Functional analysis of the cag pathogenicity island in Helicobacter pylori isolates from patients with gastritis, peptic ulcer, and gastric cancer

Helicobacter pylori is the causative agent of a variety of gastric diseases, but the clinical relevance of bacterial virulence factors is still controversial. Virulent strains carrying the cag pathogenicity island (cagPAI) are thought to be key players in disease development. Here, we have compared cagPAI-dependent in vitro responses in H. pylori isolates obtained from 75 patients with gastritis, peptic ulcer, and gastric cancer (n = 25 in each group). AGS gastric epithelial cells were infected with each strain and assayed for (i) CagA expression, (ii) translocation and tyrosine phosphorylation of CagA, (iii) c-Src inactivation, (iv) cortactin dephosphorylation, (v) induction of actin cytoskeletal rearrangements associated with cell elongation, (vi) induction of cellular motility, and (vii) secretion of interleukin-8. Interestingly, we found high but similar prevalences of all of these cagPAI-dependent host cell responses (ranging from 56 to 80%) among the various groups of patients. This study revealed CagA proteins with unique features, CagA subspecies of various sizes, and new functional properties for the phenotypic outcomes. We further showed that induction of AGS cell motility and elongation are two independent processes. Our data corroborate epidemiological studies, which indicate a significant association of cagPAI presence and functionality with histopathological findings in gastritis, peptic ulcer, and gastric cancer patients, thus emphasizing the importance of the cagPAI for the pathogenicity of H. pylori. Nevertheless, we found no significant association of the specific H. pylori-induced responses with any particular patient group. This may indicate that the determination of disease development is highly complex and involves multiple bacterial and/or host factors.     

Molecular basis of the functional podocin-nephrin complex: mutations in the NPHS2 gene disrupt nephrin targeting to lipid raft microdomains

Hereditary nephrotic syndrome is a heterogeneous disease, characterizedby heavy proteinuria and renal failure. Mutations of NPHS1 orNPHS2, the genes encoding for nephrin and podocin, lead to earlyonset of heavy proteinuria, and rapid progression to end-stagerenal disease, suggesting that both proteins are essential forthe integrity of the glomerular filter. Podocin is a stomatinprotein family member with a predicted hairpin-like structurelocalizing to the insertion site of the slit diaphragm of podocytes,the visceral glomerular epithelial cells of the kidney. Herewe investigate the pathomechanisms of different disease-causingpodocin mutations. We show that wild-type podocin is targetedto the plasma membrane, and forms homo-oligomers involving thecarboxy and amino terminal cytoplasmic domains. The associationof podocin with specialized lipid raft microdomains of the plasmamembrane was a prerequisite for recruitment of nephrin intorafts. In contrast, disease-causing mutations of podocin (R138Qand R138X) failed to recruit nephrin into rafts either becausethese mutants were retained in the endoplasmic reticulum (R138Q),or because they failed to associate with rafts (R138X) despitetheir presence in the plasma membrane. None of the mutants didaugment nephrin signaling, suggesting that lipid raft targetingfacilitates nephrin signaling. Our findings demonstrate thatthe failure of mutant podocin to recruit nephrin into lipidrafts may be essential for the pathogenesis of NPHS2. ^* To whom correspondence should be addressed. Tel: +49 7612703559;Fax: +49 7612706362; Email: benzing{at}med1.ukl.uni-freiburg.de      

Use of gene chip technology for the characterisation of the regulation of renal transport processes and of nephrotoxicity in rats

Gene expression profiling using microarrays (rat-specific array RG-U34A, Affymetrix, U.S.A.) was employed for the investigation of: (1) hormonal regulation of renal function and (2) nephrotoxicity. For this purpose about 8,800 genes were analysed in kidney and, additionally, in liver tissue.

Ad 1.) Kidney functions develop during postnatal life. Thus, in vivo transport and accumulation of p-aminohippurate (PAH) was investigated on renal cortical slices (RCS) from 10- and 55-day-old rats. The animals were treated with dexamethasone (DEXA; 60 μg/100 g b.wt./day) for 3 days, which caused a significant reduction in the accumulation of PAH in 10-day-old rats (42 ± 5% whereas it was only slightly reduced in 55-day-old rats (70 ± 8%). To further clarify the regulation of renal function by DEXA, results were compared with those obtained previously after in vitro stimulation with DEXA. RCS were incubated for 24 hours in DEXA-containing medium (10⁻⁹ M). Under these conditions DEXA significantly increased the PAH uptake capacity in RCS obtained from 10- and 55-day-old rats up to 126 and 136%, respectively. Thus a stimulation of tubular transport capacity is possible in vitro. The effect of DEXA treatment on the gene expression of the kidney (in vivo) was moderate. Focussing especially on transporters, ion channels, ATPases, glucuronyltransferases, glutathione-S-transferase and cytochrome P450, the expression of only few genes were significantly changed (3 to 50-fold up- or down-regulation). Moreover, distinct age differences were found after in vivo administration of DEXA. The investigation of in vitro effects of DEXA is currently been performed.

Ad 2.) The kidney is threatened by nephrotoxins because of its ability to accumulate them. We used a single administration of uranyl nitrate (UN; 0.5 mg/100 g b.wt.) as a model for chronic renal failure (CRF). Clearance experiments were performed 10 weeks after UN administration (maximal symptoms of CRF) in adult female rats. As expected, UN induced interstitial cicatrices with reduced GFR and diminished PAH transport capacity. Despite the impressive morphological and functional changes in the kidney after exposure to UN, the gene expression profiles in the kidneys were only minimally affected: we found significantly changed expression levels for only 20 genes (5 genes were up-regulated [e.g. transgelin], 15 down-regulated [among these the Na-K-Cl-symporter, insulin-like growth factor, kallikrein, and ornithine decarboxylase). The lack of agreement between gene expression data and the nephrotoxic effects of UN can probably be explained by the long time interval between dosing and the assessment of the effect. The results confirm that primary genomic responses are likely to be strongest transiently after exposure and then decrease in intensity.     

Matrix metalloproteinase-9 deficiency impairs host defense mechanisms against Streptococcus pneumoniae in a mouse model of bacterial meningitis

Recent reports of human immunodeficiency virus-1 (HIV-1) infection of astrocytes suggest a role for astrocytes in HIV encephalitis. In this study, we infected a human astrocytoma cell line with a pathogenic simian HIV (SHIV_50OLNV) and examined growth patterns and immunomodulatory genes. Approximately 1% of uninfected cells in culture expressed glial fibrillary acid protein (GFAP) whereas 40% of the cells expressed GFAP at 7 days post-inoculation along altered growth patterns. Using targeted cytokine cDNA arrays, we found that SHIV_50OLNV infection resulted in the up-regulation of several genes including metalloproteinase bone morphogenic protein 1 and chemokines monocyte chemoattractant protein 1 and stromal cell derived factor 1. These data suggest that astrocytic activation, altered morphology and up-regulation of immunomodulatory genes in response to SHIV infection may participate in initiation of inflammation and trafficking of infected monocytes/macrophages into the central nervous system, potentiating the development of HIV encephalitis.     

Distinct CD4+-T-cell responses to live and heat-inactivated Aspergillus fumigatus conidia

Aspergillus fumigatus is an important fungal pathogen that causes invasive pulmonary disease in immunocompromised hosts. Respiratory exposure to A. fumigatus spores also causes allergic bronchopulmonary aspergillosis, a Th2 CD4+-T-cell-mediated disease that accompanies asthma. The microbial factors that influence the differentiation of A. fumigatus-specific CD4+ T lymphocytes into Th1 versus Th2 cells remain incompletely defined. We therefore examined CD4+-T-cell responses of immunologically intact mice to intratracheal challenge with live or heat-inactivated A. fumigatus spores. Live but not heat-inactivated fungal spores resulted in recruitment of gamma interferon (IFN-gamma)-producing, fungus-specific CD4+ T cells to lung airways, achieving A. fumigatus-specific frequencies exceeding 5% of total CD4+ T cells. While heat-inactivated spores did not induce detectable levels of IFN-gamma-producing, A. fumigatus-specific CD4+ T cells in the airways, they did prime CD4+ T-cell responses in draining lymph nodes that produced greater amounts of interleukin 4 (IL-4) and IL-13 than T cells responding to live conidia. While immunization with live fungal spores induced antibody responses, we found a marked decrease in isotype-switched, A. fumigatus-specific antibodies in sera of mice following immunization with heat-inactivated spores. Our studies demonstrate that robust Th1 T-cell and humoral responses are restricted to challenge with fungal spores that have the potential to germinate and cause invasive infection. How the adaptive immune system distinguishes between metabolically active and inactive fungal spores remains an important question.     

Identification and characterization of a conserved,stage-specific gene product of Plasmodium falciparum recognized by parasite growth inhibitory antibodies

We have identified a novel conserved protein of Plasmodium falciparum, designated D13, that is stage-specifically expressed in asexual blood stages of the parasite. The predicted open reading frame (ORF) D13 contains 863 amino acids with a calculated molecular mass of 99.7 kDa and displays a repeat region composed of pentapeptide motives. Northern blot analysis with lysates of synchronized blood stage parasites showed that D13 is highly expressed at the mRNA level during schizogony. The first N'-terminal 138 amino acids of D13 were expressed in Escherichia coli and the purified protein was used to generate anti-D13 monoclonal antibodies (MAbs). Using total lysates of blood stage parasites and Western blot analysis, these MAbs stained one single band of approximately 100 kDa, corresponding to the predicted molecular mass of ORF D13. Western blot analysis demonstrated further that D13 is expressed during schizogony, declines rapidly in early ring stages and is undetectable in trophozoites. D13 protein is localized in individual merozoites in a distinct area, as demonstrated by indirect immunofluorescence analysis. After subcellular fractionation, D13 was confined to the pelleted fraction of the parasite lysate and its extraction by alkaline carbonate buffer treatment indicated that D13 is not a membrane-integral protein. Inclusion of certain anti-D13 MAbs into in vitro cultures of blood stage parasites resulted in considerable reduction in parasite growth. The N'-terminal domain encompassing 158 amino acids is 94 and 95%, respectively, identical at the amino acid level between Plasmodium knowlesi, Plasmodium yoelii, and P. falciparum. In summary, we describe a novel stage-specifically expressed, highly conserved gene product of P. falciparum that is recognized by parasite growth inhibitory antibodies.     

Enhancement of murine macrophage binding of and response to bacterial lipopolysaccharide (LPS) by LPS-binding protein.

We have studied the effects of highly purified rabbit lipopolysaccharide (LPS)-binding protein (LBP) on the ability of murine bone marrow-derived macrophages to respond to bacterial LPS. Macrophage responses studied include the secretion of tumor necrosis factor alpha, production of arginine-derived nitrite (NO2-), and killing of an intracellular pathogen, Leishmania enriettii. Macrophages from either CBA or LPS-hyporesponsive C3H/HeJ mice exhibited significantly greater sensitivity to LPS in the presence of LBP. Furthermore, both CBA and C3H/HeJ macrophages demonstrated an LBP-dependent enhancement of LPS binding. These results suggest that C3H/HeJ macrophages are capable of binding LPS-LBP complexes and support the hypothesis that hyporesponsiveness in this strain involves a step subsequent to LPS binding. Furthermore, these findings provide additional evidence of the important role played by the acute-phase plasma protein LBP in modifying host response to LPS.