Differential effects of Staphylococcal enterotoxin B-mediated immune activation on intestinal defensins.

In the small intestine members of both the alpha-defensin (DEFA5 and DEFA6) and beta-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 alpha-defensins was reversed by dexamethasone. DEFA5 and DEFB2 peptide secretion levels were altered in parallel with mRNA. Interferon-gamma and interleukin (IL)-1beta exhibited a dose-dependent down-regulation of alpha-defensin mRNA, IL-6 significantly down-regulated only DEFA6; in contrast, tumour necrosis factor-alpha 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 alpha-defensin expression noted in inflammatory T cell-mediated enteropathies.

A potential role for interleukin-18 in inhibition of the development of Cryptosporidium parvum.

Accumulating 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 alpha-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.

Paneth cell granule depletion in the human small intestine under infective and nutritional stress.

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.

Identification and molecular characterization of a bovine G3 rotavirus which causes age-independent diarrhea in cattle.

G3 rotaviruses have been reported rarely in cattle, and none have been characterized. We report the first genomic characterization of a bovine G3 rotavirus, CP-1, which had been biologically characterized in vivo and shown to cause age-independent diarrhea. CP-1 was a G3 rotavirus as its VP7 had 92 to 96% deduced amino acid identity to those of G3 rotaviruses. However, initially, CP-1 was identified as a G10 rotavirus by RT-PCR even though the CP-1 VP7 had only 81 to 85% deduced amino acid identity to those of G10 rotaviruses. Rotavirus CP-1 was of P[5] specificity, a type common in cattle, and had a bovine NSP1 and NSP4. These results added another animal species to those in which G3 rotaviruses have been found, characterized a bovine rotavirus which caused age-independent diarrhea in calves, and raised the possibility that bovine G3 rotaviruses may be misdiagnosed as G10 rotaviruses.

Rotavirus cross-species pathogenicity: molecular characterization of a bovine rotavirus pathogenic for pigs.

Rotaviruses which cause disease in heterologous animal species have been reported but the molecular basis of cross-species infectivity and disease is not established. We report the molecular characterization of a cloned rotavirus, PP-1, which was originally obtained from cattle and which had been biologically characterized in vivo in two target animal species, gnotobiotic pigs and calves. In pigs, PP-1 caused severe clinical disease but in experimental calves it replicated subclinically. PP-1 was characterized as a G3 reassortant with a porcine VP4 and NSP4 but a bovine NSP1. The PP-1 VP4 had 96 to 97% deduced amino acid identity to P[7] porcine rotaviruses and P[7] specificity was confirmed with VP4-specific monoclonal antibodies. Sequence analysis of the PP-1 NSP1 showed 94 to 99.6% deduced amino acid identity to bovine rotaviruses but the NSP4 protein had 94 to 98% identity to the NSP4 genotype B porcine rotaviruses. G-typing PCR initially classified PP-1 as a G10 rotavirus but sequence analysis revealed 92 to 96% identity of the PP-1 VP7 with porcine, simian, and human G3 rotaviruses. These results, combined with the in vivo properties of PP-1 in the two target species, supported the concept that species-specific VP4 and NSP4, but not NSP1, are required to induce rotavirus disease, at least in calves and pigs. The results illustrate experimentally that rotaviruses circulating in one animal species can pose a risk to another by the emergence of a pathogenic reassortant rotavirus under appropriate conditions.

Elimination of viruses, phages, bacteria and Cryptosporidium by a new generation Aquaguard point-of-use water treatment unit.

The elimination of human viruses, phages, bacteria and Cryptosporidium oocysts by a new generation commercial Aquaguard purifier for the domestic treatment of drinking water, has been evaluated. The unit basically consists of a candle prefilter, activated carbon filter and ultraviolet irradiation compartment. Drinking water seeded with selected laboratory test strains of resistant micro-organisms was passed through the unit. Similar tests were carried out with sewage-contaminated river water and secondary treated waste water containing naturally occurring organisms. Test procedures were based on internationally accepted principles for the evaluation of point-of-use water treatment units, including a standard test protocol of the United States Environmental Protection Agency. Reduction in numbers of seeded test organisms at several log levels higher than those expected in water for which the unit is intended, was determined by the cultivation of viable organisms. In the case of seeded viruses and Cryptosporidium parvum oocysts the qualitative absence of nucleic acid was determined by the reverse transcriptase polymerase chain reaction (RT-PCR). At the design flow rate of one litre per minute, numbers of polio, hepatitis A, adeno types 2 and 41, rota SA11, human rota and astro viruses, as well as somatic and MS2 coliphages, and Escherichia coli, Streptococcus faecalis, Clostridium perfringens, total coliform bacteria, enterococci, heterotrophic bacteria and C. parvum oocysts, were reduced by more than 99.999% in all waters tested. This efficiency conforms to specifications for such units. The quality of the treated water was well within microbiological limits of international specifications for drinking water.

Determinants of rotavirus host range restriction--a heterologous bovine NSP1 gene does not affect replication kinetics in the pig.

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.

Measurement of antibody avidity for hepatitis C virus distinguishes primary antibody responses from passively acquired antibody.

A new IgG antibody avidity test for hepatitis C virus (HCV) has been developed and was validated using sera from 12 renal dialysis patients infected with HCV. In primary HCV infection low avidity antibody (mean avidity index 24%) was detected within 50 days of seroconversion whereas in long-term infection (at least 300 days after seroconversion), the mean avidity index was high (88%); in five patients, the avidity index was shown to increase rapidly as time elapsed after primary infection, whereas immunosuppressive therapy was found to delay maturation of the immune response in two further patients. The assay was then employed to confirm that a spurious outbreak of primary HCV infection in eight bone marrow transplant patients was explicable by passive acquisition of high avidity anti-HCV after intravenous immunoglobulin therapy. It is concluded that this avidity test will have an important role in the investigation of HCV infection in patients.