Claudia Azucena Palafox-Sánchez

Oxidized low-density lipoprotein and β2-glycoprotein I in patients with systemic lupus erythematosus and increased carotid intima-media thickness: implications in autoimmune-mediated atherosclerosis

Oxidative stress and LDL modification (oxLDL) are early pro-atherogenic events. OxLDL binds β2GPI producing immunogenic oxLDL/β2GPI complexes. Antibodies to these complexes have been associated with arterial thrombosis in patients with systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS). Circulating oxLDL/β2GPI complexes, IgG and IgM antibodies to these complexes were measured by ELISA in 30 SLE patients asymptomatic for cardiovascular disease (mean age 31 years) and 27 age/sex matched healthy controls. Carotid intima-media thickness (IMT) was measured by ultrasound in all patients and controls. Forty-seven percent of SLE presented plaques (median IMT of 0.65 ± 0.12 mm) while only 7% of the controls had plaques (median IMT of 0.50 ± 0.04 mm, P < 0.001). Median optical density (OD450nm) for oxLDL/β2GPI complexes in SLE was 0.244 ± 0.07, higher than controls (0.174 ± 0.09, P < 0.001). Median OD for IgG anti-oxLDL/β2GPI antibodies was also higher in SLE (0.297 ± 0.26) compared to controls (0.194 ± 0.07, P < 0.001) while the median OD for IgM antibodies in SLE (0.444 ± 0.46) was not different than controls (0.326 ± 0.22, P = 0.267). There was no correlation between IMT and oxLDL/β2GPI complexes, IgG or IgM antibodies, possibly reflecting the complex interrelationship between these serologic elements and tissue factors in the arterial wall. These results support the hypothesis that oxLDL/β2GPI complexes and IgG (not IgM) anti-oxLDL/β2GPI antibodies contribute to the development of autoimmune-mediated atherosclerosis

Oxidized Low-Density Lipoprotein/β2-Glycoprotein I Complexes and Autoantibodies to oxLig-1/β2-Glycoprotein I in Patients with Systemic Lupus Erythematosus and Antiphospholipid Syndrome

Oxidized low-density lipoprotein (oxLDL) interacts with β 2 -glycoprotein I (β 2 -GPI) via oxLDL-derived specific ligands (oxLig-1) forming complexes. The prevalence and significance of oxLDL/β 2 -GPI complexes and antibodies to oxLig-1/β 2 -GPI were evaluated in patients with systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS). The oxLDL/β 2 -GPI complex was 69% positive (above mean + 3 SD of control subjects) in 97 consecutive patients with SLE, 62% in 40 patients with SLE with secondary APS, and 60% in 50 control patients with SLE without APS. IgG anti–oxLig-1/β 2 -GPI antibody was positive in 31 (32%) of 97 consecutive patients with SLE, in 26 (65%) of 40 patients with SLE with secondary APS, and in 6 (19%) of 32 control patients with SLE. Anti–oxLig1/β 2 -GPI antibodies were 93.7% specific with a positive predictive value of 90.0% for APS, better than anticardiolipin antibodies (80.0% specific, 71.4% predictive value). These results confirm that oxLDL/β 2 GPI complexes are common in SLE and suggest a possible immunogenic role in APS. In contrast, IgG anti–oxLig-1/β 2 -GPI antibodies not only are associated with but also are clinically useful risk factors for APS. Vascular thromboembolic events, pregnancy morbidity (miscarriages and fetal loss), and thrombocytopenia in association with the presence of elevated serum levels of antiphospholipid antibodies are common clinical features of the antiphospholipid syndrome (APS). APS is classified as primary if there is no coexisting autoimmune disease or secondary when present in the context of an autoimmune disorder. There is considerable evidence to suggest a pathogenic role of antiphospholipid antibodies in the development of these clinical features. 1-3 Antiphospholipid antibodies are a heterogeneous group of autoantibodies characterized by their reactivity to anionic phospholipids, phospholipid/protein complexes, and certain proteins presented on suitable surfaces in the absence of phospholipids, ie, activated cell membranes and oxygenated polystyrene. 4-6 Several plasma proteins that participate in coagulation and interact with anionic phospholipids have been reported to function as antiphospholipid cofactors, eg, β 2 -glycoprotein I (β 2 -GPI), prothrombin, protein C, protein S, and annexin V. β 2 -GPI is the most extensively studied of the cofactors and has been shown to be a relevant antigenic target for antiphospholipid antibodies. 7,8 β 2 -GPI is a 50-kd, single-chain polypeptide composed of 326 amino acid residues, arranged in 5 homologous repeats known as complement control protein domains. β 2 -GPI’s fifth domain contains a patch of positively charged amino acids that likely represents the binding region for phospholipids. 9-11

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 +1858C/T PTPN22 gene polymorphism confers genetic susceptibility to rheumatoid arthritis in Mexican population from the Western Mexico.

INTRODUCTION Rheumatoid arthritis (RA) is a common autoimmune disease with a complex genetic background. The PTPN22 gene encodes lymphoid tyrosine phosphatase LYP, a potent negative regulator of T cell activation. Polymorphic variants of this gene have previously been associated with various autoimmune disorders. The +1858C/T single-nucleotide polymorphism (SNP) (rs2476601), in the exon 14 of the PTPN22 gene has been associated with susceptibility to RA in several population. OBJECTIVE The aim of this work was to investigate whether the +1858C/T of the PTPN22 gene is associated with susceptibility to RA in Western Mexico population. METHODS A total of 309 unrelated RA patients, classified according to American College of Rheumatology (ACR) 1987 criteria, as well as 347 controls residents from Western Mexico were recruited for this study. The DNA samples were genotyped for +1858C/T PTPN22 gene SNP using the PCR-RFLP technique. Antibodies to cyclic citrullinated peptides (anti-CCP) were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS The frequency of +1858T risk allele was significantly increased in patients with RA compared with controls (p=0.001, OR=2.83, 95%CI=1.50-5.32). To confirm this results we established a comparison between subjects carrying of CT+TT genotypes versus those carrying CC genotype, between both groups (p=0.004, OR=2.65, 95%CI=1.33-5.36). Nevertheless, we not observed association of the +1858C/T PTPN22 gene SNP with clinical activity and functional disability in RA patients. Likewise, the +1858T variant in RA patients seropositive for anti-CCP antibodies, increased the risk for RA (p=0.008, OR=2.5, 95%CI=1.3-5.0) when we compared with controls; however, in the group of seronegative patients, no was found significant difference (p=0.1, OR=2.5, 95%CI=0.9-7.2). CONCLUSIONS Our results support the association of the +1858T risk allele of the +1858C/T PTPN22 polymorphism with susceptibility to RA and confirm that, in combination with anti-CCP antibodies, this SNP influence the autoimmune processes towards a development of RA in Mexican population.

Association of BAFF, APRIL serum levels, BAFF-R, TACI and BCMA expression on peripheral B-cell subsets with clinical manifestations in systemic lupus erythematosus.

Objective B-cell-activating factor (BAFF) and a proliferation-inducing ligand (APRIL) signaling pathways regulate B-cell survival through interactions with their receptors BAFF-R, TACI and BCMA. We evaluated the association of these ligands/receptors on B-cell subsets according to clinical manifestations of systemic lupus erythematosus (SLE). Methods BAFF and APRIL serum concentrations were measured in 30 SLE patients by enzyme-linked immunosorbent assay. The BAFF-R, TACI and BCMA expression was analyzed on each B cell subset (CD19 + CD27-CD38–/ + naïve; CD19 + CD27 + CD38–/ + memory; CD19 + CD27-CD38 + + immature and CD19 + CD27 + CD38 + + plasma cells) by flow cytometry, and compared among patients with different clinical manifestations as well as healthy controls (HCs). Results Serum BAFF and APRIL levels were high in SLE patients and correlated with the Mex-SLEDAI disease activity index (r = 0.584; p = 0.001 and r = 0.456; p = 0.011, respectively). The SLE patients showed an increased proportion of memory and plasma B cells (p < 0.05). BAFF-R, TACI and BCMA expression in SLE patients was decreased in almost all B cell subsets compared to HCs (p < 0.05). A lower BCMA expression was associated with severe disease activity, glomerulonephritis, serositis and hemolytic anemia (p < 0.01). BCMA expression showed a negative correlation with Mex-SLEDAI score (r = –0.494, p = 0.006). Conclusions Decreased BCMA expression on peripheral B cells according to severe disease activity suggests that BCMA plays an important regulating role in B-cell hyperactivity and immune tolerance homeostasis in SLE patients.

Associations of Killer Cell Immunoglobulin- Like Receptor Genes with Rheumatoid Arthritis

Objective: Rheumatoid Arthritis (RA) is an autoimmune and chronic inflammatory disease of unknown etiology. Killer cell immunoglobulin-like receptors are expressed on the surface of natural killer cells and CD28null T-cells, both present in synovial membrane of RA. Therefore we evaluated the associations of KIR genes with RA. Methods: 16 KIR genes were genotyped in 100 healthy subjects (HS) and 100 RA patients from Western Mexico using PCR-SSP. Differences in KIR genotypes and gene frequencies were assessed using the X2 test. Results: Gene frequency of KIR2DL3 was lower in RA than in HS (p = 0.0019), whereas KIR2DL2 and KIR2DS2 were higher in RA than HS (p = 0.0004 and p = 0.0487, respectively). In addition were identified 38 genotypes (from G1-G38) in both studied groups, and the genotype frequencies of G1, G6 and G14 showed significant differences (p = 0.0001, p = 0.0208 and p = 0.0300, respectively). Conclusions: The presence of KIR2DL2, KIR2DS2 and absence of KIR2DL3 are associated with RA. Moreover, two genotypes BX are associated with RA. These results suggest that KIRs can be involved in RA susceptibility.

Expression of ICAM1 and VCAM1 Serum Levels in Rheumatoid Arthritis Clinical Activity. Association with Genetic Polymorphisms

To investigate the association of sICAM-1 and sVCAM-1 with ICAM1 721G>A and VCAM1 1238G>C polymorphisms and rheumatoid arthritis (RA) clinical activity, sixty RA patients and 60 healthy non-related subjects (HS) matched for age and sex were recruited. Soluble adhesion molecules were determined by ELISA technique. Rheumatoid factor (RF), C reactive protein (CRP) and the erythrocyte sedimentation rate (ESR) were measured by routine methods. Disability and clinical activity was measured with Spanish-HAQ-DI and DAS28 scores, respectively. The ICAM1 and VCAM1 polymorphism were identified using the PCR-RFLP procedure. Inter-group comparison showed increased levels of sICAM-1 and sVCAM-1 in RA patients (284 and 481 ng/mL) versus HS (132 and 280 ng/mL); in the RA group, significant correlations between sVCAM-1 and RF (r = 0.402), ESR (r = 0.426), Spanish-HAQ-DI (r = 0.276), and DAS28 (r = 0.342) were found, whereas sICAM-1 only correlated with RF (r = 0.445). In RA patients, a significant association with the 721A allele of ICAM1 polymorphism (p = 0.04), was found. In addition, the allele impact (G/A + A/A) of this polymorphism was confirmed, (p = 0.038, OR = 2.3, C.I. 1.1–5.0). sVCAM-1 and sICAM-1 serum levels reflected the clinical status in RA, independently of the ICAM1 and VCAM1 polymorphism. However, the ICAM1 721A allele could be a genetic marker to RA susceptibility.

Comparative analysis of autoantibodies targeting peptidylarginine deiminase type 4, mutated citrullinated vimentin and cyclic citrullinated peptides in rheumatoid arthritis: associations with cytokine profiles, clinical and genetic features.

Antibodies against cyclic citrullinated peptides (anti‐CCP) are widely used for diagnosis of rheumatoid arthritis (RA). We performed a comparative analysis of antibodies targeting the citrullinating enzyme peptidylarginine deiminase type 4 (anti‐PAD4) and mutated citrullinated vimentin (anti‐MCV) with anti‐CCP autoantibodies in RA patients and examined their relationships with clinical parameters, cytokine profiles and the PADI4 gene. Autoantibodies were examined by enzyme‐linked immunosorbent assay (ELISA) in sera of 170 RA patients and 103 controls. Cytokine profiles were measured using a multiplex system. PADI4 polymorphisms (89G > A, 90T > C and 92G > C) were genotyped by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP). Anti‐PAD4, anti‐MCV and anti‐CCP autoantibodies were detected in 24, 61 and 74% of RA patients, respectively. Positive correlations were observed between anti‐PAD4 and disease duration; anti‐CCP and erythrocyte sedimentation rate (ESR); anti‐MCV and ESR and C‐reactive protein. Anti‐MCV antibodies were associated with high disease activity score 28 (DAS‐28) in early RA. Concentrations of T helper type 1 (Th1) [tumour necrosis factor (TNF)‐α, interleukin (IL)‐12, IL‐2, IL‐1β], Th2 (IL‐4, IL‐6, IL‐10, IL‐13) and Th17 (IL‐17) cytokines were higher in RA than in controls. Th2 and, to a lesser extent, Th1‐related cytokines, showed positive correlations with anti‐MCV and anti‐CCP. The GTG haplotype in PADI4 was associated with anti‐CCP and anti‐MCV, but not anti‐PAD4 antibodies. In conclusion, anti‐PAD4 antibodies are detected mainly in established RA, which is in contrast to the early detection of antibodies against citrullinated peptide/proteins (ACPAs). Among autoantibodies, anti‐MCV appear to perform better as markers of disease activity. Furthermore, anti‐CCP and anti‐MCV are associated genetically with the citrullinating enzyme PAD4 and are related strongly to Th1 and Th2 cytokines, suggesting a feed‐forward loop between cytokines and ACPA production.

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.

A Functional Ser413/Ser413 PAI-2 Polymorphism Is Associated With Susceptibility and Damage Index Score in Systemic Lupus Erythematosus

Systemic lupus erythematosus in some cases is characterized for development of thrombotic events with a significantly increased risk of mortality. The frequencies and clinical associations of Ser413/Cys413 PAI-2 polymorphism in 40 systemic lupus erythematosus, 50 rheumatoid arthritis patients, and 100 healthy subjects were investigated. The Ser413/Ser413 genotype frequency was 53% (lupus), 36% (rheumatoid arthritis), and 35% (healthy subjects). The Ser413 allele was associated with systemic lupus erythematosus (P = .04, odds ratio = 1.76, 95% confidence interval = 1.01-3.06). In all, 4 patient carriers of Ser413/Ser413 genotype, developed thrombotic events. The lupus patients identified with Ser 413/Ser413 genotype showed an increased damage (57%), compared with Ser413/Cys413 and Cys413/Cys413 genotypes, with significant difference (P = .03). These findings suggest an association of Ser413/Ser413 genotype with greater damage index score and Ser413 allele with systemic lupus erythematosus. Besides, PAI-2 polymorphism could be related with thrombotic phenomena in systemic lupus erythematosus