Common Polymorphisms Linked to Obesity and Cardiovascular Disease in Europeans and Asians are Associated with Type 2 Diabetes in Mexican Mestizos.

Background and objectives: Type 2 diabetes (T2D) is a major problem of public health in Mexico. We investigated the influence of five polymorphisms, previously associated with obesity and cardiovascular disease in Europeans and Asians, on T2D in Mexican Mestizos. Materials and Methods: A total of 1358 subjects from 30 to 85 years old were genotyped for five loci: CXCL12 rs501120; CDNK2A/B rs1333049; HNF-1α rs2259816; FTO rs9939609; and LEP rs7799039. We used logistic regressions to test the effect of each locus on T2D in two case⁻control groups with obesity and without obesity. Also, linear regression models on glucose and glycated hemoglobin (HbA1c) were carried out on the whole sample, adjusted by age, gender, and body mass index. Results: The CXCL12 rs501120 C allele (OR = 1.96, p = 0.02), the FTO rs9939609 A allele (OR = 2.20, p = 0.04) and the LEP rs7799039 A allele (OR = 0.6, p = 0.03) were significantly associated with T2D in obesity case⁻control group. No significant association was found in the non-obesity case⁻control group. The linear regression model showed that CDNK2A/B rs1333049 C allele (ß = 0.4, p = 0.03) and FTO rs9939609 A allele (ß = 0.5, p = 0.03), were significantly associated with HbA1c, but no association was found among the loci with the glucose levels. Conclusions: Polymorphisms previously linked with obesity and cardiovascular events were also associated with T2D and high levels of HbA1c. Furthermore, we must point at the fact that this is the first report where polymorphisms CXCL12 rs501120 and LEP rs7799039 are associated with T2D in subjects with obesity.

Elevated levels of enteric IgA in an unimmunised mouse model of Hyper IgM syndrome derived from gut-associated secondary lymph organs even in the absence of germinal centres.

Introduction: Patients with Human Hyper IgM syndromes (HIGM) developed pulmonary and gastrointestinal infections since infancy and most patients have mutations in the CD40 ligand (CD40L) gene. Most HIGM patients compared to healthy subjects have higher/similar IgM and lower IgG, and IgA serum concentrations but gut antibody concentrations are unknown. CD40L on activated T-cells interacts with CD40 on B-cells, essential for the formation of germinal centres (GCs) inside secondary lymphoid organs (SLOs), where high-affinity antibodies, long-lived antibody-secreting plasma cells, and memory B-cells, are produced. C57BL6-CD40 ligand deficient mice (C57BL6-cd40l -/-), are a model of HIGM, because serum immunoglobulin concentrations parallel levels observed in HIGM patients and have higher faecal IgA concentrations. In mice, TGFß and other cytokines induce IgA production. Aims: To compare and evaluate B-cell populations and IgA-producing plasma cells in peritoneal lavage, non-gut-associated SLOs, spleen/inguinal lymph nodes (ILN), and gut-associated SLOs, mesenteric lymph nodes (MLN)/Peyer´s patches (PP) of unimmunised C57BL6-cd40l -/- and C57BL6-wild-type (WT) mice. Material and methods: Peritoneal lavages, spleens, ILN, MLN, and PP from 8-10 weeks old C57BL6-cd40l -/- and WT mice, were obtained. Organ cryosections were analysed by immunofluorescence and B-cell populations and IgA-positive plasma cell suspensions by flow cytometry. Results: In unimmunised WT mice, GCs were only observed in the gut-associated SLOs, but GCs were absent in all C57BL6-cd40l -/- SLOs. PP and MLN of C57BL6-cd40l -/- mice exhibited a significantly higher number of IgA-producing cells than WT mice. In the spleen and ILN of C57BL6-cd40l- /- mice IgA-producing cells significantly decreased, while IgM-positive plasma cells increased. C57BL6-cd40l -/- B-1 cells were more abundant in all analysed SLOs, whereas in WT mice most B-1 cells were contained within the peritoneal cavity. C57BL6-cd40l -/- B-cells in MLN expressed a higher TGFß receptor-1 than WT mice. Mouse strains small intestine microvilli (MV), have a similar frequency of IgA-positive cells. Discussion: Together our results confirm the role of PP and MLN as gut inductive sites, whose characteristic features are to initiate an IgA preferential immune response production in these anatomical sites even in the absence of GCs. IgA antibodies play a pivotal role in neutralising, eliminating, and regulating potential pathogens and microorganisms in the gut.

A Novel Adult Murine Model of Typical Enteroaggregative Escherichia coli Infection Reveals Microbiota Dysbiosis, Mucus Secretion, and AAF/II-Mediated Expression and Localization of ß-Catenin and Expression of MUC1 in Ileum.

Typical enteroaggregative Escherichia coli (tEAEC) is a diarrheagenic E. coli pathotype associated with pediatric and traveler's diarrhea. Even without diarrhea, EAEC infections in children also lead to increased gut inflammation and growth shortfalls. EAEC strain's defining phenotype is the aggregative adherence pattern on epithelial cells attributable to the aggregative adherence fimbriae (AAF). EAEC only causes diarrhea in humans; therefore, not much is known of the exact intestinal region of infection and damage or its interactions with intestinal enterocytes in vivo and in situ. This study aimed to develop a new tEAEC mouse model of infection, characterize the microbiota of infected mice, and evaluate in situ the expression of host adherence and surface molecules triggering EAEC infection and the role of the EAEC AAF-II in adherence. Six-week-old C57BL/6 mice, without previous antibiotic treatment, were orally challenged with EAEC 042 strain or EAEC 042 AAF-II mutant (ΔAAF/II) strain, or DAEC-MXR strain (diffusely adherent E. coli clinical isolate), and with saline solution (control group). Paraffin sections of the colon and ileum were stained with H&E and periodic acid-Schiff. ZO-1, ß-catenin, MUC1, and bacteria were analyzed by immunofluorescence. EAEC-infected mice, in comparison with DAEC-MXR-infected and control mice, significantly lost weight during the first 3 days. After 7 days post-infection, mucus production was increased in the colon and ileum, ZO-1 localization remained unaltered, and morphological alterations were more pronounced in the ileum since increased expression and apical localization of ß-catenin in ileal enterocytes were observed. EAEC-infected mice developed dysbiosis 21 days post-infection. At 4 days post-infection, EAEC strain 042 formed a biofilm on ileal villi and increased the expression and apical localization of ß-catenin in ileal enterocytes; these effects were not seen in animals infected with the 042 ΔAAF/II strain. At 3 days post-infection, MUC1 expression on ileal enterocytes was mainly detectable among infected mice and colocalized with 042 strains on the enterocyte surface. We developed a novel mouse model of EAEC infection, which mimics human infection, not an illness, revealing that EAEC 042 exerts its pathogenic effects in the mouse ileum and causes dysbiosis. This model is a unique tool to unveil early molecular mechanisms of EAEC infection in vivo and in situ.

Bacteriostatic effect of Echeveria extracts on diarrheagenic E. coli pathotypes and non-cytotoxicity on human Caco-2 cells.

INTRODUCTION: Diarrheagenic Escherichia coli pathotypes are important aetiological agents of diarrhoeal illness among children from less developed areas, worldwide. Diarrheagenic E. coli pathotypes strains are increasingly becoming drug resistant, thus effective and accessible therapeutic alternatives are required for their treatment; herbal extracts may be a potential alternative. AIMS: to evaluate Echeveria craigiana, E. kimnachii, and E. subrigida methanol extracts antibacterial effect on six diarrheagenic E. coli reference strains and on human colorectal adenocarcinoma cells viability and cytokine production. METHODOLOGY: Diarrheagenic E. coli pathotypes reference strains: typical enteropathogenic E2348/69, enterotoxigenic H10407, enterohaemorrhagic O157:H7/EDL933, enteroinvasive E11, diffusely adherent C18451-A, and enteroaggregative 042 E. coli. E craigiana, E. kimnachii, and E. subrigida leaves, collected at Sinaloa, Mexico, were freeze-dried and macerated in methanol solvent. Antibacterial activity was determined by a novel method developed in our laboratory, bacterial oxygen consumption by polarographic oxygen electrode technique and membrane integrity by two methods (live/dead and protein leakage assays). Colorectal adenocarcinoma cells viability by MTT assay and cytokine production using a Cytometric Bead Array kit. RESULTS: Extracts concentrations of 100 µg/mL and 5-hour incubation, reduced more than 93% the growth of all diarrheagenic E. coli pathotypes tested strains and significantly decreased bacterial oxygen consumption, like bacteriostatic antibiotics. After 24-hour incubation methanol extracts had a differential antibacterial effect on each diarrheagenic E. coli pathotypes strain. Echeveria extracts did not have any effect on viability and cytokine production of colorectal adenocarcinoma cells. CONCLUSIONS: Echeveria methanol extracts have a bacteriostatic effect on all diarrheagenic E. coli pathotypes strains, thus potentially they could be used as antibacterial agents on diarrheagenic E. coli pathotypes-contaminated products and on patients with diarrheagenic E. coli pathotypes infections.

Isolation of Staphylococcus aureus, Uropathogenic Escherichia coli, and Nontuberculous Mycobacteria Strains from Pasteurized Cheeses and Unpasteurized Cream Sold at Traditional Open Markets in Mexico City.

ABSTRACT: Fresh cheeses and cream are important garnishes of traditional Mexican food, often purchased at street or itinerant open markets or tianguis. However, there is scarce information regarding the microbiological quality of cheeses and cream sold in tianguis. For 2 years, three dairy stalls from three tianguis in Mexico City were visited once each season, trading practices were registered, and 96 dairy products were purchased. In total 72 fresh pasteurized cheeses that were hand-cut to order (24 Panela, 24 Canasto, and 24 Doble Crema) and 24 unpasteurized Crema de Rancho samples were collected. All dairy products remained without refrigeration for 8 h. Based on the National Guidelines limits, 87.5% of cheeses and 8% of Crema de Rancho samples were of low microbiological quality, and 1 sample of each type of cheese and 3 samples of Crema de Rancho exceeded the guidelines limits for Staphylococcus aureus. All dairy products were negative for Salmonella, Listeria monocytogenes, and all diarrheagenic Escherichia coli pathotypes, including Shiga toxin-producing E. coli. Among the 96 dairy samples, the prevalence of uropathogenic E. coli (UPEC) and of mycobacteria strains were determined because food items contaminated with these strains have been associated with urinary tract infections and mycobacteriosis, respectively. UPEC strains were isolated from 43% of cut-to-order cheeses and 29% of Crema de Rancho samples. Nontuberculous mycobacteria (NTM) strains were identified in 12.5% of Doble Crema cheese samples and 21% of Crema de Rancho samples. From the eight NTM-positive samples, 10 strains were identified (3 strains of Mycolicibacterium fortuitum, 2 of Mycobacteroides abscessus, 2 of Mycobacteroides chelonae, 2 of Mycolicibacterium porcinum, and 1 of Mycolicibacterium rhodesiae). All produced biofilms, and 70% had sliding motility (both virulence traits). Trading practices of cut-to-order pasteurized cheeses and unpasteurized Crema de Rancho in tianguis increase the risk of microbiological contamination of these products, including with human pathogens, and their consumption may cause human illness.

Human caliciviruses detected in Mexican children admitted to hospital during 1998-2000, with severe acute gastroenteritis not due to other enteropathogens.

Few studies exist regarding the frequency of human caliciviruses as single etiologic agents in sporadic cases, or in outbreaks occurring in children hospitalized for acute gastroenteritis. In this study, a total of 1,129 children of <5 years of age and hospitalized due to acute diarrhea were enrolled from three main hospitals in Mexico City during a period of 3 years (March 1998 to December 2000). After analyzing all fecal samples for several enteropathogens, 396 stools that remained negative were further screened for human caliciviruses by RT-PCR using a primer set specific to norovirus and sapovirus. Human caliciviruses were detected in 5.6% (22/396) of the children. The minimum incidence rate for 1999 were 5.3% (7/132) for 1999 and 7.8% (13/167) for 2000, since only fecal specimens that tested negative to other enteric pathogens were examined. Positive samples were further characterized using specific GI and GII primers and sequencing. Norovirus GII was detected in 19/22 samples, most of them were GII/4, while sapovirus GI/2 was detected in one sample. Associations between the presence of human calicivirus and clinical and epidemiological data revealed that diarrhea occurred with a seasonal pattern, and that children hospitalized due to human calicivirus disease scored an average of 13 +/- 3.2 (SD) points on the Vesikari scale, which corresponded to severe episodes. These results highlight that human caliciviruses, by themselves, are enteropathogens of acute severe diarrhea among young Mexican children requiring hospitalization and that their detection is important in order to reduce the diagnosis gap.

Association of diarrheagenic Escherichia coli Pathotypes with infection and diarrhea among Mexican children and association of atypical Enteropathogenic E. coli with acute diarrhea.

Seventy-six children < or =2 years old were prospectively followed for 1 year in a peri-urban community of Mexico City to determine asymptomatic infection and acute diarrhea associated with diarrheagenic Escherichia coli pathotypes (DEPs). By use of a pathogen-specific multiplex PCR, DEPs were sought in 795 stool samples, of which 125 (16%) were positive for DEP; of these, 4 represented shedding episodes and 4 parasite coinfections. Most single-DEP infections (85/117) were asymptomatic (P < 0.001), and of the 32 DEP diarrhea episodes, 41% were associated with atypical enteropathogenic E. coli (aEPEC), 37.5% with enterotoxigenic E. coli, 9% with typical EPEC, 9% with enteroinvasive E. coli, and 3% with Shiga toxin-producing E. coli strains. Among the 76 children, 54 had at least one stool positive for DEP, of which 23 experienced a DEP-associated diarrhea episode. In the last group of children, DEP infection was significantly associated with a diarrhea episode (relative risk [RR] = 2.5; 95% confidence interval [CI], 1.79 to 3.57; P < 0.001), with ETEC (RR = 2.30; 95% CI, 1.49 to 3.54; P = 0.003) and aEPEC (RR = 1.92; 95% CI, 1.23 to 3.0; P = 0.019) being the pathotypes associated with diarrhea. aEPEC-associated diarrhea episodes were frequently in the <12-month age group (RR = 2.57; 95% CI, 1.05 to 6.27; P = 0.04). aEPEC infections were distributed all year round, but associated diarrheal episodes were identified from April to October, with a May-June peak (rainy season). Most ETEC infections and diarrhea episodes characteristically occurred during the summer (rainy season), with a diarrhea peak in August. Of all DEPs, only aEPEC was associated with acute diarrhea episodes lasting 7 to 12 days (P = 0.019). DEPs are important causes of community-acquired enteric infection and diarrhea in Mexican children.

Longitudinal Analysis of the Microbiological Quality of Raw Cow's Milk Samples Collected from Three Small Family Dairy Farms in Mexico over a 2-Year Period.

In Mexico, the total milk production that family dairy farms (FDF) contribute is ca. 35%, but this milk is not evaluated for microbiological quality. Forty percent of the milk and dairy products consumed by Mexicans is unpasteurized. In total, 24 raw cow's milk samples from three FDF (one sample per each season from each FDF for two sequent years) were characterized for the presence of food quality indicator organisms, Staphylococcus aureus, Salmonella enterica, Listeria monocytogenes, and Mycobacterium spp., by standard procedures. Escherichia coli presence was also evaluated by a direct count method and diarrheagenic E. coli (DEC) by molecular methods. On the basis of Mexican guidelines for raw milk entering production, 42% of samples exceeded the aerobic mesophilic bacteria limits. A total of 83% raw milk samples were positive for total coliforms, 54% for fecal coliforms, and 46% for E. coli. Forty-three E. coli isolates were selected and characterized for the presence of 11 DEC loci; of theses, 40 isolates were negative for all DEC loci, and 3 isolates, all collected from the same sample, were Shiga toxin 2 (stx2) positive and O157 antigen negative, and one stx2 isolate was resistant to 6 of the 16 antibiotics tested. None of the 24 raw milk samples were positive for Salmonella enterica, L. monocytogenes, or staphylococcal enterotoxin. S. aureus was isolated from nine samples, of which only three samples harbored resistant isolates. From three samples, four nontuberculous mycobacterial isolates were recovered (Mycobacteroides chelonae, Mycobacteroides porcinum, and two Mycobacteroides abscessus). All four isolates produced biofilm and had sliding motility, and three isolates (M. porcinum and two M. abscessus) were resistant to the two antibiotics tested (clarithromycin and linezolid). FDF provide raw milk to a large proportion of the Mexican population, but its consumption could be harmful to health, emphasizing the need to implement national microbiological quality guidelines for raw milk intended for direct human consumption.

Serum uric acid levels are associated with homeostasis model assessment in obese nondiabetic patients: HOMA and uric acid.

BACKGROUND: Hyperuricemia leads to insulin resistance, whereas insulin resistance decreases renal excretion of uric acid. The aim of this study was to evaluate whether there is a correlation between serum uric acid levels with homeostatic model assessment (HOMA) 1 in nondiabetic patients. METHODS: We evaluated 88 nondiabetic patients, in whom uric acid levels were measured, in all of them HOMA of ß-cell function (HOMA 1B) and HOMA of insulin resistance (HOMA 1IR) scores were performed. Uric acid and the HOMA 1 values were correlated using the Pearson coefficient. RESULTS: We did not find any correlation between uric acid levels with both HOMA 1B (r = 0.102, p = 0.343), nor with HOMA 1IR (r = 0.158, p = 0.117). When patients were analyzed by sex, we found a significant correlation with HOMA 1IR (0.278, p = 0.01), but not with HOMA 1B (0.138, p = 0.257) in women. We found a correlation with HOMA 1B in men (r = 0.37, p = 0.044), but not with HOMA 1IR: 0.203, p = 0.283. The analysis performed based on body mass index did not show correlation in the patients with normal weight, (HOMA 1B r = 0.08, p = 0.5, HOMA 1IR = 0.034, p = 0.793), nor in the patients who were overweight (HOMA 1B: r = 0.05, p = 0.76, HOMA 1IR r = 0.145, p = 0.43). However, a significant correlation between uricemia with both HOMA 1B (0.559, p < 0.001), and HOMA 1IR (0.326, p < 0.05), was observed in obese patients. CONCLUSION: Our results suggest that serum uric acid levels seem to be associated with insulin resistance in women, and in obese patients, but not in nonobese men. Uric acid also modifies ß-cell function in men and in obese patients.

CD40 Ligand Deficient C57BL/6 Mouse Is a Potential Surrogate Model of Human X-Linked Hyper IgM (X-HIGM) Syndrome for Characterizing Immune Responses against Pathogens.

Individuals with X-HIGM syndrome fail to express functional CD40 ligand; consequently they cannot mount effective protective antibody responses against pathogenic bacteria. We evaluated, compared, and characterized the humoral immune response of wild type (WT) and C57-CD40L deficient (C57-CD40L(-/-)) mice infected with Citrobacter rodentium. Basal serum isotype levels were similar for IgM and IgG3 among mice, while total IgG and IgG2b concentrations were significantly lower in C57-CD40L(-/-) mice compared with WT. Essentially IgG1 and IgG2c levels were detectable only in WT mice. C57-CD40L(-/-) animals, orally inoculated with 2 × 10(9) CFU, presented several clinical manifestations since the second week of infection and eventually died. In contrast at this time point no clinical manifestations were observed among C57-CD40L(-/-) mice infected with 1 × 10(7) CFU. Infection was subclinical in WT mice inoculated with either bacterial dose. The serum samples from infected mice (1 × 10(7) CFU), collected at day 14 after infection, had similar C. rodentium-specific IgM titres. Although C57-CD40L(-/-) animals had lower IgG and IgG2b titres than WT mice, C57-CD40L(-/-) mice sera displayed complement-mediated bactericidal activity against C. rodentium. C. rodentium-infected C57-CD40L(-/-) mice are capable of producing antibodies that are protective. C57-CD40L(-/-) mouse is a useful surrogate model of X-HIGM syndrome for studying immune responses elicited against pathogens.

C57-CD40 ligand deficient mice: a potential model for enterotoxigenic Escherichia coli (H10407) colonization.

Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrheal disease in humans, calves and pigs. In humans, these infections mainly occur in developing countries leading to a high diarrheal morbidity and infant mortality and to travellers' diarrhea. ETEC strains constitute a phenotypically and genetically diverse pathotype with as common characteristics the production of heat-labile (LT) and/or heat-stable enterotoxins (ST) as well as of one or more fimbrial colonization factors. Despite the global importance of these pathogens, a broadly ETEC protective vaccine is not yet available, partially due to the lack of a suitable animal model for human ETEC. Such model would allow to test more ETEC molecules as potential vaccine candidates. The C57-CD40 ligand deficient (C57-cd40l(-/-)) mouse has been successfully used to develop infection models of intestinal pathogens, but little is known about its humoral immune response. Therefore, the aims of this study were to characterize the humoral immune response of C57 and C57-cd40l(-/-) mice and to determine the persistence of ETEC H10407 and two of its variants after oral inoculation. The serum IgM, IgG and IgA and faecal IgG and IgA concentrations, of twelve mice per mouse strain (C57 and C57-cd40l(-/-)), were determined by ELISA. All serum immunoglobulins and the faecal IgG concentration were significantly lower in C57-cd40l(-/-) than in C57 mice. In contrast the faecal IgA concentration was significantly higher in the C57-cd40l(-/-) mice. This high intestinal IgA concentration might be a compensatory T cell-independent production of IgA production. Both mouse strains were orally inoculated with 5×10(8) ETEC H10407 (LT(+), ST-colonization factor antigen I (CFA/I)(+)) and ETEC in animal faeces was established by culture followed by st and lt loci identification by PCR until day 14 post infection. Most C57 mice eliminated the strain within 3 days whereas infection remained in C57-cd40l(-/-) mice until day 14. Subsequently both mouse strains were inoculated with ETEC H10407 variants and followed up until day 113. Likewise C57 mice eliminated both ETEC variants within 4 days. All C57-cd40l(-/-) mice had eliminated the LT(-) variant at day 31, whereas the ST-CFA/I(-) variant remained in mice stools until day 113. These observations suggest that C57-cd40l(-/-) mice are permissive for ETEC H10407 colonization.

Impact of vitamin A on selected gastrointestinal pathogen infections and associated diarrheal episodes among children in Mexico City, Mexico.

BACKGROUND: The overall effect of vitamin A supplementation on diarrheal disease in community trials may result from its effect on specific diarrheal pathogens. METHODS: We conducted a placebo-controlled, double-blind trial of the prophylactic effect of vitamin A on gastrointestinal pathogen infections and clinical symptoms among 188 children in Mexico City, Mexico, from January 1998 to May 1999. Children 6-15 months of age were randomly assigned to receive either a vitamin A supplement (for children <12 months of age, 20,000 international units [IU] of retinol; for children > or =12 months of age, 45,000 IU of retinol) every 2 months or a placebo and were followed for up to 15 months. Stool samples, collected semimonthly, were screened for enteropathogenic Escherichia coli (EPEC), enterotoxigenic E. coli (ETEC), Shiga toxin-producing E. coli (STEC), enteroinvasive E. coli (EIEC), and Giardia lamblia. RESULTS: Vitamin A supplementation reduced the prevalence of EPEC infections (rate ratio [RR], 0.52 [95% confidence interval {CI}, 0.23-0.86]) and led to shorter durations of both EPEC and ETEC infections. Supplementation also reduced the prevalence of EPEC-associated diarrhea (RR, 0.41 [95% CI, 0.16-1.00]), EPEC-associated fever (RR, 0.15 [95% CI, 0.02-0.98]), and G. lamblia-associated fever (RR, 0.27 [95% CI, 0.13-0.80]). Finally, children who received vitamin A supplementation had shorter durations of EPEC-associated diarrhea than did children who did not receive supplementation but had longer durations of G. lamblia-associated diarrhea. CONCLUSIONS: These results suggest that the effect of vitamin A supplementation on clinical outcomes may be pathogen dependent.

Single multiplex polymerase chain reaction to detect diverse loci associated with diarrheagenic Escherichia coli.

We developed and tested a single multiplex polymerase chain reaction (PCR) that detects enterotoxigenic, enteropathogenic, enteroinvasive, and Shiga toxin-producing Escherichia coli. This PCR is specific, sensitive, and rapid in detecting target isolates in stool and food. Because of its simplicity, economy, and efficiency, this protocol warrants further evaluation in large, prospective studies of polymicrobial substances.