In addition to their bridging function between innate and adaptive immunity, dendritic cells (DCs) may also contribute to primary resistance against infection. Here we analyzed the role of DCs during infection with Listeria monocytogenes by performing systemic in vivo depletion of these cells. We showed that CD8alpha(+) DCs were crucial for L. monocytogenes spreading and proliferation in the spleen. Efficient and rapid uptake of L. monocytogenes by CD8alpha(+) DCs required the small GTPase Rac1 and is a general characteristic of this DC subpopulation in filtering particles out of the blood. Thus, CD8alpha(+) DCs appear to play an important role for efficient bacterial entry into the spleen, which is of relevance for subsequent immune responses.

Background-The development of macrolide resistance in Helicobacter pylori is considered an essential reason for failure of antibiotic eradication therapies. The predominant mechanism of resistance to macrolides, particularly clarithromycin, is based on three defined mutations within 23S rRNA, resulting in decreased binding of the antibiotic to the bacterial ribosome. Aim-To develop an rRNA based whole cell hybridisation method to detect Helicobacter species in situ within gastric tissue, simultaneously with its clarithromycin resistance genotype. Methods-A set of fluorescent labelled oligonucleotide probes was developed, binding either to H pylori 16S rRNA or 23S rRNA sequences containing specific point mutations responsible for clarithromycin resistance. After hybridisation and stringent washing procedures, labelling of intact single bacteria was monitored by fluorescence microscopy. The new approach was compared with PCR based assays, histology, and microbiological culture. Results-In comparison with the phenotypic resistance measurement by E test, the genotypic clarithromycin resistance correlated perfectly (100%) for 35 H pylori isolates analysed. In a set of gastric biopsy specimens (27) H pylori infection was confirmed by histology (17/27) and correctly detected by whole cell hybridisation. Five clarithromycin resistant strains were identified in gastric tissue specimens directly. Furthermore, non-cultivable coccoid forms of H pylori were easily detectable by whole cell hybridisation. Conclusions-Whole cell hybridisation of rRNA holds great promise for cultivation independent, reliable, and rapid (three hours) genotypic determination of clarithromycin resistance in H pylori. Compared with PCR techniques it is independent of nucleic acid preparations, not prone to inhibition, and allows semiquantitative visualisation of the bacteria within intact tissue samples. (Gut 2000;46:608-614)

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The aim of the present study was to evaluate the Helicobacter pylori ClariRes assay (Ingenetix, Vienna, Austria) for the detection of H. pylori infection and the simultaneous clarithromycin susceptibility testing of the H. pylori isolates in stool samples from 100 symptomatic children. The results obtained by this novel biprobe real-time PCR method were directly compared with the results obtained from histological examination of gastric biopsy specimens, culturing, the [ 13 C]urea breath test, and a monoclonal antibody-based stool antigen enzyme immunoassay (EIA). Fecal specimens from all 54 children who were shown to be noninfected by "gold standard" tests gave true-negative PCR results (specificity, 100%). Of the remaining 46 individuals with a positive H. pylori status, 29 were found to be positive by real-time PCR (sensitivity, 63%). For these 29 cases, the H. pylori ClariRes assay confirmed all results from phenotypic clarithromycin susceptibility testing by Etest. In summary, this investigation demonstrates that detection of Helicobacter DNA in stool samples by real-time PCR is a difficult task and that this method cannot replace the stool antigen EIA (sensitivity, 95.7%) for the accurate diagnosis of H. pylori infection in children.

Certain metal ions are known to be potent sensitizers, but the self proteins modified by metal ions and the self peptides recognized by 'metal-specific' T cells are unknown. In humans and mice treatment with gold anti-rheumatic drugs, containing Au(I), may lead to allergic and autoimmune side effects. Human and murine T cells do not react to Au(I), however, but to the reactive metabolite Au(III). Here we show that alteration by Au(III) of a model antigen, bovine ribonuclease (RNase)A, results in T cell sensitization to cryptic peptides of this protein. Upon immunization of mice with Au(III)-pretreated RNase [RNase/Au(III)], CD4+ T cell hybridomas specific for RNase/Au(III) were obtained in addition to those recognizing the immunodominant peptide RNase 74-88; the latter also were obtained after immunization with native RNase. RNase/Au(III)-specific T cell hybridomas reacted against RNase/Au(III) and RNase denatured by S-sulfonation of cysteine residues, but not against native RNase, or RNase pretreated with Au(I), A1(III), Cu(II), Fe(II), Fe(III), Ni(II), Mn(II), or Zn(II). Using a panel of overlapping, synthetic RNase peptides which were devoid of gold or gold-induced modifications, epitope mapping revealed that RNase/Au(III)-specific T cell hybridomas recognized the cryptic peptides 7-21 and 94-108, respectively. Comparison of the proliferative response of bulk CD4+ T cells, prepared from splenocytes after immunization with either RNase/Au(III) or native RNase, revealed that Au(III) pretreatment of RNase led to a markedly enhanced response to the two cryptic peptides while it did not influence the response to the immunodominant peptide. The cryptic peptides were also presented after preincubation of bone marrow-derived macrophages with RNase and Au(I), but not with RNase alone, suggesting that oxidation of Au(I) to Au(III) and subsequent protein alteration by Au(III) can happen in mononuclear phagocytes. We conclude that Au(III) alteration of proteins alters antigen processing and, thus leads to presentation of cryptic peptides. This mechanism may shed light on the development of allergic and autoimmune side effects of Au(I) anti-rheumatic drugs. In addition, it might provide a general mechanism of how metal ions act as T cell sensitizers.

Helicobacter pylori encodes three histidine kinases and five response regulators belonging to the family of two-component regulatory systems which are involved in transcriptional control. Here we demonstrate that isogenic mutants of H. pylori P76 with deletions of the response regulator open reading frame (ORF) HP1365 and ORFs HP244, HP165, and HP1364 encoding histidine kinases are unable to colonize the stomachs of BALB/c mice, suggesting an essential role of these systems in the regulation of important virulence properties of H. pylori. Furthermore, we demonstrate that the genes under the control of the P HP1408 and P HP119 promoters which are regulated by the two-component system HP166-HP165 are not essential for single mutant colonization of mice but are required under competitive colonization conditions.

The gram-negative bacterial pathogen Helicobacter pylori is a major cause of peptic ulcer disease and a risk factor for gastric cancer in humans. Adapted H. pylori strains, such as strain SS1, are able to infect mice and are a useful model for gastric colonization and vaccination studies. In this study we used a streptomycinresistant derivative of H. pylori SS1 to analyze the colonization behavior and the success of vaccination in wild-type (wt) and various knockout mice of the BALB/c and C57BL/6J genetic backgrounds. We here report that BALB/c interleukin-4 knockout (IL-4 ؊/؊ ) mice are weakly overcolonized compared to the wt strain but that the IL-12 ؊/؊ knockout results in a strong overcolonization (500%). Unexpectedly, in the C57BL/6J background the same knockouts behaved in diametrically opposed manners. The IL-4 ؊/؊ mutation caused a 50% reduction and the IL-12 ؊/؊ knockout caused a 95% reduction compared to the wt colonization rate. For C57BL/6J mice we further analyzed the IL-18 ؊/؊ and Toll-like receptor 2 knockout mutations, which showed reductions to 66 and 57%, respectively, whereas mice with the IL-10 ؊/؊ phenotype were hardly infected at all (5%). In contrast, the tumor necrosis factor receptor knockout (p55 ؊/؊ and p55/75 ؊/؊ ) mice showed an overcolonization compared to the C57BL/6J wt strain. With exception of the low-level infected C57BL/6J IL-10 ؊/؊ and IL-12 ؊/؊ knockout mice, all knockout mutants were accessible to a prophylactic vaccination and their vaccination behavior was comparable to that of the wt strains.

Bacterial ghosts are empty cell envelopes, which may be generated by the controlled expression of the PhiX174 lysis gene E in gram-negative bacteria to obtain vaccine candidates. We describe here the application of this technology to Helicobacter pylori.