Alejandra Guadalupe García-Zapién

Influence of CRP, IL6, and TNFA gene polymorphisms on circulating levels of C-reactive protein in Mexican adolescents.

BACKGROUND AND AIMS Obesity correlates with a chronic and low-grade inflammation status. C-reactive protein (CRP) measurement has been used as an independent risk marker for future cardiovascular events. CRP level shows interindividual variability due to environmental and genetic factors. The aim of this study was to assess the association of functional polymorphisms on CRP, IL6, and TNFA genes with serum CRP levels in Mexican mestizo adolescents. METHODS Body mass index (BMI), serum high-sensitivity C-reactive protein (hsCRP) levels, and genotypes for CRP+1444C>T, IL6-174G>C, and TNFA-308G>A polymorphisms were obtained from 418 unrelated Mexican adolescents. Genetic association with hsCRP levels was evaluated by means of a dominant genetic model with uni- and multivariate analysis. RESULTS Genotype frequencies for all three polymorphisms were according to Hardy-Weinberg equilibrium (HWE). CRP+1444T, TNFA-308A, and IL6-174C allele frequencies were 37, 7, and 10%, respectively. CRP+1444T was associated with higher mean CRP levels independent of age, gender and BMI (β = 0.21; 95% confidence interval [95% CI] = 0.02-0.39); p = 0.030). IL6-174C was associated with low CRP levels in the overweight group (p = 0.005). IL6-174G>C and TNFA-308G>A allele frequencies observed from this Mexican sample were similar to data for other Mexican populations. CONCLUSIONS The CRP+1444C>T polymorphism was associated with CRP levels in Mexican adolescents and could be used as a genetic marker for the early detection of individuals at risk for developing obesity-related conditions such as cardiovascular disease or type 2 diabetes mellitus in early adulthood.

Tumor Necrosis Factor-Alpha Gene Promoter −308G/A and −238G/A Polymorphisms in Mexican Patients with Type 2 Diabetes Mellitus

The association between some Tumor necrosis factor-alpha (TNF-α) promoter polymorphisms and Type 2 diabetes mellitus (T2DM) remains controversial. Ethnic differences may play a role in these conflicting results. The aim of this study was to investigate the association between −308G/A and −238G/A polymorphisms located in the promoter region of the TNF-α gene and T2DM in Mexican mestizo patients. Nine hundred four individuals (259 patients with T2DM and 645 controls) were genotyped for the −308G/A and −238G/A polymorphisms by PCR—RFLP. We found that the −238A allele increased the risk of developing T2DM in Mexican patients (OR = 1.57, 95% CI: 1.07–2.29; p = 0.018). Moreover, we found that the frequency of the GA haplotype (created by the −308G and −238A alleles) was significantly increased in patients with T2DM when compared with controls (OR = 1.56, 95% CI: 1.05–2.31; p = 0.026). Our results suggest that the −238G/A polymorphism and a specific haplotype (GA) are genetic risk factors for the development of T2DM in Mexican population.

Association Analysis Between –308G/A and –238G/A TNF-Alpha Gene Promoter Polymorphisms and Insulin Resistance in Mexican Women With Gestational Diabetes Mellitus

Background Gestational diabetes mellitus (GDM) is characterized by insulin resistance. It has been described that tumor necrosis factor α (TNF-α) plays a key role in the pathogenesis of insulin resistance; moreover, increased levels of this proinflammatory cytokine have been reported in women with GDM. Therefore, this study was aimed to assess the presence of associations between the –308G/A and –238G/A polymorphisms and specific haplotypes of the TNF-α gene promoter region and insulin resistance in Mexican women with GDM. Methods This study included 51 women with GDM and 44 pregnant women with normal glucose tolerance. Measurements of anthropometric parameters and biochemical estimations were performed. We genotyped the TNF-α –308G/A and –238G/A polymorphisms using polymerase chain reaction–restriction fragment length polymorphism analysis. Results The genotype and allele frequencies of both polymorphisms did not differ significantly between the women with GDM and the controls. However, we found that the frequency of the AG haplotype was significantly increased in the patients with GDM compared with controls (P = 0.019; odds ratio, 4.11; 95% confidence interval, 1.31–12.85). In patients with GDM, we observed that insulin levels and homeostasis model assessment of insulin resistance were significantly higher in women bearing the G/G genotype than in carriers of the G/A and A/A genotypes of the –308G/A polymorphism (P = 0.022 and P = 0.043, respectively). Conclusions Our results suggest that the G/G genotype of the TNF-α –308G/A polymorphism increases insulin levels and insulin resistance in women with GDM and that the AG haplotype is a genetic risk factor for GDM in our study population.

Screening of R122H and N29I mutations in the PRSS1 gene and N34S mutation in the SPINK1 gene in Mexican pediatric patients with acute and recurrent pancreatitis.

Objectives The study’s objective was to assess the association between the PRSS1 R122H and N29I and the SPINK1 N34S mutations and acute pancreatitis (AP) and recurrent pancreatitis in Mexican pediatric patients. Methods The N34S and R122H mutations were detected using polymerase chain reaction–restriction fragment length polymorphism, and the N29I mutation was detected using allele-specific polymerase chain reaction in 92 pancreatitis patients (58 AP and 34 recurrent pancreatitis patients) and 144 controls. Results We found 1 mutated allele in 4 (4.3%) of 92 pancreatitis patients and none in the controls. All 4 patients bearing mutations had AP, with a frequency of 6.8% (4/58). Three (5.2%) of 58 patients were heterozygous for the N34S mutation, and 1 (1.7%) of 58 patients was heterozygous for the N29I mutation. The comparison between the AP and control groups revealed both a significant number of patients carrying any mutations in the screened genes (P = 0.008) and bearing the N34S mutation (P = 0.023). Moreover, we found that the N34S G allele increased the risk of developing AP (odds ratio, 10.3; confidence interval, 1.1–248.8). Conclusions Patients bearing the N34S G allele exhibited a 10-fold increased risk of developing AP compared with controls, suggesting that the SPINK1 N34S mutation represents an etiologic risk factor for AP in our Mexican pediatric patients.

Congo Red Effect on Cyst Viability and Cell Wall Structure of Encysting Entamoeba invadens

BACKGROUND The cell wall of Entamoeba invadens cysts is composed of chitin microfibrils as the main structural component. It has been demonstrated in yeast that the chitin cell wall assembly is altered by dyes such as Congo red (CR) and Calcofluor. METHODS The purpose of this work was to study the cell wall assembly under the effect of CR dye on encysting E. invadens by means of light and electron microscopy, after the amebas were subjected to the effect of 100-2,000 micrograms CR/mL. Experiments were performed either in BI-S-33 or in mLG media. RESULTS Trophozoite growth was not inhibited by 100-1,000 micrograms/mL CR after 8 days of incubation in BI-S-33 medium. However, low levels of growth were observed with 2,000 micrograms/mL of dye. No significant differences in morphologically viable (hyaline) cyst production occurred after 24-48 h, when 100 micrograms CR/mL was used, while the highest concentration of CR (2,000 micrograms/mL) resulted in a significant decrease of hyaline cyst yield; dead cysts prevailed in cultures, particularly at 72 h of CR treatment. Differentiation of amebas incubated in the presence of 500-2,000 micrograms/mL CR produced abnormal chitin deposits, rendering irregularly thick or double cell walls, as shown by transmission and scanning electron microscopy. Cyst cultures obtained under 100 micrograms/mL CR produced as many trophozoites as did the control when they were incubated in BI-S-33, but only low numbers of trophozoites were found in culture cysts obtained under higher CR doses. CONCLUSION Our results suggest that CR affects E. invadens encystment, alters the cell wall formation, and also affects the cyst viability.

Serum Levels of Glutathione Peroxidase 3 in Overweight and Obese Subjects from Central Mexico

BACKGROUND AND AIMS Overweight and obesity are considered complex entities in which there are alterations in the concentration of antioxidant enzymes. It has been reported that glutathione peroxidase 3 (GPx3), an extracellular enzyme involved in the reduction of both hydro- and lipoperoxides, shows changes both in gene expression and protein concentration in animal models for type 2 diabetes (T2D) and obesity, but the variability of GPx3 levels in different human populations and under different health conditions are currently unclear. We undertook this study to determine the GPx3 levels in overweight and obese subjects from central Mexico. METHODS Biochemical profile (serum glucose, insulin and lipid profile) and GPx3 concentrations were determined in 28 healthy subjects (control) and 133 subjects who were overweight or obese (OW-OB). RESULTS The OW-OB group had a higher concentration of triacylglycerides (TAG) compared with the control group (201.2 ± 88.7 vs. 100.3 ± 46.4 mg/dL, p <0.05) and the TAG/high density lipoprotein-cholesterol (HDL-C) index (5.6 ± 2.8 vs. 2.1 ± 1.2, p <0.05), whereas the concentration of HDL-C decreased (38.2 ± 8.7 vs. 50.1 ± 14.5 mg/dL, p <0.05). Serum GPx3 was significantly higher in the OW-OB group than in the control group (175.4 ± 25.4 vs. 143.5 ± 23.1 ng/dL). GPx3 concentration correlated with insulin sensitivity (IS) and the TAG/HDL-C index (Rho = -0.2336 and Rho = 0.2275) (p <0.01). CONCLUSIONS The TAG/HDL-C index and serum GPx3 concentration increased in the OW-OB group. In addition, GPx3 had a significant correlation with IS, weight, and the TAG/HDL-C index.

Glutathione Peroxidase 3 Serum Levels and GPX3 Gene Polymorphisms in Subjects with Metabolic Syndrome

BACKGROUND AND AIMS Glutathione peroxidase 3 (GPx3) plays a main role in removing hydro- and lipoperoxides from the body. Changes in concentration and several single-nucleotide polymorphisms (SNP) at the GPX3 gene have been associated with vascular diseases, but the relationship of GPx3 with metabolic syndrome (MetS) remains unexplored. We undertook this study to determine the association of GPx3 serum levels and several GPX3 SNPs with the presence of MetS in Mexican subjects. METHODS Clinical, biochemical, and anthropometric evaluation were conducted in 426 subjects assigned to three groups: control (n = 42); risk group (RG, n = 200), and MetS group (n = 184). Insulin sensitivity (IS) and cardiovascular risk were determined by the QUICKI and TG/HDL-C index, respectively. Serum GPx3 was determined by enzyme immunoassay and polymorphisms within GPX3 gene were identified by nucleotide sequencing. RESULTS MetS group showed low IS and increased cardiovascular risk with respect to controls as well as higher GPx3 serum levels (172.9 ± 32.2 vs. 145.6 ± 24.8 ng/dL; p <0.05). Only three of the ten GPX3 SNPs screened were polymorphic with two haplotypes observed (CCT and TTA-rs8177404, rs8177406, and rs8177409), indicating tight linkage disequilibrium in this genetic region. No differences for either genotype or allele frequencies among groups were observed, but rs8177409 (allele T) was associated with cardiovascular risk (odds ratio [OR], 4.5; p = 0.0125). CONCLUSION This study shows that serum levels of GPx3 are increased in subjects with MetS and that rs8177409 SNP was associated with cardiovascular risk in a Mexican population.

Low Prevalence of Interleukin-6 Haplotypes Associated with a Decreased Risk of Type 2 Diabetes in Mexican Subjects with a Family History of Type 2 Diabetes

BACKGROUND AND AIMS There is evidence that family history of type 2 diabetes (FHT2D) and single nucleotide polymorphisms (SNP) on the IL-6 gene promoter region are separately associated with the risk of developing type 2 diabetes. However the relationship between adult Mexican subjects with FHT2D and genotypes/haplotypes for IL-6 gene has not been explored. The aim of the present work was to study the prevalence of IL-6 -598G>A-572G>C-174G>C haplotypes among subjects with FHT2D and to determine whether their presence influences the relationship between FHT2D and risk factors for diabetes. METHODS Two hundred fifty eight nondiabetic subjects participated in this study; 153 with and 105 without FHT2D. Polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) was used for genotyping. Logistic regression analysis was employed to assess the impact of IL-6 haplotypes on FHT2D per se and hyperinsulinemia and insulin resistance as risk factors for diabetes. RESULTS Subjects with FHT2D showed a higher prevalence of hyperinsulinemia and insulin resistance (IR) than those without FHT2D (14.4 vs. 5.7%, p = 0.029, and 14.2 vs. 7.0% p = 0.050, respectively). Lower prevalence of -598 -572-174 (AGC)-haplotype (19%) in subjects with FHT2D was observed as well as a lower prevalence of hyperinsulinemia and IR among AGC haplotype carriers (12 and 14%, respectively). The relationship between FHT2D and IR was modified by the presence of AGC haplotype (from OR, 2.70; 95% CI, 0.99-7.36; p = 0.050 OR, 30.08; 95% CI, 0.58-1,568.06; p = 0.092). CONCLUSIONS IL-6 -598/-572/-174 (AGC) haplotype has a low prevalence among first-degree relatives of subjects with type 2 diabetes. Our results suggest that this haplotype is associated with decreased risk of type 2 diabetes in Mexican subjects with FHT2D.

Analysis of cytokine gene polymorphisms in Mestizo and native populations from Mexico

To determine whether the well‐known genetic structure of the Mexican population observed with other multiallelic markers can be detected by analyzing functional polymorphisms of cytokine and other inflammatory‐response‐related genes.

Comparative Study of Entamoeba invadens Differentiation in Three Different Axenic Encystation Media

Differentiation of Entamoeba invadens trophozoites to cysts in axenic conditions has been achieved by means of several procedures. The axenic encystation medium (AEM) (1) was the first simple and reliable medium available to induce massive encystation of E. invadens trophozoites. This allowed the study of several aspects of amebic biology, such as cell wall composition, cell cycle, drug effects, and changes in cell surface composition during encystation. However, low cyst yields were reported by some researchers, thus impairing reproducibility of the experiments. The presence of variable numbers of cysts in E. invadens cultures at stationary phase of growth was the clue to test the role of various carbon sources—or their absence—on the initiation of encystation, resulting in the implementation of low-glucose (LG) medium for massive encystation (2). Larger numbers of cysts than of inoculated trophozoites were attained with this medium, whose efficiency and reproducibility have been corroborated in our laboratory (3). Avron et al. (4) reported E. invadens encystation in low osmotic pressure medium by using several semipermeable solutes in solutions ranging from 60–160 mosm/kg. High cyst yields were obtained with some solutes. It is now clear that hypotonicity and/or lack of carbon sources are able to induce encystation in E. invadens. However, quantitative evaluation of these three media is necessary to identify the best medium to produce high yields of viable mature, tetranucleate, detergent-resistant cysts under similar conditions of amebic strain, serum lot, chemicals, incubation temperature, and handling. In this study, we quantitatively evaluated E. invadens differentiation in AEM, LG, and MgSO 4 -serum media. Materials and Methods