Association of obesity, diabetes, and hypertension with arsenic in drinking water in the Comarca Lagunera province (north-central Mexico).

Chronic endemic regional hydroarsenicism (CERHA) is a global issue that affects over 200 million people exposed to arsenic (As) in drinking water. This includes 1.75 million individuals residing in La Comarca Lagunera, a region in north-central Mexico. Arsenic levels in this region typically exceeds the WHO guideline of 10 µg L-1. Biochemical alterations related to the human As metabolism may increase the risk of overweight and obesity (O&O), type 2 diabetes (T2D), and hypertension (AHT). In our study, we investigated the role of As in drinking water as a risk factor for these metabolic diseases. We focused on populations with historically moderate (San Pedro) and low (Lerdo) drinking water As levels and people with no historical evidence of As water contamination. The exposure assessment to As was based on measurements of the drinking water (medians 67.2, 21.0, 4.3 µg L-1) and urinary As concentrations in women (9.4, 5.3, 0.8 µg L-1) and men (18.1, 4.8, 1.0 µg L-1). A significant correlation between As in drinking water and urine evidenced the As exposure in the population (R2 = 0.72). Adjusted odds ratios with 95% confidence intervals evidenced higher chances of being diagnosed with T2D (1.7, 1.2-2.0) and AHT (1.8, 1.7-1.9) in individuals living in San Pedro than those in Lerdo. Still, there was no significant association with obesity. Individuals living in CERHA towns were found to have a higher risk of obesity (1.3-1.9), T2D (1.5 to 3.3), and AHT (1.4 to 2.4) compared to those residing in non-CERHA towns. Finally, obesity is more probable in women [inverse of OR and 95%CI 0.4 (0.2-0.7)] compared to men, while men is more likely to be diagnosed with T2D [OR = 2.0 (1.4-2.3)] and AHT [OR = 2.0 (1.5-2.3)] than women, independently of the municipality.

Production and characterization of monoclonal antibodies to E1 Tor toxin co-regulated pilus of Vibrio cholerae.

Murine monoclonal antibodies (MAbs) against Vibrio cholerae toxin co-regulated pilus (TCP) were generated using conventional hybridoma procedures. Four hybridomas were obtained and two characterized. Hybridomas 10E10E1 and 4D6F9 secreted antibodies of the IgG2a and IgG1 isotypes, respectively, that reacted with a 24-kDa antigen corresponding to the product of the El Tor tcpA gene fused to a six Histidine tail. Additionally, MAbs produced by 4D6F9 selectively recognized the major pilin subunit (TcpA) of El Tor and O139 vibrios in western immunoblot, while MAbs from 10E10E1 also cross-reacted with classical TcpA. Furthermore, vibrios expressing TCP on their surface selectively inhibited binding of the antibodies secreted by both hybridomas to TcpA-coated microtiter plates. Thus, the MAbs reported in this work detected the structural subunit of the pilus either denatured or assembled on the bacterial surface.

Differential interleukin-8 response of intestinal epithelial cell line to reactogenic and nonreactogenic candidate vaccine strains of Vibrio cholerae.

In this study, we analyzed whether attachment of Vibrio cholerae vaccine strains to human intestinal epithelial cells can induce an interleukin-8 (IL-8) response. The IL-8 transcripts were detected by PCR amplification of reverse-transcribed mRNA, and the gene product secretion was measured by an enzyme-linked immunosorbent assay. Infection of monolayers of the undifferentiated HT29-18N2 cell line with reactogenic (JBK70 and 81) and nonreactogenic (CVD103HgR and 638) vaccine strains of V. cholerae resulted in markedly higher IL-8 expression by epithelial cells exposed to reactogenic strains than by cells exposed to the nonreactogenic strains. Additionally, epithelial cells produced IL-8 transcripts following stimulation with cholera vaccine strains in a concentration-dependent manner. These results represent a new insight into the inflammatory component of reactogenicity and could be used as a predictive marker of vaccine reactogenicity prior to human testing.

Preliminary assessment of the safety and immunogenicity of a new CTXPhi-negative, hemagglutinin/protease-defective El Tor strain as a cholera vaccine candidate.

Vibrio cholerae 638 (El Tor, Ogawa), a new CTXPhi-negative hemagglutinin/protease-defective strain that is a cholera vaccine candidate, was examined for safety and immunogenicity in healthy adult volunteers. In a double-blind placebo-controlled study, no significant adverse reactions were observed in volunteers ingesting strain 638. Four volunteers of 42 who ingested strain 638 and 1 of 14 who received placebo experienced loose stools. The strain strongly colonized the human small bowel, as evidenced by its isolation from the stools of 37 of 42 volunteers. V. cholerae 638, at doses ranging from 4 x 10(7) to 2 x 10(9) vibrios, elicited significant serum vibriocidal antibody and anti-Ogawa immunoglobulin A antibody secreting cell responses.

Cholera toxin differentially regulates nitric oxide synthesis, tumor necrosis factor-alpha production and respiratory burst in murine macrophages.

The aim of this study was to determine if cholera toxin can modulate the expression of several macrophage effector functions. The effect of cholera toxin on the induction of NO synthesis, production of tumor necrosis factor-alpha and induction of respiratory burst was examined in the J774.A2 macrophage cell line. Pre-incubation of cell cultures with cholera toxin significantly down-regulated lipopolysaccharide-induced NO synthesis and phorbol-12-myristate-13-acetate-induced respiratory burst. Concomitant addition of cholera toxin and lipopolysaccharide to cell cultures enhanced the production of tumor necrosis factor-alpha. These effects were abrogated when cholera toxin was inactivated by heat or treated with a specific monoclonal antibody.

Replicating function of the RS1 element associated with Vibrio cholerae CTX phi prophage.

The RS1 element associated with Vibrio cholerae CTX phi prophage was cloned from an E1 Tor biotype Vibrio cholerae strain. We used the recA- vaccine strain Peru-15, that lacks the target for RS-mediated site-specific integration, to show that RS1 promotes autonomous replication of a suicide vector. A linker insertion in the rstR open reading frame abolished autonomous replication in Peru-15 but not in a strain containing an RS1 in the chromosome. An AT-rich region containing cis-acting elements involved in autonomous replication was identified by deletion. This region was sufficient to support autonomous replication in a strain containing an RS1 in the chromosome. DNA sequence analysis of a region present in RS1 and not RS2 revealed the presence of putative binding sites for host proteins involved in plasmid replication. These results indicate that RS1 contains a replicon distinct from RS2 which could be involved in replicative recombination events associated with tandem amplification of the CTX element.

Production and characterization of a monoclonal antibody against mannose-sensitive hemagglutinin of Vibrio cholerae.

We have generated murine monoclonal antibodies (MAb) against Vibrio cholerae mannose-sensitive hemagglutinin (MSHA) using conventional hybridoma procedures. Seven hybridomas were obtained and one characterized. Hybridoma 2F12/F1 secreted an antibody of the IgG3 type that reacted with a 17-kDa antigen corresponding to the product of the mshA gene. This MAb inhibited mannose-sensitive agglutination of chicken erythrocytes by EL tor and O139 vibrios. Vibrios expressing MSHA activity inhibited binding of the antibody secreted by 2F12/F1 to MSHA-coated microtiter plates.

Promoter activities in Vibrio cholerae ctx phi prophage.

Comparison of cholera toxin (CT) production directed by different gene constructs and S1 nuclease mapping revealed the presence of a ctxB-specific promoter within the ctxA coding sequence. Initiation of transcription in this region occurred in wild-type El Tor and classical biotype choleragenic vibrios. We propose that transcription from the ctxB-specific promoter and a stronger ribosomal binding site on the ctxB mRNA synergistically contribute to achieve the correct (5B:1A) subunit stoichiometry. Plasmid pB, a CT promoterless vector expressing only CTB, was used to detect promoter activity by restoration of A-subunit synthesis. Promoter activity expressed in vitro and in vivo was detected upstream of the zonula occludens toxin gene, suggesting that this factor could be produced in vivo to contribute to fluid accumulation. No promoter activity was detected in vitro and in vivo upstream from the accessory cholera enterotoxin gene.

Tagging a Vibrio cholerae El Tor candidate vaccine strain by disruption of its hemagglutinin/protease gene using a novel reporter enzyme: Clostridium thermocellum endoglucanase A.

The celA gene encoding Clostridium thermocellum endoglucanase A was expressed in Vibrio cholerae on its own promoter and used to tag a candidate El Tor biotype cholera vaccine strain. Colonies of the tagged strain could be unequivocally distinguished by overlaying them with CM-cellulose indicator agar and Congo Red staining. Expression of celA did not affect growth of V. cholerae in vitro and in vivo. The celA gene was inserted in the chromosomal hap locus encoding V. cholerae hemagglutinin/protease, a putative "detachase", to create a hap- mutant that could be identified and scored by its halo of cellulolytic activity. The inactivation of hap had a positive effect on colonization in the infant mice model. The above results indicate that celA is a suitable marker gene for V. cholerae and hap is an appropriate locus for insertion of foreign DNA in vaccine development. Inactivation of hap, by increasing the duration of adherence, might decrease excretion of the resulting vaccine vector strain and thus increase its immunogenicity.

Genetic manipulation of Vibrio cholerae for vaccine development: construction of live attenuated El Tor candidate vaccine strains.

The recent spread of El Tor cholera to America augments the need for an effective, safe and economical vaccine. In the present paper we describe the construction of live attenuated V. Cholerae strains by specifically deleting the genes encoding cholera toxin and other putative toxins from the bacterial chromosome. To maximize the likelihood of exposing protective antigens relevant to currently circulating vibrios we selected for genetic manipulation recent epidemic V. cholerae isolates from Peru. The mutant strains did not produce cholera toxin in vitro and in vivo. Deletion of the virulence cassette was accompanied by marked attenuation in the infant mouse cholera model. A selected El Tor Ogawa candidate vaccine strain was refractory to acquisition of foreign genes by conjugation with toxigenic vibrios.

Generation of murine triomas secreting bi-specific monoclonal antibodies that recognize HBsAG ad and ay subtypes.

We report the generation of murine triomas by fusing splenocytes from mice previously immunized with HBsAg ay-subtype and a hybridoma, secreting anti-HBsAg ad-subtype monoclonal antibody, which was rendered HGPRT- by induced mutagenesis with N-methyl-N'nitro-N-nitrosoguanidine. The fusion yielded a 83.8% of hybrids showing the antigen specificity of the parental hybridoma and a 16.1% of bi-specific monoclonal antibodies. One of them, coded as 1C8A5, showing a heavy chain isotype (IgG1/IgG2b) was used as capture reagent in an ultramicro-ELISA. As little as 0.78 I.U. of both HBsAg ad- and ay-subtypes could be realiably detected.