F A el-Zaatari

To analyze the pathogenesis of the neurotropic murine coronavirus JHMV, we used monoclonal antibodies to the E2 viral glycoprotein to select antigenic variant viruses. Monoclonal antibodies J.7.2 and J.2.2 were shown to bind to topographically distinct regions of the E2 molecule, and the variants selected with the two antibodies demonstrated very different disease pictures in mice. Variants selected with J.7.2 were, like the parental virus, highly virulent and caused an acute encephalitic illness. By contrast, J.2.2-selected variants predominantly caused a subacute paralytic disease clinically and extensive demyelination histologically. Antigenic differences among the variants and parental virus were readily demonstrable with anti-E2 monoclonal antibodies. However, no differences between the viruses could be shown in binding studies with monoclonal antibodies directed against either E1 or N, the other two JHMV structural proteins. Since only J.2.2 selected demyelinating variants with reduced neurovirulence, it is likely that this monoclonal antibody recognizes a subregion of the E2 molecule that is particularly important in JHMV pathogenesis.

To investigate whether the oral cavity is a potential reservoir and possible sanctuary for Helicobacter pylori, supragingival and subgingival plaques were analyzed by a Helicobacter genus-specific reverse transcriptase-polymerase chain reaction based on the sequence data of H. pylori 16S rRNA. The amplified 500-bp DNA fragment was identified by ethidium bromide staining after agarose gel electrophoresis and by Southern hybridization. Twenty-five dyspeptic patients were studied. Histologic examination of gastric biopsy specimens revealed that 18 had H. pylori gastritis and 7 did not. For seven of the 18 (38.8%) patients with proven H. pylori gastritis, H. pylori was also identified in their dental plaque. None of the patients without H. pylori gastritis had H. pylori in their dental plaque. The detection of H. pylori in dental plaque suggests that this H. pylori colonization is not restricted to the gastric mucosa and that this ecological niche may serve as a possible sanctuary which may be responsible for reinoculation of the stomach after topical anti-H. pylori therapies such as bismuth.

Thirty one cultures were established in MG3 medium from the intestinal tissues of 29 patients, including 18 with Crohn's disease, five with ulcerative colitis, and six non-inflammatory bowel disease controls. All cultures grew either acid fast bacilli or uncharacterized spheroplasts. Pellets from these cultures were coded and assayed blind for M paratuberculosis and M avium subsp silvaticum using IS900- and IS902-PCR (polymerase chain reaction) assays, respectively. IS900 and IS902 are multicopy DNA insertion elements specific for these two organisms. Six Crohn's disease cultures and a single non-inflammatory bowel disease control were positive for M paratuberculosis. A further six cultures were positive for M avium subsp silvaticum, of which two each were from Crohn's disease, ulcerative colitis, and non-inflammatory bowel disease controls. The intensity of the IS900-PCR signals indicated very low numbers of M paratuberculosis organisms and bore no relation to visible spheroplastic or bacillary mycobacterial growth. The results suggest that M paratuberculosis isolated from man exists in a form which hardly replicates if at all when cultured in MG3 medium in vitro, and are consistent with the involvement of this known animal enteric pathogen in a proportion of chronic enteritis in man.

The objective of these experiments was to examine the ability of Helicobacter pylori to stimulate interleukin-10 (IL-10) or IL-12 and select for either Th1 or Th2 cells. Gastric biopsy specimens were collected from patients who were categorized with respect to the presence of H. pylori and gastric disease as well as their age, gender, medications, and other factors. As Th1 and Th2 cells are selected by IL-12 and IL-10, respectively, biopsy specimens were screened for mRNA and protein for these cytokines. Although mRNA for IL-12 and IL-10 was detected in biopsy specimens obtained from both infected and uninfected patients, IL-12 protein predominated. Levels of IL-10 and IL-12 in gastric tissue did not change in response to infection. Moreover, gamma interferon (IFN-gamma)-producing T cells were found in both the infected and the uninfected gastric mucosa. Stimulation of peripheral blood leukocytes from either infected or uninfected donors with various concentrations of live or killed H. pylori induced immunoreactive IL-12 and IL-10. After stimulation with live H. pylori, IL-12 levels increased more than 30-fold, whereas IL-10 levels increased only 2- to 5-fold, compared to cells stimulated with medium alone. Interestingly, killed H. pylori induced significantly more IL-10 (P < 0.05) than live H. pylori, while recombinant urease only induced IL-10. These results demonstrate that live H. pylori selectively stimulates the induction of IL-12 and Th1 cells that produce IFN-gamma, whereas preparations used in oral vaccines induce more IL-10 and may favor Th2 cell responses.

Metronidazole (Mtz) is a critical ingredient of modern multidrug therapies for Helicobacter pylori infection. Mtz resistance reduces the effectiveness of these combinations. Although null mutations in a rdxA gene that encodes oxygen-insensitive NAD(P)H nitroreductase was reported in Mtz-resistant H. pylori, an intact rdxA gene has also been reported in Mtz-resistant H. pylori, suggesting that additional Mtz resistance mechanisms exist in H. pylori. We explored the nature of Mtz resistance among 544 clinical H. pylori isolates to clarify the role of rdxA inactivation in Mtz resistance and to identify another gene(s) responsible for Mtz resistance in H. pylori. Mtz resistance was present in 33% (181 of 544) of the clinical isolates. There was marked heterogeneity of resistance, with Mtz MICs ranging from 8 to >/=256 microg/ml. rdxA inactivation resulted in Mtz MICs of up to 32 microg/ml for 6 Mtz-sensitive H. pylori strains and 128 microg/ml for one Mtz-sensitive strain. Single or dual (with rdxA) inactivation of genes that encode ferredoxin-like protein (designated fdxB) and NAD(P)H flavin oxidoreductase (frxA) also increased the MICs of Mtz for sensitive and resistant strains with low to moderate levels of Mtz resistance. fdxB inactivation resulted in a lower level of resistance than that from rdxA inactivation, whereas frxA inactivation resulted in MICs similar to those seen with rdxA inactivation. Further evidence for involvement of the frxA gene in Mtz resistance included the finding of a naturally inactivated frxA but an intact rdxA in an Mtz-resistant strain, complementation of Mtz sensitivity from an Mtz-sensitive strain to an Mtz-resistant strain or vice versa by use of naturally inactivated or functional frxA genes, respectively, and transformation of an Mtz-resistant Escherichia coli strain to an Mtz sensitive strain by a naturally functional frxA gene but not an inactivated frxA gene. These results are consistent with the hypothesis that null mutations in fdxB, frxA, or rdxA may be involved in Mtz resistance.