Genetic Variant of DNAM-1 rs763361 C>T Is Associated with Ankylosing Spondylitis in a Mexican Population.

DNAM-1 (CD226) is an activating receptor expressed in CD8+ T cells, NK cells, and monocytes. It has been reported that two SNPs in the DNAM-1 gene, rs763361 C>T and rs727088 G>A, have been associated with different autoimmune diseases; however, the role of DNAM-1 in ankylosing spondylitis has been less studied. For this reason, we focused on the study of these two SNPs in association with ankylosing spondylitis. For this, 34 patients and 70 controls were analyzed using endpoint PCR with allele-specific primers. Our results suggest that rs763361 C>T is involved as a possible protective factor under the CT co-dominant model (OR = 0.34, 95% CI = 0.13-0.88, p = 0.022) and the CT + TT dominant model (OR = 0.39, 95% CI = 0.17-0.90, p = 0.025), while rs727088 G>A did not show an association with the disease in any of the inheritance models. When analyzing the relationships of the haplotypes, we found that the T + A haplotype (OR = 0.31, 95% CI = 0.13-0.73, p = 0.0083) is a protective factor for developing the disease. In conclusion, the CT and CT + TT variants of rs763361 C>T and the T + A haplotype were considered as protective factors for developing ankylosing spondylitis.

Hematological indices as indicators of inflammation induced by exposure to pesticides.

Pesticide toxicity, both acute and chronic, is a global public health concern. Pesticides are involved in abnormal inflammatory responses by interfering with the normal physiology and metabolic status of cells. In this regard, inflammatory indices aggregate index of systemic inflammation (AISI), monocyte-to-high-density lipoprotein ratio, monocyte-to-lymphocyte ratio (MLR), neutrophil-to-lymphocyte platelet ratio (NLPR), neutrophil-to-lymphocyte ratio, platelet-to-lymphocyte ratio, systemic immune inflammation index, and systemic inflammation response index (SIRI) have been used as predictive markers of inflammatory status in several diseases and also in acute poisoning events. This study aimed to determine systemic inflammation indices and their relationship with pesticide exposure from urban sprayers in 302 individuals categorized into three groups (reference group and moderate and high exposure groups). The data suggest that the AISI, MLR, NLPR, and SIRI indices were significantly higher in the exposed groups compared with the reference group. In conclusion, this study proposes that inflammation indices warrant further attention in order to assess their value as early biomarkers of acute and chronic pesticide intoxication.

Association of MIR3117 and MIR612 Genes Polymorphisms with Childhood Acute Lymphoblastic Leukemia in the Mexican Population.

INTRODUCTION: Acute lymphoblastic leukemia (ALL) is the most common childhood cancer in the world, which is associated with a wide spectrum of factors that play an important role in epidemiology, risk stratification, and therapeutic intervention. Several studies have shown the role of microRNAs (miRNAs) in the development of the disease. Genetic variations such as single-nucleotide polymorphisms (SNPs) in miRNAs can alter their function and lead to alter the expression of their target genes. OBJECTIVE: The aim of this study was to evaluate the association of rs12402181 in MIR3117 and rs12803915 in MIR612 with the risk of childhood preB-ALL in Mexican population. MATERIAL AND METHODS: DNA from 148 children (<18 years old) diagnosed with preB-ALL and 172 samples from participants in control group were included in the present study. Genotyping of the rs12402181 and rs12803915 polymorphisms was carried out by Real-Time PCR. To estimate the risk factor, the multiple genetic models co-dominant, dominant, and recessive were determined in both polymorphisms. RESULTS: In dominant genetic model from rs12402181, a high risk of susceptibility to ALL was observed (OR = 2.03, 95% CI = 1.27-3.22, p = 0.003). In the analysis adjusted for gender, a significant increase in the risk of ALL was maintained (OR = 2.03, 95% CI = 1.28-3.24, p = 0.003). The rs12803915 polymorphism was no associated with the risk of susceptibility to preB-ALL in any of the genetic models using in this study. CONCLUSIONS: Our data indicated that the A allele of the rs12402181 polymorphism may be considered as a genetic biomarker of preB-ALL susceptibility. Likewise, it was identified that the A allele of the rs12402181 polymorphism is an independent risk factor for ALL.

Particulate matter (PM10) induces in vitro activation of human neutrophils, and lung histopathological alterations in a mouse model.

The epidemiological association between exposure to particulate matter (PM10) and various respiratory and cardiovascular problems is well known, but the mechanisms driving these effects remain unclear. Neutrophils play an essential role in immune defense against foreign agents and also participate in the development of inflammatory responses. However, the role of these cells in the PM10 induced inflammatory response is not yet fully established. Thus, this study aims to evaluate the effect of PM10 on the neutrophil-mediated inflammatory response. For this, neutrophils from healthy adult human donors were in vitro exposed to different concentrations of PM10. The cell viability and cytotoxic activity were evaluated by MTT. LDH, propidium iodide and reactive oxygen species (ROS) were quantified by flow cytometry. Interleukin 8 (IL-8) expression, peptidyl arginine deiminase 4 (PAD4), myeloperoxidase (MPO), and neutrophil elastase (NE) expression were measured by RT-PCR. IL-8 was also quantified by ELISA. Fluorescence microscopy was used to evaluate neutrophil extracellular traps (NETs) release. The in vivo inflammatory responses were assessed in BALB/c mice exposed to PM10 by histopathology and RT-PCR. The analysis shows that PM10 exposure induced a cytotoxic effect on neutrophils, evidenced by necrosis and LDH release at high PM10 concentrations. ROS production, IL-8, MPO, NE expression, and NETs release were increased at all PM10 concentrations assessed. Neutrophil infiltration in bronchoalveolar lavage fluid (BALF), histopathological changes with inflammatory cell infiltration, and CXCL1 expression were observed in PM10-treated mice. The results suggest that lung inflammation in response to PM10 could be mediated by neutrophils activation. In this case, these cells migrate to the lungs and release pro-inflamatory mediators, including ROS, IL-8, and NETs. Thus, contributing to the exacerbation of respiratory pathologies, such as allergies, infectious and obstructive diseases.

Deleted genes associated with obesity in Mexican patients diagnosed with nonalcoholic fatty liver disease.

AIM: Nonalcoholic fatty liver disease (NAFLD) is a complex metabolic condition in which both lifestyle and genetic factors have a pathogenic role. The LEP gene encodes leptin, which regulates appetite, body weight, and several metabolic functions. Proopiomelanocortin (POMC), regulates food intake and energy balance. The aim of the study was to determine partial or complete deletions of genes associated with obesity in patients diagnosed with NAFLD. MATERIAL AND METHODS: Blood samples and DNA from 43 individuals diagnosed with NAFLD by ultrasonographic technique (Fibroscan) were obtained. The partial or complete deletions of genes were determined by MLPA (Multiplex Ligation-dependent Probe Amplification) using the SALSA probemix P220-B2 Obesity only on 43 individuals. Fifty blood samples from healthy individuals were included. RESULTS: Eleven out of 43 individuals analyzed by MLPA presented some deletion of the genes analyzed: six were female and five were male. The partial or complete deletion of the LEPR and POMC genes was observed in eight patients (18.6%), SIM1 in six patients (13.9%), GRIK2 and SH2B1 in two patients (4.7%), SEZGL2 in four patients (9.3%), and MCR4 in one patient (2.3%). CONCLUSION: Partial deletion was observed in LEPR, POMC, SIM1, GRIK2, SH2B1, SEZGL2, and MCR4 genes in 26% of the cases, and we suggest that these alterations probably has a potential relationship for the development of NAFLD.

The influence of the -94 Ins/Del ATTG polymorphism of NFkB on the anti-CCP antibody levels in patients with rheumatoid arthritis.

ABSTRACT: Rheumatoid arthritis (RA) is an autoimmune disease characterized by an inflammatory process that affects mainly synovial tissue in joints, and by the production of cyclic citrullinated peptides (anti-CCP) antibodies. In the inflammatory process the regulation of the nuclear factor kappa B (NFkB) transcription factor activation is a key point in the production of inflammatory cytokines. On the other hand, polymorphisms in several genes could contribute to the promotion of the inflammatory process observed in RA, and the association of the rs28362491 polymorphism in the NFkB gene with RA has been studied in different population. Therefore, it could be one of the interest targets to analyze their association with RA in a Mexican population.This is a case-control study to determine the influence of rs28362491 in the NFkB gene on RA and on clinical features of this disease, such as anti-CCP antibody levels, Disease Activity Score, and Health Assessment Questionnaire-Disability Index.The genotype of rs28362491 in the NFkB gene was determined in 140 RA patients and 135 healthy controls using the polymerase chain reaction-restriction fragment length polymorphism method with the enzyme PflMI. The following clinical variables were also determined: anti-CCP levels, Disease Activity Score, and Spanish version of the Health Assessment Questionnaire Disability-Index.Although no association of the polymorphism as a risk/protection factor with RA was found, the RA patients who carried the Ins/Ins genotype showed higher anti-CCP levels, while those with the Del/Del genotype showed higher Spanish version of the Health Assessment Questionnaire-Disability Index levels, compared to the other genotypes.The NFkB -94 Ins/Del ATTG (rs28362491) polymorphism is, therefore, associated with higher levels of anti-CCP antibodies, though no significant association as a risk or protection factor in RA cases was identified.

High frequency of the risk allele of rs4132601 and rs11978267 from the IKZF1 gene in indigenous Mexican population.

BACKGROUND: IKZF1 is a relevant gene associated with the pathogenesis of acute lymphoblastic leukemia, and the rs4132601 (T>G) and rs11978267 (A>G) polymorphisms have been associated with the development of this disease in several populations. The aim of this study was to determine the allelic and genotypic frequencies of the rs4132601 and rs11978267 polymorphisms in two indigenous Mexican groups (Cora and Huichol) and Mestizo populations from Nayarit, Mexico, and compare them with the frequencies of both polymorphisms in other populations of the world. METHODS: One hundred, 116, and 100 subjects from the Mestizo, Huichol, and Cora populations, respectively, all of them residents of the state of Nayarit, Mexico, were analyzed. The frequencies of rs4132601 and rs11978267 were determined by allelic discrimination using TaqMan assays. RESULTS: The allelic frequencies of rs4132601 were as follows: Mestizo group T = 0.74, G = 0.26; Cora T = 0.745, G = 0.255; and Huichol T = 0.47, G = 0.53. In the case of the rs11978267 polymorphism, the allelic frequencies were Mestizo A = 0.745, G = 0.255; Cora A = 0.735, G = 0.265; and Huichol A = 0.457, G = 0.543. For each population, both polymorphisms were in Hardy-Weinberg equilibrium. CONCLUSION: The Huichol population from Nayarit presented the highest frequencies of the risk allele reported to date in the whole world for both rs4132601 and rs11978267 polymorphisms.

Neutrophil extracellular traps and inflammatory response: Implications for the immunopathogenesis of ankylosing spondylitis.

AIM: Ankylosing spondylitis (AS) pathogenesis has focused on the adaptive immune response; however, innate immune responses may also play a role in the inflammatory response of AS. Dysregulated neutrophil activation can induce tissue damage and contribute to the pathogenesis of immune-related diseases. Hence, the aim of this study was to assess the effect of immune complexes formed with the p30 of Salmonella typhimurium and anti-p30 antibodies present in the sera of AS patients and controls in inducing the release of neutrophil extracellular traps (NETs) and the secretion of pro-inflammatory cytokines. METHODS: We collected polymorphonuclear leukocytes (PMNs) from healthy donors. The PMNs isolated were stimulated with p30 alone or in immunocomplexes formed with antibodies presents in sera of AS patients or control subjects. Then, the NETs were analyzed by fluorescence microscopy. Concentrations of interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-1ß, IL-8 and IL-10, were determined using the Cytometric Bead Array kit. RESULTS: Significant difference was observed in the release of NETs between the neutrophils stimulated with p30 + AS (70.52 ± 16.24) those unstimulated neutrophils (9.94 ± 12.12; P = .0095), stimulated with phorbol 12-myristate 13-acetate (39.78 ± 14.50; P = .0190), stimulated with control serum (CS) (10.85 ± 5.33; P = .0082) and serum of AS patient (10.28 ± 6.15; P = .0087). The stimulation of neutrophils with p30 alone induced a relatively low production of IL-6 (64.5 pg/mL), IL-8 (2658.3 pg/mL), IL-1ß (31.11 pg/mL), and TNF-α (3.8 pg/mL), compared to p30 + AS and p30 + CS groups. CONCLUSION: Our results show that neutrophils release NETs and pro-inflammatory cytokines in response to p30 in immunocomplexes. These findings could improve our understanding of the role of innate immunity in the initiation and/or maintenance of inflammatory responses, and in the progression of AS.

The TNFA -857C/T Polymorphism: Association with Rheumatoid Arthritis and Anti-CCP Levels in a Mexican Population.

Rheumatoid arthritis (RA) is a chronic inflammatory disease whose association with SNPs has led to the identification of biomarkers in different populations. To determine the association of the -857C/T SNP of the TNFA gene with RA and clinical parameters, 233 RA patients and 237 healthy controls were included in this study. The -857C/T polymorphism was determined using the TaqMan® system and clinical features were also determined. We found that the -857C/T SNP was in Hardy-Weinberg equilibrium. Our results showed no association of the -857C/T SNP with RA; however, RA patients carrying the TT genotype showed lower anti-CCP levels than other groups. Therefore, the TT genotype could be a risk factor for developing anti-CCP-negative RA. Our results suggest that the T allele of the TNFA -857C/T SNP exerts an influence on anti-CCP levels and could be a candidate marker for anti-CCP-negative RA.

1,25-Dihydroxyvitamin D3 induces formation of neutrophil extracellular trap-like structures and modulates the transcription of genes whose products are neutrophil extracellular trap-associated proteins: A pilot study.

Neutrophils are components of the innate immune system that participate in controlling infectious diseases through microbicidal mechanisms such as phagocytosis, degranulation and the release of neutrophil extracellular traps (NETs). NETs are DNA structures that are released through the decondensation and spreading of chromatin and the adherence of various proteins, including neutrophil elastase (NE), myeloperoxidase (MPO) and peptidyl arginine deiminase 4 (PDA4). Since NETs recovered after treatment of activated polymorphonuclear neutrophils can enhance IL-1ß and IFN-α production by LPS-activated macrophages, they are thought to be keys to the host's defenses and inflammation. 1,25(OH)2D3 has been shown to play an important role in modulating neutrophils activation and in preventing infections. Therefore, the aim of this study was to assess the effect of 1,25(OH)2D3 in modulating induction of the release of NETs and in regulating the transcription of genes whose products in human neutrophils are NETs-associated proteins, TLRs and interferon. We observed that 1,25(OH)2D3 induced production of NETs-like structures while also upregulating NE/PAD4/COX-3/GAPDH mRNA levels. Additionally, we found an increase in TLR7 and type I interferon (IFN) mRNA levels as a result of neutrophil activation by 1,25(OH)2D3. Since the transcription of genes whose products constitute NETs-associated proteins are differentially-regulated by 1,25(OH)2D3, we proposed that this might restrict the spread of pathogens, such as virus, by inducing NETs, the expression of TLR7 and secretion of IFN-α. Our results suggest the potential importance of this hormone in preventing infections by inducing NETs formation.

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.

Understanding the molecular mechanisms of NETs and their role in antiviral innate immunity.

Polymorphonuclear neutrophils (PMNs) are the most abundant cells in the context of innate immunity; they are one of the first cells to arrive at the site of viral infection constituting the first line of defense in response to invading pathogens. Indeed, neutrophils are provided with several defense mechanisms including release of cytokines, cytotoxic granules and the last recently described neutrophil extracellular traps (NETs). The main components of NETs are DNA, granular antimicrobial peptides, and nuclear and cytoplasmic proteins, that together play an important role in the innate immune response. While NETs were first described as a mechanism against bacteria and fungi, recently, several studies are beginning to elucidate how NETs are involved in the host antiviral response and the prominent characteristics of this new mechanism are discussed in the present review.

Over-expression of TLR4-CD14, pro-inflammatory cytokines, metabolic markers and NEFAs in obese non-diabetic Mexicans.

INTRODUCTION: Obesity is the world's most important public health problem. Adipose tissue contributes significantly to increase pro-inflammatory mediators whose cascade begins with the union of TLR4 to its microbial ligands (TLR: Toll Like Receptors). It has been reported recently that NEFAs (Non-Esterified Fatty Acids) bind to this receptor as agonists. The purpose of our study was to determine the levels of expression of TLR4-CD14, the pro-inflammatory cytokines, the metabolic markers and the NEFAs in a group of adult, non-diabetic obese Mexicans. METHOD: A group of 210 adult middle-class Mexican non-diabetic obese patients was evaluated: 105 normal weight individuals, and 105 non-diabetic obese. On both groups, the following was tested in each patient: TLR4-CD14 receptors on monocytes in peripheral blood, inflammatory profile, HOMA-IR (Homeostasis Model Assessment-Insulin Resistance), NEFAs and each individual's anthropometric profile. RESULTS: Obesity is strongly associated with the expression of TLR4 (77%, MFI (Mean Fluorescence Index) 7.70) and CD14 (86% MFI 1.61) with 66% double positives (p = 0.000). These figures contrast with those for the normal weight individuals that constituted the control group: TLR4 (70% MFI 6.41) and CD14 (84% MFI 1.25) with 59% double positives. As for cytokine concentration, non- diabetic obese individuals vs the normal weight/thin, the numbers were: IL-1ß = 2.0 vs 2.5 pg/ml (p = NS), IL-6 = 36 vs 28 pg/ml (p = 0.030), IL-8 = 27 vs 27 pg/ml (p = NS), IL-10 = 8.4 vs 6.8 pg/ml (p = NS), TNF-α =31 vs 15 pg/ml (p = 0.000) respectively. Insulin levels were 12.1 vs 19.7 mcU/ml (p = 0.000) and the NEFAs were much higher in the obese vs normal weight/thin individuals (p = 0.000). CONCLUSION: Adipose tissue used to be thought of as mere storage of fats and energy, but it has been revealed to be an important neuro-immune-endocrine organ. Immune cells, stimulated by NEFAs, produce pro-inflammatory cytokines, which have a direct effect on oxidating radicals that directly target the release of noradrenalin. This in turn, reactivates the vicious cycle of low-grade chronic inflammation, as is now described in obesity.

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.