Paula Sandrin-Garcia

Polimorphisms in Inflammasome Genes Are Involved in the Predisposition to Systemic Lupus Erythematosus

Recent findings provide evidence of inflammasome critical role in the predisposition to autoimmune disorders. The involvement of inflammasome in the pathogenesis of systemic lupus erythematosus (SLE) has been hypothesized even if no significant association within inflammasome genes mutations or polymorphisms and lupus has been reported yet. We analyzed 14 single nucleotide polymorphisms (SNPs) within 7 inflammasome genes (NLRP1, NLRP3, NLRC4, AIM2, CARD8, CASP1, IL1B) in 144 patients affected by systemic lupus erythematosus and in 158 healthy controls from Southern Brazilian (state of São Paulo) with the aim of disclosing the possible role of inflammasome genes in the susceptibility of SLE. Our results demonstrated that NLRP1 rs2670660 SNP and the NLRP1 rs12150220-rs2670660 A-G haplotype were associated with SLE in our study population, and in particular with the development of nephritis, rash and arthritis. These findings are concordant with previously reported association of NLRP1 with vitiligo and type-1 diabetes underlining once more the involvement of NALP1 inflammasome in the pathogenesis of autoimmune disorders.

Gene expression profiles in human lymphocytes irradiated in vitro with low doses of gamma rays

Abstract Fachin, A. L., Mello, S. S., Sandrin-Garcia, P., Junta, C. M., Donadi, E. A., Passos, G. A. S. and Sakamoto-Hojo, E. T. Gene Expression Profiles in Human Lymphocytes Irradiated In Vitro with Low Doses of Gamma Rays. Radiat. Res. 168, 650–665 (2007). The molecular mechanisms underlying responses to low radiation doses are still unknown, especially in normal lymphocytes, despite the evidence suggesting specific changes that may characterize cellular responses. Our purpose was to analyze gene expression profiles by DNA microarrays in human lymphocytes after in vitro irradiation (10, 25 and 50 cGy) with γ rays. A cytogenetic analysis was also carried out for different radiation doses. G0 lymphocytes were irradiated and induced to proliferate for 48 h; then RNA samples were collected for gene expression analysis. ANOVA was applied to data obtained in four experiments with four healthy donors, followed by SAM analysis and hierarchical clustering. For 10, 25 and 50 cGy, the numbers of significantly (FDR ≤ 0.05) modulated genes were 86, 130 and 142, respectively, and 25, 35 and 33 genes were exclusively modulated for each dose, respectively. We found CYP4X1, MAPK10 and ATF6 (10 cGy), DUSP16 and RAD51L1 (25 cGy), and RAD50, REV3L and DCLRE1A (50 cGy). A set of 34 significant genes was common for all doses; while SERPINB2 and C14orf104 were up-regulated, CREB3L2, DDX49, STK25 and XAB2 were down-regulated. Chromosome damage was significantly induced for doses ≥10 cGy (total aberrations) and ≥50 cGy (dicentrics/ rings). Therefore, low to moderate radiation doses induced qualitative and/or quantitative differences and similarities in transcript profiles, reflecting the type and extent of DNA lesions. The main biological processes associated with modulated genes were metabolism, stress response/DNA repair, cell growth/differentiation, and transcription regulation. The results indicate a potential risk to humans regarding the development of genetic instability and acquired diseases.

Promiscuous gene expression in the thymus: The root of central tolerance

The thymus is a complex organ with an epithelium formed by two main cell types, the cortical thymic epithelial (cTECs) and medullary thymic epithelial cells (mTECs), referred to as stroma. Immature thymocytes arising from the bone marrow, macrophages and dendritic cells also populate the thymus. Thymocytes evolve to mature T cells featuring cell differentiation antigens (CDs), which characterize the phenotypically distinct stages, defined as double-negative (DN), double positive (DP) and single positive (SP), based on expression of the coreceptors CD4 and CD8. The thymus is therefore implicated in T cell differentiation and during development into T cells thymocytes are in close association with the stroma. Recent evidence showed that mTECs express a diverse set of genes coding for parenchymal organ specific proteins. This phenomenon has been termed promiscuous gene expression (PGE) and has led to the reconsideration of the role of the thymus in central T cell tolerance to self-antigens, which prevents autoimmunity. The evidence of PGE is causing a reanalysis in the scope of central tolerance understanding. We summarize the evidence of PGE in the thymus, focusing particularly the use of cDNA microarray technology for the broad characterization of gene expression and demarcation of PGE emergence during thymus ontogeny.

Mannose binding lectin gene (MBL2) functional polymorphisms are associated with systemic lupus erythematosus in southern Brazilians

Susceptibility to systemic lupus erythematosus (SLE) has been associated with immunologic, environmental, and genetic factors. To uncover a possible association between MBL2 gene polymorphisms and SLE, we analyzed functional polymorphisms in the promoter and first exon of the MBL2 gene in 134 Brazilian SLE patients and 101 healthy controls. Genotype and allele frequencies of MBL2 A/O polymorphism were significantly different between patients and controls, and the O allele was associated with an increased risk of SLE. An association between low mannose binding lectin (MBL) producer combined genotypes and increased risk for SLE was also reported. Furthermore, when stratifying SLE patients according to clinical and laboratory data, an association between the A/O genotype and nephritic disorders and between the X/Y genotype and antiphospholipid syndrome was evident. Combined genotypes responsible for low MBL production were more frequently observed in SLE patients with nephritis. Our results indicate MBL2 polymorphisms as possible risk factors for SLE development and disease-related clinical manifestations.

Comprehensive gene expression profiling in lungs of mice infected with Mycobacterium tuberculosis following DNAhsp65 immunotherapy

The continued increase in tuberculosis (TB) rates and the appearance of extremely resistant Mycobacterium tuberculosis strains (XDR‐TB) worldwide are some of the great problems of public health. In this context, DNA immunotherapy has been proposed as an effective alternative that could circumvent the limitations of conventional drugs. Nonetheless, the molecular events underlying these therapeutic effects are poorly understood.

Vitamin D receptor (VDR) gene polymorphisms and susceptibility to systemic lupus erythematosus clinical manifestations

Systemic lupus erythematosus (SLE) is an autoimmune disorder with heterogeneous clinical manifestations and target tissue damage. Currently, several genes have been associated with SLE susceptibility, including vitamin D receptor (VDR), which is a mediator of immune responses through the action of vitamin D. Polymorphisms in the VDR gene can impair the vitamin D (D3) function role, and since SLE patients show deficient D3 blood levels, it leads to a possible connection to the disease’s onset. In our study we searched for an association between VDR polymorphisms and risk of developing SLE, as well as the disease’s clinical manifestations. We enrolled 158 SLE patients and 190 Southeast Brazilian healthy controls, genotyped for five Tag single nucleotide polymorphisms (SNPs), covering most of the VDR gene region. We found an association between VDR SNPs and SLE for the following clinical manifestations: rs11168268 and cutaneous alterations (p = 0.036), rs3890733 (p = 0.003) rs3890733 and arthritis (p = 0.001), rs2248098 and immunological alterations (p = 0.040), rs4760648 and antibody anti-dsDNA (p = 0.036). No association was reported between VDR polymorphisms and SLE susceptibility.

Polymorphisms in STK17A gene are associated with systemic lupus erythematosus and its clinical manifestations.

Systemic lupus erythematosus (SLE) is an autoimmune disorder with several clinical manifestations. SLE etiology has a strong genetic component, which plays a key role in diseases predisposition, as well as participation of environmental factors, such and UV light exposure. In this regard, we investigated whether polymorphisms in STK17A, a DNA repair related gene, encoding for serine/threonine-protein kinase 17A, are associated with SLE susceptibility. A total of 143 SLE patients and 177 healthy controls from Southern Brazil were genotyped for five STK17A TagSNPs. Our results indicated association of rs7805969 SNP (A and G/A genotype, OR=1.40 and OR=1.73, respectively) with SLE predisposition and the following clinical manifestations: arthritis, cutaneous and immunological alterations. When analyzing haplotypes distribution, we found association between TGGTC, TAGTC and AAGAT haplotypes and risk to develop SLE. When considering clinical manifestations, the haplotypes TGGTT and TAGTC were associated with protection against cutaneous alterations and the haplotype TAGTC to hematological alterations. We also observed association between SLE clinical manifestations and ethnicity, with the European-derived patients being more susceptible to cutaneous and hematological alterations.

Vitamin D receptor (VDR) gene polymorphisms and age onset in type 1 diabetes mellitus

Abstract Vitamin D receptor is a mediator of immune responses through the action of vitamin D, which is capable of regulate the insulin secretion by the pancreas. Since polymorphisms in the vitamin D receptor (VDR) gene might modulate vitamin D function, and thus immunologic response, VDR is possibly able to influence the predisposition to type 1 diabetes mellitus (T1DM). The aim of this work was to perform an association study among VDR polymorphisms and T1DM susceptibility, as well as the correlation with the disease onset. Two hundred and four T1DM patients and 217 controls, from Northeast Brazil, were genotyped for five tagSNPs, covering the whole VDR gene. Our results indicated an association between rs1540339 and rs4760648 SNPs (p = 0.02 and p = 0.03, respectively) and T1DM. No association was found with T1DM onset and age at diagnose. To our knowledge, this is the first association study in T1DM where the whole VDR gene was analyzed, and our results indicate that VDR polymorphisms could be important for T1DM susceptibility, but do not seem to be associated to age at disease onset.

Interleukin-18, interleukin-12B and interferon-γ gene polymorphisms in Brazilian patients with rheumatoid arthritis: a pilot study

Polymorphisms in interleukin (IL)-18, IL-12 and interferon (IFN)-γ genes are associated with different levels of cytokines expression and have been associated with rheumatoid arthritis (RA). IL-18 +105 A/C, IL-12B +1188 A/C and IFN-γ +874 T/A polymorphisms were analyzed by restriction fragment length polymorphism-polymerase chain reaction (PCR) and amplification refractory mutation system PCR from 90 RA patients and 186 healthy individuals. There were significant differences to IL-18 +105 A/C polymorphism between the RA and control groups (odds ratio = 3.77; P < 0.0001). Individual carriers of the variant allele C had a 3.77-fold increased risk of for RA (P = 0.0032). No association was observed for IL-12B and IFN-γ polymorphisms. Our finds suggest a possible role for IL-18 polymorphism in the RA susceptibility in studied population.

Shared and unique gene expression in systemic lupus erythematosus depending on disease activity.

Patients presenting with active systemic lupus erythematosus (SLE) manifestations may exhibit distinct pathogenetic features in relation to inactive SLE. Also, cDNA microarrays may potentially discriminate the gene expression profile of a disease or disease variant. Therefore, we evaluated the expression profile of 4500 genes in peripheral blood lymphocytes (PBL) of SLE patients. We studied 11 patients with SLE (seven with active SLE and four with inactive SLE) and eight healthy controls. Total RNA was isolated from PBL, reverse transcribed into cDNA, and postlabeled with Cy3 fluorochrome. These probes were then hybridized to a glass slide cDNA microarray containing 4500 human IMAGE cDNA target sequences. An equimolar amount of total RNA from human cell lines served as reference. The microarray images were quantified, normalized, and analyzed using the R environment (ANOVA, significant analysis of microarrays, and cluster‐tree view algorithms). Disease activity was assessed by the SLE disease activity index. Compared to the healthy controls, 104 genes in active SLE patients (80 repressed and 24 induced) and 52 genes in nonactive SLE patients (31 induced and 21 repressed) were differentially expressed. The modulation of 12 genes, either induced or repressed, was found in both disease variants; however, each disease variant had differential expression of different genes. Taken together, these results indicate that the two lupus variants studied have common and unique differentially expressed genes. Although the biological significance of the differentially expressed genes discussed above has not been completely understood, they may serve as a platform to further explore the molecular basis of immune deregulation in SLE.