti-beta 2GPI IgM levels (6 vs. 0%; P = 0.040) and was greater than in the control group (6 vs. 1%; P = 0.034). The studied polymorphisms did not influence the plasma concentrations of beta 2GPI. These results suggest that Leu247Val and Trp316Ser SNPs may represent genetic risk factors for anti-beta 2GPI antibody production in MB patients.

Autologous Hematopoietic Stem Cell Transplantation for Childhood Autoimmune Disease

Autologous and allogeneic hematopoietic stem cell transplantation (HSCT) can be used in the management of patients with autoimmune disorders. Experience gained in adults has helped to better define the conditioning regimens required and appropriate selection of patients who are most likely to benefit from autologous HSCT. The field has been shifting toward the use of safer and less intense nonmyeloablative regimens used earlier in the disease course before patients accumulate extensive irreversible organ damage. This article reviews the experience of using autologous HSCT in treating the most common childhood autoimmune and rheumatic diseases, primarily juvenile idiopathic arthritis, systemic lupus erythematosus, and diabetes mellitus.

Hematopoietic SCT modulates gut inflammation in experimental inflammatory bowel disease

Hematopoietic SCT (HSCT) and high-dose chemotherapy are being explored as therapy for various human refractory immune-mediated conditions, including inflammatory bowel diseases (IBD). Nevertheless, the exact immunological mechanisms by which the BM cells (BMCs) or immunosuppression provide remission from these diseases is not yet clear. In this work, we investigated the role of these therapies in the modulation of gut mucosal inflammation in an experimental model of IBD. Colitis was induced in mice by 2,4,6-trinitrobenzenesulfonic acid and after CY was administered (200 mg/kg) alone (CY group) or followed by BMCs infusion (HSCT group). Animals were followed for 60 days. Both HSCT and CY reduced the histopathological features of colitis significantly. Infused cells were localized in the gut, and a marked decrease of CD4(+) leukocytes in the inflammatory infiltrate on days +7 and +14 and of CD8(+) cells on day +7 was found in both treatments allied to impressive reduction of proinflammatory Th1 and Th17 cytokines. Although chemotherapy alone was the best treatment regarding the induction of immunosuppressive molecules, only HSCT resulted in increased survival rates compared with the control group. Our findings indicate that high-dose CY followed by HSCT is effective in the modulation of mucosal immunity and in accelerating immune reconstitution after BMT, thus providing valuable tools to support the development and understanding of novel therapeutic strategies for IBD. Bone Marrow Transplantation (2010) 45, 1562-1571; doi:10.1038/bmt.2010.6; published online 15 March 2010

Long-Term Outcome and Prognostic Factors of Juvenile Dermatomyositis: A Multinational, Multicenter Study of 490 Patients

Objective. To investigate the long-term outcome and prognostic factors of juvenile dermatomyositis (DM) through a multinational, multicenter study. Methods. Patients consisted of inception cohorts seen between 1980 and 2004 in 27 centers in Europe and Latin America. Predictor variables were sex, continent, ethnicity, onset year, onset age, onset type, onset manifestations, course type, disease duration, and active disease duration. Outcomes were muscle strength/endurance, continued disease activity, cumulative damage, muscle damage, cutaneous damage, calcinosis, lipodystrophy, physical function, and health-related quality of life (HRQOL). Results. A total of 490 patients with a mean disease duration of 7.7 years were included. At the cross-sectional visit, 41.2-52.8% of patients, depending on the instrument used, had reduced muscle strength/endurance, but less than 10% had severe impairment. Persistently active disease was recorded in 41.2-60.5% of the patients, depending on the activity measure used. Sixty-nine percent of the patients had cumulative damage. The frequency of calcinosis and lipodystrophy was 23.6% and 9.7%, respectively. A total of 40.7% of the patients had decreased functional ability, but only 6.5% had major impairment. Only a small fraction had decreased HRQOL. A chronic course, either polycyclic or continuous, consistently predicted a poorer outcome. Mortality rate was 3.1%. Conclusion. This study confirms the marked improvement in functional outcome of juvenile DM when compared with earlier literature. However, many patients had continued disease activity and cumulative damage at followup. A chronic course was the strongest predictor of poor prognosis. These findings highlight the need for treatment strategies that enable a better control of disease activity over time and the reduction of nonreversible damage.

The Provisional Paediatric Rheumatology International Trials Organisation/American College of Rheumatology/European League Against Rheumatism disease activity core set for the evaluation of response to therapy in juvenile dermatomyositis: A prospective validation study

Objective. To validate a core set of outcome measures for the evaluation of response to treatment in patients with juvenile dermatomyositis (DM). Methods. In 2001, a preliminary consensus-derived core set for evaluating response to therapy in juvenile DM was established. In the present study, the core set was validated through an evidence-based, large-scale data collection that led to the enrollment of 294 patients from 36 countries. Consecutive patients with active disease were assessed at baseline and after 6 months. The validation procedures included assessment of feasibility, responsiveness, discriminant and construct ability, concordce in the evaluation of response to therapy between physicians and parents, redundancy, internal consistency, and ability to predict a therapeutic response. Results. The following clinical measures were found to be feasible, and to have good construct validity, discriminative ability, and internal consistency; furthermore, they were not redundant, proved responsive to clinically important changes in disease activity, and were associated strongly with treatment outcome and thus were included in the final core set: 1) physician`s global assessment of disease activity, 2) muscle strength, 3) global disease activity measure, 4) parent`s global assessment of patient`s well-being, 5) functional ability, and 6) health-related quality of life. Conclusion. The members of the Paediatric Rheumatology International Trials Organisation, with the endorsement of the American College of Rheumatology and the European Leauge Against Rheumatism, propose a core set of criteria for the evaluation of response of therapy that is scientifically and clinically relevant and statistically validated. The core set will help standardize the conduct and reporting of clinical trials and assist practitioners in deciding whether a child with juvenile DM has responded adequately to therapy.

The genetic structure of 3 ` untranslated region of the HLA-G gene: polymorphisms and haplotypes

The HLA-G gene is predominantly expressed at the maternal-fetal interface. It has been associated with maternal-fetal tolerance and in the inhibition of cytotoxic T lymphocyte and natural killer cytolytic functions. At least two variations in the 3` untranslated region (UTR) of HLA-G locus are associated with HLA-G expression levels, the 14-bp deletion/insertion polymorphism and the +3142 single-nucleotide polymorphism (SNP). However, this region has not been completely characterized yet. The variability of the 3`UTR of HLA-G gene and its haplotype structure were characterized in 155 individuals from Brazil, as well as HLA-G alleles associated with each of the 3`UTR haplotype. The following eight variation sites were detected: the 14-bp polymorphism and SNPs at the positions +3003T/C, +3010C/G, +3027A/C, +3035C/T, +3142G/C, +3187A/G and +3196C/G. Similarly, 11 different 3`UTR haplotypes were identified and several HLA-G alleles presented only one 3`UTR haplotype. In addition, a high linkage disequilibrium among the variation sites was detected, especially among the 14-bp insertion and the alleles +3142G and +3187A, all previously associated with low mRNA availability, demonstrating that their effects are not independent. The detailed analyses of 3`UTR of the HLA-G locus may shed some light into mechanisms underlying the regulation of HLA-G expression. Genes and Immunity (2010) 11, 134-141; doi: 10.1038/gene.2009.74; published online 1 October 2009

Mutations involved in Aicardi-Goutieres syndrome implicate SAMHD1 as regulator of the innate immune response

Aicardi-Goutieres syndrome is a mendelian mimic of congenital infection and also shows overlap with systemic lupus erythematosus at both a clinical and biochemical level. The recent identification of mutations in TREX1 and genes encoding the RNASEH2 complex and studies of the function of TREX1 in DNA metabolism have defined a previously unknown mechanism for the initiation of autoimmunity by interferon-stimulatory nucleic acid. Here we describe mutations in SAMHD1 as the cause of AGS at the AGS5 locus and present data to show that SAMHD1 may act as a negative regulator of the cell-intrinsic antiviral response.

Enhanced programmed death 1 (PD-1) and PD-1 ligand (PD-L1) expression in patients with actinic cheilitis and oral squamous cell carcinoma

PD-1 and PD-L1 can be involved in tumor escape, and little is known about the role of these molecules in oral tumors or pre-malignant lesions. In the present study, we investigated the expression of PD-1 and PD-L1 in the blood and lesion samples of patients with actinic cheilitis (AC) and oral squamous cell carcinoma (OSCC). Our results showed that lymphocytes from peripheral blood and tissue samples exhibited high expression of PD-1 in both groups analyzed. Patients with AC presented higher percentage as well as the absolute numbers of CD4(+)PD-1(+) and CD8(+)PD-1(+) lymphocytes in peripheral blood mononuclear cells (PBMC) than healthy individuals, while patients with OSCC presented an increased frequency of CD8(+)PD1(+) in PBMC when compared with controls. On the other hand, increased frequency of CD4(+) and CD8(+) T cells expressing PD-1(+) accumulate in samples from OSCC, and the expression of PD-L1 was intense in OSCC and moderate in AC lesion sites. Lower levels of IFN-gamma and higher levels of TGF-beta were detected in OSCC samples. Our data demonstrate that PD-1 and PD-L1 molecules are present in blood and samples of AC and OSCC patients. Further studies are required to understand the significance of PD-1 and PD-L1 in oral tumors microenvironment.

The broad effects of the functional IL-10 promoter-592 polymorphism: modulation of IL-10, TIMP-3, and OPG expression and their association with periodontal disease outcome

Periodontal diseases are infectious diseases, in which periodontopathogens trigger chronic inflammatory and immune responses that lead to tissue destruction. It occurs through the generation of metalloproteinases and the activation of bone resorption mechanisms. Anti-inflammatory cytokines such as IL-10 seem to attenuate periodontal tissue destruction through the induction of tissue inhibitors of metalloproteinases (TIMPs) and the inhibitor of osteoclastogenesis osteoprotegerin (OPG). A high individual variation in levels of IL-10 mRNA is verified in periodontitis patients, which is possibly determined by genetic polymorphisms. In this study, the IL-10 promoter -592C/A single nucleotide polymorphism ( SNP), which is associated with a decrease in IL-10 production, was analyzed by RFLP in 116 chronic periodontitis (CP) patients and 173 control (C) subjects, and the IL-10, TIMPs, and OPG mRNA expression levels in diseased gingival tissues were determined by real-time-PCR. The IL-10-592 SNP CA (P=0.0012/OR=2.4/CI:1.4-4.1), AA (P=0.0458/OR=2.3/CI:1.1-4.9), and CA+AA (P=0.0006/OR=2.4/CI: 1.4-3.4) genotypes and the allele A (P=0.0036/OR=1.7/CI:1.2-2.4) were found to be significantly more prevalent in the CP group when compared with control subjects. Both CA and AA genotypes were associated with lower levels of IL-10, TIMP-3, and OPG mRNA expression in diseased periodontal tissues and were also associated with disease severity as mean pocket depth. Taken together, the results presented here demonstrate that IL10-592 SNP is functional in CP, being associated with lower levels of IL-10 mRNA expression, which is supposed to consequently decrease the expression of the downstream genes TIMP-3 and OPG, and influence periodontal disease outcome. J. Leukoc. Biol. 84: 1565-1573; 2008.

Stem cells in the aetiopathogenesis and therapy of rheumatic disease

Animal models of autoimmune disease and case reports of patients with these diseases who have been involved in bone marrow transplants have provided important data implicating the haemopoietic stem cell in rheumatic disease pathogenesis. Animal and human examples exist for both cure and transfer of rheumatoid arthritis, systemic lupus erythematosus (SLE) and other organ-specific diseases using allogeneic haemopoietic stem cell transplantation. This would suggest that the stem cell in these diseases is abnormal and could be cured by replacement of a normal stem cell although more in vitro data are required in this area. Given the morbidity and increased mortality in some patients with severe autoimmune diseases and the increasing safety of autologous haemopoietic stem cell transplantation (HSCT), pilot studies have been conducted using HSCT in rheumatic diseases. It is still unclear whether an autologous graft will cure these diseases but significant remissions have been obtained which have provided important data for the design of randomized trials of HSCT versus more conventional therapy. Several trials are now open to accrual under the auspices of the European Bone Marrow Transplant Group/European League Against Rheumatism (EBMT/EULAR) registry. Future clinical and laboratory research will need to document the abnormalities of the stem cell of a rheumatic patient because new therapies based on gene therapy or stem cell differentiation could be apllied to these diseases. With increasing safety of allogeneic HSCT it is not unreasonable to predict cure of some rheumatic diseases in the near future.

Deformação miocárdica analisada por 2D strain em indivíduos com lúpus eritematoso sistémico

Mestrado em Tecnologia de Diagnóstico e Intervenção Cardiovascular. Área de especialização: Ultrassonografia Cardiovascular.