Jorge Ramón Padilla-Gutiérrez

Macrophage migration inhibitory factor (MIF): Genetic evidence for participation in early onset and early stage rheumatoid arthritis

Macrophage migration inhibitory factor (MIF) is an upstream pro-inflammatory cytokine that is associated with the pathogenesis of autoimmune inflammatory diseases including rheumatoid arthritis (RA). Two polymorphisms in the upstream region exist in the MIF gene and are associated with RA susceptibility or severity in different populations. In this case-control study, we investigated whether MIF polymorphisms are associated with RA susceptibility or activity in a western Mexican population .The relationship of MIF levels with clinical features of disease also was assessed. Genotyping of the -794 CATT5-8 (rs5844572) and the -173 G>C (rs755622) polymorphisms was performed by PCR and PCR-RFLP respectively on 226 RA patients and 210 healthy subjects. Serum MIF levels were determined by ELISA. We found a significant association between the -794 CATT5-8 6,7 MIF genotype with RA. Moreover, we detected an association between the -794 CATT7 allele with early onset RA. The -794 CATT7 and -173(*)C alleles, which are in linkage disequilibrium, were associated with high disease activity on RA patients. A positive correlation between circulating MIF levels and C-reactive protein, erythrocyte sedimentation rate, rheumatoid factor, anti-citrullinated protein/peptides antibodies and TNFα was detected. MIF levels appear to be associated with disease progression rather than disease activity, which is distinct from the established relationship between disease activity and TNFα levels. In conclusion, the MIF gene and protein are associated with RA in a western Mexican population, with a main contribution onto early onset and early stages of disease.

The +49A>G CTLA-4 polymorphism is associated with rheumatoid arthritis in Mexican population.

BACKGROUND The Cytotoxic T lymphocyte antigen (CTLA-4) is one of the major susceptibility genes associated with autoimmune diseases. Susceptibility to rheumatoid arthritis (RA) is determined by both environmental and genetic factors. The genetic contribution approaches 50-60%. The association between RA with the +49A>G CTLA-4 polymorphism in the Mexican population was investigated. METHODS The polymerase chain reaction-restriction fragment was used to amplify the +49A>G CTLA-4 polymorphism in RA patients and healthy subjects (HS). RESULTS We analyzed the association between the +49A>G CTLA-4 polymorphism and RA. The G allele frequency was higher in RA patients than HS (46.8 vs 37.7%, OR=1.45, p=0.01). RA patients carrying the A/G genotype were significantly more likely to be positive to CRP and RF. There was no evidence of an association between SNP genotypes and the clinical characteristics of rheumatoid arthritis. CONCLUSIONS The +49A>G CTLA-4 polymorphism is a genetic marker of susceptibility for RA in western Mexican population.

Macrophage migration inhibitory factor: Association of -794 CATT5-8 and -173 G>C polymorphisms with TNF-α in systemic lupus erythematosus

Macrophage migration inhibitory factor (MIF) is an upstream immunoregulatory cytokine associated with the pathogenesis of autoimmune inflammatory diseases. There is evidence that MIF functions in a positive feedback loop with TNF-α that could perpetuate the inflammatory process in systemic lupus erythematosus (SLE). In this case-control study we investigated whether commonly occurring functional MIF polymorphisms are associated with SLE as well as with MIF and TNF-α serum levels in a Mexican-Mestizo population. Genotyping of the -794 CATT5-8 (rs5844572) and -173 G>C (rs755622) MIF polymorphisms was performed by PCR and PCR-RFLP, respectively in 186 SLE patients and 200 healthy subjects. MIF and TNF-α serum levels were determined by ELISA. A significant increase of MIF and TNF-α levels was found in SLE patients. According to a genetic model, we found a significant association of genotypes carrying the -794 CATT7 and -173(∗)C risk alleles with susceptibility to SLE and with a significant increase of TNF-α. In conclusion, MIF gene polymorphisms are associated with SLE susceptibility and with an increase of TNF-α serum levels in a Mexican-Mestizo population.

Circulating TNFRI and TNFRII levels correlated with the disease activity score (DAS28) in rheumatoid arthritis

Objective: To measure levels of soluble tumour necrosis factor alpha (TNFα) receptor type I (sTNFRI) and type II (sTNFRII) in order to correlate them with C-reactive protein (CRP), rheumatoid factor (RF), erythrocyte sedimentation rate (ESR), and disease activity score (DAS28) in RA patients. Methods: We recruited 41 RA patients classified according to American College of Rheumatology (ACR) criteria and 38 healthy subjects (HS). sTNFRI and sTNFRII were measured using an enzyme-linked immunosorbent assay (ELISA) kit. Clinical activity in RA patients was evaluated using the Disease Activity Score using 28 joint counts (DAS28). The statistical analysis was realized using SPSS version 10.0. Results: Soluble TNFRI and TNFRII levels were higher in RA patients (p = 0.04 and 0.001, respectively) than HS. Serum levels of sTNFRI correlated with sTNFRII (r = 0.699, p < 0.0001). sTNFRII correlated with DAS28 (r = 0.375, p = 0.017), RF (r = 0.505, p = 0.004), and ESR (r = 0.323, p = 0.042). Conclusion: The increased levels of both sTNFRI and sTNFRII suggest a secondary event related to the inflammatory state observed in RA, whereas the correlation of sTNFRII with RF, ESR, and DAS28 reflects the preferential TNFRII shedding induced by TNFα. sTNFRII may be useful as an additional inflammatory marker in RA.

Association of the -1031T>C polymorphism and soluble TNF-α levels with Acute Coronary Syndrome.

INTRODUCTION Inflammation has gained a pivotal role in the pathophysiology of Acute Coronary Syndrome (ACS). TNF-α is a pro-inflammatory cytokine that could be a potential biomarker in ACS due to its multiple functions. The rs1799964 TNFA polymorphism (-1031T>C) has been associated with a decrease in gene transcription and cytokine levels. OBJECTIVE To determine the association of rs1799964 TNFA polymorphism and TNF-α soluble levels in ACS. METHODS A total of 251 patients diagnosed with ACS and 164 individuals without cardiovascular diseases classified as the reference group (RG), were included. The rs1799964 polymorphism was genotyped by PCR-RFLP. Soluble protein levels were determined by ELISA. Statistical analyses were performed using chi square and U-Mann Whitney tests. RESULTS The genotype and allele frequencies were different between ACS and RG (OR=0.317, p=0.01; OR=0.688, p=0.03 respectively). ACS patients had higher soluble TNF-α levels compared with the RG (31.08 vs 23.00pg/mL, p<0.001); according genotype significant differences were observed (T/T: 24.06 vs T/C: 34.95pg/mL, p=0.0001) in patients. In the RG, T/T carriers showed discrete lower levels than C/C genotype (22.14 vs 27.83pg/mL, p=0.04). CONCLUSIONS The -1031C allele of the TNFA polymorphism confers protection for the development of ACS. The T/C genotype carriers had higher TNF-α serum levels compared to the T/T genotype in ACS. In addition, the -1031T>C TNFA polymorphism was associated with dyslipidemia in ACS in a Western Mexican population.

Association between the -794 (CATT)5-8 MIF gene polymorphism and susceptibility to acute coronary syndrome in a western Mexican population.

The macrophage migration inhibitory factor (MIF) is related to the progression of atherosclerosis, which, in turn, is a key factor in the development of acute coronary syndrome (ACS). MIF has a CATT short tandem repeat (STR) at position −794 that might be involved in its expression rate. The aim of this study was to investigate the association between the −794 (CATT)5–8   MIF gene polymorphism and susceptibility to ACS in a western Mexican population. This research included 200 ACS patients classified according to the criteria of the American College of Cardiology (ACC) and 200 healthy subjects (HS). The −794 (CATT)5–8   MIF gene polymorphism was analyzed using a conventional polymerase chain reaction (PCR) technique. The 6 allele was the most frequent in both groups (ACS: 54% and HS: 57%). The most common genotypes in ACS patients and HS were 6/7 and 6/6, respectively, and a significant association was found between the 6/7 genotype and susceptibility to ACS (68% versus 47% in ACS and HS, resp., P = 0.03). We conclude that the 6/7 genotype of the MIF −794 (CATT)5–8 polymorphism is associated with susceptibility to ACS in a western Mexican population.

Aberrant expression of interleukin-10 in rheumatoid arthritis: Relationship with IL10 haplotypes and autoantibodies

HighlightsIn rheumatoid arthritis there is an exacerbated up‐regulation of IL‐10 at the transcriptional level.Increased IL‐10 levels are associated with seropositivity for RF and anti‐CCP antibodies.GCC haplotype was associated with higher IL‐10 serum levels compared with the ACC and ATA haplotypes in RA patients.There was no significant association between IL10 haplotypes and risk of RA in a Mexican population. Abstract Interleukin 10 (IL‐10) is an immunomodulatory cytokine that plays a central role in the pathogenesis of autoimmune diseases. Different studies consistently show increased IL‐10 serum levels in rheumatoid arthritis (RA) and it appears to be caused by genetic variants. Three polymorphisms situated at positions −1082, −819 and −592 of IL10 gene and its major haplotypes have been associated with regulating IL10 promoter activity. In this study, we evaluated whether IL10 haplotypes are associated with mRNA expression and IL‐10 serum levels as well as susceptibility to RA in a Western Mexican population. A total of 240 RA patients and 240 control subjects (CS) were included. Genotyping of IL10 polymorphisms was performed by PCR and PCR‐RFLP, respectively. IL10 mRNA expression was determined by real‐time PCR and IL‐10 serum levels were measured using an ELISA kit. IL10 mRNA expression was 50‐fold higher in RA patients than CS (p < 0.001), while IL‐10 serum levels did not show differences between groups. However, high IL‐10 serum levels were positively related to a higher seropositivity for rheumatoid factor (FR) and anti‐CCP antibodies (p < 0.05). No significant differences between the distribution of haplotype frequencies were observed between both study groups, but GCC haplotype was associated with higher IL‐10 serum levels compared with the ACC and ATA haplotypes in RA patients (p < 0.05). In addition, patients carrying ATA and GCC haplotypes showed higher mRNA expression than ACC (5.4‐fold and 8.8‐fold, respectively) and surprisingly, this trend was reversed in the controls, although it was not significant. In conclusion, our findings suggest that IL10 (GCC, ACC, and ATA) haplotypes may not be a susceptibility marker for RA in a population from Western Mexico. Nevertheless, independently of the presence of these variants, there is an aberrant overexpression of IL10 gene in RA, and it may play an important role in the pathogenesis of RA.

The 14 bp Del/Ins HLA-G Polymorphism Is Related with High Blood Pressure in Acute Coronary Syndrome and Type 2 Diabetes Mellitus

Immunologic and inflammatory processes are involved in the pathogenesis of acute coronary syndrome (ACS) and type 2 diabetes mellitus (DM2). Human leukocyte antigen-G (HLA-G) is a negative regulator of the immune response. This study evaluates the 14 bp Del/Ins HLA-G polymorphism in ACS and DM2. Three hundred and seventy individuals from Western Mexico were recruited and categorized into three groups: ACS (86), DM2 without coronary complications (70), and healthy subjects (214). Genotyping of the 14 bp Del/Ins HLA-G polymorphism was performed by PCR and Native-PAGE. The most common risk factors were hypertension and overweight in ACS and DM2, respectively. The genetic distribution of the 14 bp Del/Ins HLA-G polymorphism showed no significant differences between groups (P ≥ 0.23). Nonetheless, the Ins/Ins genotype was associated with high blood pressure (HBP) in the DM2 group (ORc = 1.65, P = 0.02). The genetic recessive model showed similar findings (ORc = 3.03, P = 0.04). No association was found in ACS, with a P of 0.05; nevertheless, the prevalence of Ins/Ins carriers was quite similar to that found in the DM2-HBP group. The 14 bp Del/Ins HLA-G polymorphism was not a susceptibility factor for ACS or DM2; however, the Ins/Ins genotype might have contributed to the development of HBP in the studied groups.

A new PCR-RFLP assay for -1123 G>C polymorphism in the PTPN22 gene : allele and genotype frequencies in a western Mexican population

Figure 1 –1123 PTPN22 polymorphism restriction. PTPN22 –1123G)C polymorphism was analyzed by polyacrylamide gel electrophoresis (native PAGE, 6%, 29:1), and silver staining. (A) PCR products in native PAGE (205 bp, lanes 1–4) and 50 bp ladder (lane 5). (B) PAGE showing homozygous wild type (G/G) genotype (lanes 1 and 2), heterozygous genotypes (G/C, lanes 3 and 4) and homozygous mutant (C/C) (lanes 5 and 6). Lane 7, 50 bp ladder. Jorge Ramón Padilla-Gutiérrez, Yeminia Valle, Mónica Vázquez-Del Mercado, Montserrat Maldonado and José Francisco Muñoz-Valle* 1 Instituto de Investigación en Reumatologı́a y del Sistema Músculo Esquelético, Departamento de Biologı́a Molecular y Genómica, Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Guadalajara, Jalisco, México 2 Postdoctoral Fellow in Biomedical Sciences (Immunology), Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Guadalajara, Jalisco, México

Decreased serum levels of sCD40L and IL-31 correlate in treated patients with Relapsing-Remitting Multiple Sclerosis

The CD40/CD40L system is a binding key for co-stimulation of immune cells. Soluble form of CD40L has been widely studied as marker of inflammatory and autoimmune diseases. Here we analyze serum concentrations of sCD40L, as well as 14 cytokines, in patients with Multiple Sclerosis (MS) treated with Glatiramer acetate or Interferon beta. In the healthy control group, we found in serum a highly positive correlation between sCD40L and Interleukin (IL)-31, an anti-inflammatory Th2 cytokine. Additionally, an important reduction in IL-31 and sCD40L serum levels, as well as a significant reduction in CD40 mRNA expression and complete depletion of CD40L mRNA, detected from peripheral blood cells, was found in treated patients with MS. Therefore, sCD40L and IL-31 must be taken into account as possible prognostic markers when analyzing the disease progress of MS in order to provide more personalized treatment.