José Francisco Zambrano-Zaragoza

Ankylosing spondylitis: from cells to genes.

Ankylosing spondylitis (AS) is a chronic inflammatory disease of unknown etiology, though it is considered an autoimmune disease. HLA-B27 is the risk factor most often associated with AS, and although the mechanism of involvement is unclear, the subtypes and other features of the relationship between HLA-B27 and AS have been studied for years. Additionally, the key role of IL-17 and Th17 cells in autoimmunity and inflammation suggests that the latter and the cytokines involved in their generation could play a role in the pathogenesis of this disease. Recent studies have described the sources of IL-17 and IL-23, as well as the characterization of Th17 cells in autoimmune diseases. Other cells, such as NK and regulatory T cells, have been implicated in autoimmunity and have been evaluated to ascertain their possible role in AS. Moreover, several polymorphisms, mutations and deletions in the regulatory proteins, protein-coding regions, and promoter regions of different genes involved in immune responses have been discovered and evaluated for possible genetic linkages to AS. In this review, we analyze the features of HLA-B27 and the suggested mechanisms of its involvement in AS while also focusing on the characterization of the immune response and the identification of genes associated with AS.

IgG class antibodies to heat shock-induced streptococcal antigens in psoriatic patients.

Background Psoriasis is a chronic inflammatory skin disease. Probably autoimmune in nature, and associated with streptococcal throat infections as a triggering factor. Although many groups have associated the disease with other pathogens, Streptococcus pyogenes seems to be the most important microorganism related to this disease. Therefore, it is necessary to identify the streptococcal antigens involved in the process.

Characterization of the Humoral Immune Response against Gnathostoma binucleatum in Patients Clinically Diagnosed with Gnathostomiasis

Gnathostomiasis is an emerging systemic parasitic disease acquired by consuming raw or uncooked fresh-water fish infected with the advanced third-stage larvae of Gnathostoma spp. This disease is endemic to the Pacific region of Mexico, and one of its etiologic agents has been identified as Gnathostoma binucleatum. We characterized the humoral immune response of patients clinically diagnosed with gnathostomiasis by detecting total IgM, IgE, and IgG class and subclasses against a crude extract of the parasite by Western blotting. Our results do not show differences in the antigens recognized by IgM and IgE. However, we found that the specific humoral immune response is caused mainly by IgG, specifically IgG4. We found that 43%, 65.2%, 54.1%, and 26.3% of the patients recognize the 37-kD, 33-kD, 31-kD, and 24-kDa antigens, suggesting that the 33-kD antigen is the immunodominant antigen of G. binucleatum.

The 30-kDa band from Salmonella typhimurium: IgM, IgA and IgG antibody response in patients with ankylosing spondylitis

OBJECTIVE To determine the association of Salmonella typhimurium antigens with AS by analysing the IgA, IgG and IgM antibody response to the crude lysate and the 30-kDa band from this micro-organism. METHODS Sera from 28 AS patients, 28 HLA-B27+ healthy relatives, 28 unrelated healthy subjects and 14 RA patients were included. Salmonella typhimurium proteins were electrophoretically separated and blotted onto nitrocellulose sheets for immunodetection with sera from AS patients and unrelated healthy subjects. The electroeluted 30-kDa band (p30) and a crude lysat (StCL) from S. typhimurium were used as antigen to evaluate the IgM, IgA and IgG (total and subclasses) antibody levels by ELISA. An inhibition assay was carried out to confirm the specificity of IgG response to the p30. RESULTS Twenty out of 28 AS patients (71.4%) and 4 out of 28 unrelated healthy subjects (14.3%) recognized a 30-kDa band from S. typhimurium with IgG antibodies. Six out of 28 AS patients (21.4%) and 4 out of 28 unrelated healthy subjects (14.3%) detected it with IgA antibodies. Recognition of p30 and StCL by both IgA and IgG antibodies was higher in AS patients than in control groups (P = 0.003, <0.001 and 0.003 for IgA and <0.001, 0.003 and 0.006 for IgG). Sera from AS patients have higher percentage of IgG antibodies p30 and IgG3 subclass was higher in AS patients than in control groups. No differences in the IgM response were found. CONCLUSIONS Data presented suggest the association between the p30 and AS.

Genotyping WSSV isolates from northwestern Mexican shrimp farms affected by white spot disease outbreaks in 2010-2012

White spot disease (WSD) causes high mortality in cultured shrimp throughout the world. Its etiologic agent is the white spot syndrome virus (WSSV). The genomic repeat regions ORF 75, ORF 94, and ORF 125 have been used to classify WSSV isolates in epidemiological studies using PCR with specific primers and sequencing. The present study investigated the variation in nucleotide sequences from 107, 150, and 143 isolates of WSSV collected from Litopenaeus vannamei shrimp ponds with WSD outbreaks in northwestern Mexico during the period 2010-2012, in the genomic repeat regions ORFs 75, 94, and 125, respectively. The haplotypic nomenclature for each isolate was based on the number of repeat units and the position of single nucleotide polymorphisms on each ORF. We report finding 17, 43, and 66 haplotypes of ORFs 75, 94, and 125, respectively. The study found high haplotypic diversity in WSSV using the complete sequences of ORFs 94 and 125 as independent variables, but low haplotypic diversity for ORF 75. Different haplotypes of WSSV were found from region-to-region and year-to-year, though some individual haplotypes were found in different places and in more than one growing cycle. While these results suggest a high rate of mutation of the viral genome at these loci, or perhaps the introduction of new viral strains into the area, they are useful as a tool for epidemiological surveys. Two haplotypes from some of the ORFs in the same shrimp were encountered, suggesting the possibility of multiple infections.

STAT4 rs7574865 G/T polymorphism is associated with rheumatoid arthritis and disease activity, but not with anti-CCP antibody levels in a Mexican population.

Rheumatoid arthritis (RA) is a systemic autoimmune disease in whose etiology genetic factors are known to play an important role. Among the genes associated with RA, STAT4 could be an important factor in conducting helper T cells toward the pro-inflammatory Th1 and Th17 lineages. The aim of this study is to determine the association of the STAT4 polymorphism rs7574865 with RA, disease activity, and anti-cyclic citrullinated peptide (CCP) antibody levels in a Mexican population. Genotyping was carried out using the Taqman® system from Applied Biosystems in 140 patients with RA and 150 healthy subjects. Disease activity was evaluated by a rheumatologist using the DAS28 and Spanish-HAQ-DI instruments. Anti-CCP levels were determined by ELISA. Associations of the genotypes of rs7574865 with DAS28, HAQ, and anti-CCP antibody levels with RA were determined. Findings showed that the GT and TT genotypes and the T allele from rs7574865 were all associated as risk factors for RA, independently of their anti-CCP status. An association with moderate-to-high disease activity (DAS28 ≥ 3.2) was also found. Additionally, patients with the GT or TT genotypes showed lower HAQ values than those who carried the GG genotype. No differences in anti-CCP antibody levels or DAS28 and genotypes were found. This work supports the association of the STAT4 rs7574865 polymorphism with RA and disease activity, but not with anti-CCP antibody levels in a Mexican population.

Humoral Immune Response to Salmonella Antigens and Polymorphisms in Receptors for the Fc of IgG in Patients with Ankylosing Spondylitis

Ankylosing Spondylitis (AS) is the prototype of an interrelated group of rheumatic diseases now named spondyloarthritides (SpA), otherwise known as spondyloarthropathies. Clinical features of this disease include inflammatory back pain, asymmetrical peripheral oligoarthritis, enthesitis, and specific organ involvement, such as anterior uveitis, psoriasis and chronic inflammatory bowel disease (Braun & Sieper, 2007).

Impaired neutrophil extracellular traps and inflammatory responses in the peritoneal fluid of patients with liver cirrhosis

Liver cirrhosis (LC) is an inflammatory process associated with impaired functions in adaptive and innate immune responses at both systemic and local levels, also referred as Cirrhosis‐Associated Immune Dysfunction. In this study, we evaluated the functionality of neutrophils from ascitic fluid (AF) of patients with hepatic cirrhosis by testing their ability to generate neutrophil extracellular traps (NETs) in vitro. To further determine the activation state of neutrophils, expression of the activation markers CD66b, CD69, and CD80 on these cells was analysed by flow cytometry. The inflammatory environment in AF was assessed by measured concentration of pro‐ and anti‐inflammatory cytokines. Samples were collected from 40 patients with LC, 20 of them with uncomplicated ascites (ASC) and 20 with spontaneous bacterial peritonitis (SBP). Peripheral blood (PB) neutrophils from healthy individuals were used as control (HC). Our results revealed a significant decrease in the release of NETs in neutrophils from the SBP group compared with HC. Low expression of CD69 and CD80 on neutrophils from AF of SBP patients was also observed. Comparisons of inflammatory cytokine levels in AF from the different study groups (SBP and ASC) revealed significant differences. In conclusion, we demonstrate that the development of complications, such as SBP, increases initially the inflammatory status, but chronically results in impaired neutrophil function as demonstrated by the decreased capability of NETs formation. There is also an increase in both pro‐inflammatory and anti‐inflammatory cytokines, thus predisposing for new episodes of SPB and increasing morbidity and mortality in cirrhotic patients.

pirA- and pirB-like gene identification in Micrococcus luteus strains in Mexico

Acute hepatopancreatic necrosis disease (AHPND) was first reported in China in 2009 and afterwards in Mexico in 2013. AHPND is caused by Vibrio parahaemolyticus and affects Penaeus monodon and Litopenaeus vannamei shrimp cultures. The bacterium contains the pirA- and pirB-like genes in 69- to 70-Kb plasmids, which encode the toxins that produce the disease. The aim of this study was to determine whether pirA- and pirB-like genes existed in bacterial genera distinct from Vibrio before the first cases of AHPND were documented in Mexico. Two bacterial isolates were selected from shrimp farms in Nayarit in 2006 and analysed by nested-PCR to determine the presence of pirA- and pirB-like genes. The two isolates chosen did indeed show the presence of these genes, and those findings were confirmed by sequencing. Both strains matched to the bacterial species Micrococcus luteus. Results revealed two important situations: (a) the pirA- and pirB-like genes were present in a bacterial species that has not been reported previously (Micrococcus luteus); and (b) pirA- and pirB-like bacterial genes were present in Mexico before the first AHPND outbreak was reported in China.

Proteases secreted by Gnathostoma binucleatum degrade fibronectin and antibodies from mammals

Summary Gnathostomiasis is a prevalent zoonosis in humans in some regions of the world. The genus Gnathostoma is considered an accidental parasite for humans; G. binucleatum is the endemic species in Nayarit, Mexico. This work was designed to determine the proteolytic activity of the excretory-secretory products (ESP) of advanced third-stage larvae (ADVL3) of Gnathostoma binucleatum against human fibronectin and antibodies from human and sheep. Our findings showed protease activity against human fibronectin as well as sheep and human gamma globulins of the ESP at molecular weights of 80 and 56 kDa. The proteases found in the ESP of G. binucleatum are thus candidate molecules for consideration as pathogenic elements, owing to the fact that they destroy proteins of the host tissue, which probably allows them to migrate through those tissues and degrade molecules involved in the humoral immune response.