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Because both micronutrients and antimicrobial peptides protect against diarrhea, we looked for an effect on intestinal antimicrobial peptide gene expression during a randomized controlled trial of multiple micronutrient (MM) supplementation.Consenting adults (n=287) in Lusaka, Zambia, were randomized to receive a daily MM supplement or placebo and were followed up for 3.3 years, with a crossover after 2 years. Intestinal biopsy samples were obtained at annual intervals, and messenger RNA of the intestinal antimicrobial peptides human alpha defensin (HD) 5, HD6, human beta-defensin (hBD) 1, hBD2, and LL-37 were quantified by real-time reverse-transcriptase polymerase chain reaction. Samples were also obtained during diarrhea episodes and after convalescence.There was no effect overall of treatment allocation. However, in malnourished adults (body mass indexor =18.5), HD5 mRNA was increased by 0.8 log transcripts/microg total RNA in MM recipients, compared with HD5 mRNA in placebo recipients (P=.007). During diarrhea, HD5 expression was reduced by 0.8 log transcripts in placebo recipients (P=.02) but was not reduced in MM recipients, nor was it reduced after the crossover. Correlations between HD5 and nutritional status were found that were sex-specific but not explained by serum leptin or adiponectin concentrations.Micronutrient supplementation was associated with up-regulation of HD5 only in malnourished adults. Interactions between antimicrobial gene expression and nutritional status may help to explain the increased risk of infection in individuals with malnutrition.

SummaryIn the small intestine members of both the α-defensin (DEFA5 and DEFA6) and β-defensin (DEFB1 and DEFB2) family contribute to the anti-microbial barrier against infection. The aim of this study was to determine whether Staphylococcal enterotoxin B (SEB)-mediated immune activation and proinflammatory cytokines play a role in the regulation of intestinal defensin expression. Defensin mRNA and peptide secretion was studied after ex vivo tissue culture of duodenal biopsies over 24 h. Immune (T cell and macrophage) activation was induced by SEB, and in separate experiments exogenous proinflammatory cytokines were added individually. Defensin mRNA levels were quantified by reverse transcription–polymerase chain reaction, and peptide release into culture supernatants was quantified by immuno dot blot or enzyme-linked immunosorbent assay. Increasing concentrations of SEB down-regulated DEFA5, DEFA6 and DEFB1 mRNA in a dose-dependent manner but increased DEFB2 simultaneously. The down-regulation of α-defensins was reversed by dexamethasone. DEFA5 and DEFB2 peptide secretion levels were altered in parallel with mRNA. Interferon-γ and interleukin (IL)-1β exhibited a dose-dependent down-regulation of α-defensin mRNA, IL-6 significantly down-regulated only DEFA6; in contrast, tumour necrosis factor-α and IL-4 had no significant effect. Immune cell activation and proinflammatory cytokines down-regulated the constitutively expressed DEFA5, DEFA6 and DEFB1 defensins, and up-regulated DEFB2 in intact human intestinal tissue explants in short-term culture. The effect of local immune activation on innate defence may explain the reduced α-defensin expression noted in inflammatory T cell-mediated enteropathies.

SUMMARY Paneth cells are important contributors to the intestinal antimicrobial barrier through synthesis and release of antimicrobial peptides and proteins. Animal studies indicate that Paneth cell numbers, location and granule morphology are altered by infection and zinc status. We examined human tissue to determine whether Paneth cell numbers, distribution or granule morphology are altered in infective, inflammatory and nutritional disorders. Archival sections from infective disorders (giardiasis, cryptosporidiosis, HIV, helminth infection) were compared with active inflammatory conditions (coeliac, Crohn's and graft-versus-host diseases) and histologically normal tissues. A subset of tissues was studied by electron microscopy and TUNEL staining for apoptosis. Human defensin-5 (HD5) peptide and mRNA was analysed by immunohistochemistry, in situ hybridization and quantitative reverse transcription polymerase chain reaction. Sections from a tropical population cohort study were then analysed to determine the relationship of granule depletion to infection, nutritional status and plasma zinc concentration. In HIV-related cryptosporidiosis, but not other disorders, Paneth cells were reduced in number and markedly depleted of granules. Paneth cell granule depletion was associated with reduced HD5 immunoreactivity, but this was not due to apoptosis and there was no reduction in mRNA transcripts. In the tropical population studied, depletion of granules was associated with reduced body mass index, reduced plasma zinc levels and HIV infection. Paneth cell granules in human small intestine may be depleted in response to infective and nutritional stress. We postulate that this is one mechanism through which zinc status influences host susceptibility to intestinal infection.

Defensins are thought to play a major role in the defence of small intestinal crypts against colonisation by potential pathogens. In humans two alpha-defensins, HD5 and HD6 and two beta-defensins, hBD1 and hBD2, probably contribute to the antimicrobial barrier, but there are no data to indicate how the expression of these defensin genes might vary in individuals and in populations. To begin to address this question we developed a competitive reverse transcriptase polymerase chain reaction (RT-PCR) assay to quantify HD5 and HD6 mRNA and used it to measure transcripts in small intestinal biopsy tissue from adults living in London, UK, or in Lusaka, Zambia. We also measured alpha- and beta-defensin mRNA in biopsies collected in London from different regions of the small intestine. Jejunal biopsies (n=169) from 83 adults in Lusaka contained approximately one order of magnitude less HD5 and HD6 mRNA than biopsies (n=33) obtained from 27 adults in London. HD5 and HD6 transcript levels were high throughout duodenum, jejunum and ileum. hBD1 and hBD2 mRNA were detected in some, but not all, biopsies from normal small intestine. These data suggest that alpha-defensin expression is down-regulated in tropical populations, and that there are distinct pathways regulating transcription of alpha- and beta-defensins.

The genetic basis of rotavirus host range restriction (host species specificity) is unknown but the NSP1 (fifth) gene has been implicated in some studies. We studied the replication kinetics in vivo of a NSP1 gene monoreassortant, E11, to assess the influence of a heterologous NSP1 gene on the ability to replicate in pigs. The monoreassortant possessed 10 genes from the porcine parent rotavirus SW20/21, which replicated productively in pigs, and the NSP1 gene from the bovine rotavirus UK which produced an abortive infection in pigs. Groups of up to four pigs were inoculated orally with 10(5) to 10(6) TCID50 of the monoreassortant, the porcine parent rotavirus, or the bovine parent rotavirus or were sham inoculated. The monoreassortant replicated productively in pigs with replication kinetics almost identical to the porcine parent rotavirus. During a 9-day observation period after inoculation, the number of days with virus in the faeces, the onset and duration of virus excretion, and peak titres in faeces were similar for the monoreassortant and the parent porcine rotavirus. The genetic composition of the viruses excreted in the faeces was confirmed as that of the inocula by PAGE. Thus possession of a heterologous NSP1 gene from a bovine rotavirus which failed to replicate in pigs did not produce an abortive infection or affect the replication kinetics in vivo. The genetic basis of host range restriction between porcine and bovine rotaviruses remains to be established.

Background Mortality in Zambian AIDS patients is high, especially in patients with diarrhoea, and there is still unacceptably high mortality in Zambian patients just starting anti-retroviral therapy. We set out to determine if high concentrations of serum cytokines correlate with mortality. Methods Serum samples from 30 healthy controls (HIV seropositive and seronegative) and 50 patients with diarrhoea (20 of whom died within 6 weeks) were analysed. Concentrations of tumour necrosis factor receptor p55 (TNFR p55), macrophage migration inhibitory factor (MIF), interleukin (IL)-6, IL-12, interferon (IFN)-γ and C-reactive protein (CRP) were measured by ELISA, and correlated with mortality after 6 weeks follow-up. Results Apart from IL-12, concentrations of all cytokines, TNFR p55 and CRP increased with worsening severity of disease, showing highly statistically significant trends. In a multivariable analysis high TNFR p55, IFN-γ, CRP and low CD4 count (CD4 count Conclusion High serum concentrations of TNFR p55, IFN-γ, CRP and low CD4 count correlated with disease severity and short-term mortality in HIV-infected Zambian adults with diarrhoea. These factors were better predictors of survival than BMI. Understanding the cause of TNFR p55, IFN-γ and CRP elevation may be useful in development of interventions to reduce mortality in AIDS patients with chronic diarrhoea in Africa.

SummaryAccumulating evidence suggests that intestinal epithelial cells (IECs) constitutively express the immunoregulatory cytokine interleukin (IL)-18. IECs also serve as the host cell for the intracellular parasitic protozoan Cryptosporidium parvum. In the present study, C. parvum infection of a human enterocyte cell-line HCT-8 resulted in increased expression of IL-18 mRNA as measured by quantitative reverse transcription–polymerase chain reaction (RT-PCR). IL-18 protein was detected in control uninfected cells and following infection there was increased expression as measured by enzyme-linked immunosorbent assay (ELISA). Gene expression revealed the presence of the IL-18 receptor subunits not only in cell-lines but also in freshly isolated IECs, suggesting that IL-18-mediated signalling events may contribute to epithelial host defence during infection. Recombinant IL-18 inhibited intracellular development of the parasite in HCT-8 and HT-29 cells. Increased expression of bactericidal antibiotic peptides LL-37 and α-defensin 2 by IL-18 in HCT-8 and HT-29 cells may represent one mode of action by which this pluripotent cytokine aids in limiting the development of intracellular pathogens such as C. parvum in the gastrointestinal tract.

Background. Human defensin ( HD) 5 and HD6, both Paneth cell alpha-defensins, contribute to the antimicrobial barrier against intestinal infection. We have previously demonstrated that levels of both HD5 and HD6 mRNA were reduced in adults living in urban Zambia, compared with those in adults living in London. The aim of the present study was to determine, during 2 years of follow-up, whether alpha-defensin expression in Zambian adults is related to susceptibility to diarrhea.Methods. We analyzed intestinal biopsy samples from a longitudinal cohort study conducted in 83 Zambian adults by quantitative reverse-transcription polymerase chain reaction, Western blotting, immunohistochemistry, and in situ hybridization, and we measured the incidence of diarrhea.Results. Levels of HD5 and HD6 mRNA in Paneth cells varied between participants, over time, and seasonally and were strongly correlated with mucosal architecture. Gene expression was almost exclusively restricted to Paneth cells. The median ( interquartile range) HD5 mRNA level was 6.0 ( 5.6-6.7) log(10) transcripts/mg of total RNA among 18 participants who experienced diarrhea within 2 months after biopsy-sample collection, compared with 6.8 ( 6.2-7.3) log(10) transcripts/mg of total RNA among 94 participants who did not (P = .006). A similar observation was made for HD6.Conclusions. These data indicate that intestinal alpha-defensin expression is dynamic and seasonal and suggest that susceptibility to intestinal infection is related to alpha-defensin expression.