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.

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.

Entamoeba histolytica Interaction with Enteropathogenic Escherichia coli Increases Parasite Virulence and Inflammation in Amebiasis.

Epidemiological studies suggest frequent association of enteropathogenic bacteria with Entamoeba histolytica during symptomatic infection. In this study, we sought to determine if the interaction with enteropathogenic (EPEC) or nonpathogenic Escherichia coli (strain DH5α) could modify the virulence of E. histolytica to cause disease in animal models of amebiasis. In vitro studies showed a 2-fold increase in CaCo2 monolayer destruction when E. histolytica interacted with EPEC but not with E. coli DH5α for 2.5 h. This was associated with increased E. histolytica proteolytic activity as revealed by zymogram analysis and degradation of the E. histolytica CP-A1/5 (EhCP-A1/5) peptide substrate Z-Arg-Arg-pNC and EhCP4 substrate Z-Val-Val-Arg-AMC. Additionally, E. histolytica-EPEC interaction increased EhCP-A1, -A2, -A4, and -A5, Hgl, Apa, and Cox-1 mRNA expression. Despite the marked upregulation of E. histolytica virulence factors, nonsignificant macroscopic differences in amebic liver abscess development were observed at early stages in hamsters inoculated with either E. histolytica-EPEC or E. histolytica-E. coli DH5α. Histopathology of livers of E. histolytica-EPEC-inoculated animals revealed foci of acute inflammation 3 h postinoculation that progressively increased, producing large inflammatory reactions, ischemia, and necrosis with high expression of il-1ß, ifn-γ, and tnf-α proinflammatory cytokine genes compared with that in livers of E. histolytica-E. coli DH5α-inoculated animals. In closed colonic loops from mice, intense inflammation was observed with E. histolytica-EPEC manifested by downregulation of Math1 mRNA with a corresponding increase in the expression of Muc2 mucin and proinflammatory cytokine genes il-6, il-12, and mcp-1 These results demonstrate that E. histolytica/EPEC interaction enhanced the expression and production of key molecules associated with E. histolytica virulence, critical in pathogenesis and progression of disease.

Escherichia coli enterotoxigénica y enteroagregativa: prevalencia, patogénesis y modelos múridos / Enterotoxigenic and enteroaggregative Escherichia coli: prevalence, pathogenesis and murine models

Resumen Los patotipos de Escherichia coli enterotoxigénica (ETEC) y enteroagregativa (EAEC) son importantes agentes etiológicos causantes de diarrea en niños menores de cinco años de México y países en desarrollo, en quienes causan numerosas muertes. Ambos se han asociado con retraso en el crecimiento infantil y son los principales agentes causales de la "diarrea del viajero". La patogénesis de ambas bacterias se inicia cuando estas se adhieren al epitelio intestinal mediante fimbrias, denominadas factores de colonización en las cepas ETEC aisladas de humano y fimbrias de adherencia agregativa en las cepas de EAEC. Una vez que ETEC se adhiere al enterocito produce una o ambas de sus toxinas e induce la secreción de iones de cloruro, sodio y agua al lumen intestinal, produciendo su característica diarrea acusa. EAEC se une al epitelio intestinal formando una biopelícula, induce la producción de moco, libera sus toxinas y promueve inflamación. Modelos de infección de EAEC y ETEC con ratones C57BL/6 silvestres y deficientes del ligando de CD40 (con microbiotas intactas), respectivamente, revelaron que la desnutrición y la dieta baja en cinc incrementan la infección de EAEC causando retraso en el crecimiento y que ETEC coloniza, persiste e induce respuesta inmune humoral local y sistémica.

Abstract Enterotoxigenic (ETEC) and enteroaggregative Escherichia coli (EAEC) pathotypes are important etiological agents causative of diarrhea in children younger than 5 years of age in Mexico and in developing countries, where they cause numerous deaths. Both have been associated with delayed growth in children and are the main causative agents of traveler's diarrhea. The pathogenesis of both bacteria starts by adhering to the intestinal epithelium by means of fimbriae, called colonization factors in human ETEC isolates and aggregative adherence fimbriae in EAEC isolates. Once ETEC adheres to the enterocyte, it produces one or both of its toxins and induces the secretion of chloride and sodium ions and water into the intestinal lumen, producing its characteristic watery diarrhea. EAEC binds to the intestinal epithelium forming a biofilm, induces the production of mucus, releases its toxins and promotes inflammation. EAEC and ETEC infection models with wild-type C57BL/6 and CD40 ligand-deficient mice (with intact microbiota), respectively, revealed that undernutrition and low-zinc diet increases EAEC infection, causing growth retardation, and that ETEC colonizes, persists and induces local and systemic humoral immune response.

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.

Vitamin A supplementation modifies the association between mucosal innate and adaptive immune responses and resolution of enteric pathogen infections.

BACKGROUND: The efficacy of vitamin A supplementation on diarrheal disease morbidity may reflect the divergent effects that supplementation has on pathogen-specific immune responses and pathogen-specific outcomes. OBJECTIVE: We examined how vitamin A supplementation modified associations between gut-cytokine immune responses and the resolution of different diarrheal pathogen infections. DESIGN: Stools collected from 127 Mexican children who were 5-15 mo old and enrolled in a randomized, placebo-controlled vitamin A supplementation trial were screened for enteropathogenic Escherichia coli (EPEC), enterotoxigenic E. coli (ETEC), and Giardia lamblia. Fecal concentrations of interleukin (IL)-6, IL-8, IL-4, IL-5, IL-10, monocyte chemoattractant protein 1 (MCP-1), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) were measured by using an enzyme-linked immunosorbent assay. Hazard models that incorporated categorized cytokine variables (ie, nondetectable, less than the median of detectable concentrations, and at least the median of detectable concentrations) were fit to the length of pathogen infections stratified by treatment group. RESULTS: Vitamin A-supplemented children with fecal MCP-1 or IL-8 concentrations less than the median of detectable concentrations and IL-10 concentrations of at least median concentrations had longer durations of EPEC infection than did children in the placebo group. In supplemented children, detectable fecal TNF-α or IL-6 concentrations were associated with shorter ETEC infection durations, whereas MCP-1 concentrations of at least the median were associated with longer infection durations. Children in this group who had IL-4, IL-5, or IFN-γ concentrations of at least median detectable concentrations had shorter durations of G. lamblia infection. CONCLUSION: The effect of supplementation on associations between fecal cytokine concentrations and pathogen infection resolution depends on the role of inflammatory immune responses in resolving specific pathogen infections.

Correlation between circulating adhesion molecules and resistin levels in hypertensive type-2 diabetic patients.

BACKGROUND: Endothelial dysfunction, a common feature among hypertensive and type-2 diabetic patients, has been associated with inflammation and increased concentrations of serum soluble adhesion molecules and resistin, a monocyte-macrophage- and adipocyte-derived cytokine. THE AIM OF THIS STUDY: To determine if there is a correlation between the serum concentrations of ICAM-1, VCAM-1, Eselectin and resistin in hypertensive type-2 diabetic patients. METHODS: Thirty hypertensive type-2 diabetic patients were enrolled in the study. Serum ICAM-1, VCAM-1, E-selectin and resistin concentrations were determined by ELISA and correlated with the Spearman correlation coefficient. RESULTS: The patients' serum resistin concentrations significantly correlated with VCAM-1 (r = 0.31, p= 0.05) concentrations but not with ICAM-1 (r = 0.29, p = >0.05) and E-selectin (r = 0.10, p = 0.24) concentrations. CONCLUSION: VCAM-1 and resistin may participate in the pathophysiology of vascular damage in hypertensive type-2 diabetic patients. Serum resistin concentrations may be a marker of endothelial dysfunction.

Associations between mucosal innate and adaptive immune responses and resolution of diarrheal pathogen infections.

The identification of immune response mechanisms that contribute to the control of diarrheal disease in developing countries remains an important priority. We addressed the role of fecal chemokines and cytokines in the resolution of diarrheal Escherichia coli and Giardia lamblia infections. Stools collected from 127 Mexican children 5 to 15 months of age enrolled in a randomized, double-blind, placebo-controlled, vitamin A supplementation trial were screened for enteropathogenic Escherichia coli (EPEC), enterotoxigenic E. coli (ETEC), and Giardia lamblia. Fecal concentrations of tumor necrosis factor alpha (TNF-alpha), monocyte chemoattractant protein-1 (MCP-1), interleukin-4 (IL-4), IL-5, IL-6, IL-8, IL-10, and interferon-gamma (IFN-gamma) were determined. Hazard models incorporating cytokine variables were fit to durations of asymptomatic and symptomatic pathogen infections, controlling for treatment group. Increased levels of TNF-alpha and IL-6 were associated with decreased durations of EPEC infection and increased ETEC durations. Increased IL-4 and IFN-gamma levels were associated with decreased and increased durations, respectively, of both EPEC and ETEC infections. Increased IL-10 levels were associated with increased and decreased durations of asymptomatic and symptomatic EPEC infections, respectively, and increased durations of both asymptomatic and symptomatic ETEC infections. Increased levels of MCP-1, IFN-gamma, IL-4, and IL-5 were associated with increased G. lamblia infection duration, while increased IL-8 levels were associated with decreased durations. Differences in proinflammatory and Treg cytokine levels are associated with differences in the resolution of inflammatory and noninflammatory pathogen infections.