Gene structure of the Helicobacter pylori cytotoxin and evidence of its key role in gastric disease.

The gram negative, microaerophilic bacterium Helicobacter pylori colonizes the human gastric mucosa and establishes a chronic infection that is tightly associated with atrophic gastritis, peptic ulcer, and gastric carcinoma. Cloning of the H. pylori cytotoxin gene shows that the protein is synthesized as a 140-kD precursor that is processed to a 94-kD fully active toxin. Oral administration to mice of the purified 94-kD protein caused ulceration and gastric lesions that bear some similarities to the pathology observed in humans. The cloning of the cytotoxin gene and the development of a mouse model of human gastric disease will provide the basis for the understanding of H. pylori pathogenesis and the development of therapeutics and vaccines.

Development of a mouse model of Helicobacter pylori infection that mimics human disease.

The human pathogen Helicobacter pylori is associated with gastritis, peptic ulcer disease, and gastric cancer. The pathogenesis of H. pylori infection in vivo was studied by adapting fresh clinical isolates of bacteria to colonize the stomachs of mice. A gastric pathology resembling human disease was observed in infections with cytotoxin-producing strains but not with noncytotoxic strains. Oral immunization with purified H. pylori antigens protected mice from bacterial infection. This mouse model will allow the development of therapeutic agents and vaccines against H. pylori infection in humans.

Characterisation of a monoclonal antibody and its use to purify the cytotoxin of Helicobacter pylori.

The vacuolating cytotoxin (VacA) is a major virulence factor of Helicobacter pylori which is not yet well characterised and is difficult to obtain in large quantities. Here we describe the production of a monoclonal antibody that recognises the native but not the denatured form of VacA. The antibody can be efficiently used in affinity chromatography for one-step purification of large quantities of VacA from culture supernatants. Elution at acidic pH dissociates the oligomeric molecule into monomers that reanneal in a time-dependent fashion. The purified cytotoxin is able to bind, and to intoxicate HeLa cells.

Trembler mouse Schwann cells in culture: anomalies in the synthesis of lipids and proteins.

We investigated the biochemical and growth properties of Schwann cells from the sciatic nerve of Trembler and unaffected mice in culture. Both Trembler and control cultures showed similar growth rates. The specific activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) and enzymes involved in lipid metabolism of cerebrosides and sulfatides were studied. UDP-galactose: ceramide galactosyltransferase was significantly decreased in Trembler cultures less than 21 days in vitro. No differences were found in the specific activities of cerebroside sulfotransferase, arylsulfatase A or CNP between Trembler and control cultures. Schwann cells from Trembler and control mice were labeled with [35S]methionine and the protein analyzed by two-dimensional gel electrophoresis. Our study revealed few but consistent differences in the protein pattern synthesized by the Trembler Schwann cells.

Generation of a monoclonal antibody to a defined portion of the Helicobacter pylori vacuolating cytotoxin by DNA immunization.

The capacity of mammalian cells to express proteins encoded by plasmid vectors injected as naked DNA has opened the possibility to induce an immune response against protein antigens by DNA vaccination. We have used DNA immunization to generate a strong antibody response to a defined portion of the Helicobacter pylori vacuolating cytotoxin. A high specific monoclonal antibody has been derived from the splenocytes of an immunized mouse. Our data show that DNA immunization offers the possibility to obtain monoclonal antibodies following a significant shortcut with respect to traditional immunization protocols.

The effect of alkaline pH on the cell growth of six different mammalian cells in tissue culture.

The effect of high alkaline pH on the reinitiation of cell growth was studied in six different mammalian cells. We failed to confirm the observation of Zetterberg & Engström, Proc natl acad sci US 78 (1981) 4334 [17] and Exp cell res 144 (1983) 199 [18]. Treatment of quiescent cells at pH 9.5 did not stimulate cell growth when measured by total protein/flask or increase in cell number.

The Hsp60 protein of Helicobacter pylori: structure and immune response in patients with gastroduodenal diseases.

Helicobacter pylori is a human pathogen that has been associated with gastritis, peptic ulcer and gastric carcinoma. The role of the direct action of H. pylori virulence factors and of the induction of autoreactive immunity in the development of chronic gastritis has not been clarified yet. Here we report the cloning and molecular characterization of a gene of H. pylori coding for a protein of 58 kDa, recognized by sera of patients affected by H. pylori-induced gastroduodenal diseases. This antigen is present in all the H. pylori strains tested and it belongs to the Hsp60 family of heat-shock proteins, with high homology with other bacterial and eukaryotic proteins of the same family. This class of homologous proteins has been implicated in the induction of autoimmune disorders in different systems. The presence in infected patients of anti-H. pylori Hsp60 antibodies, potentially cross-reactivity between human Hsp60 and a rabbit antiserum against H. pylori Hsp60 suggest that a role of this protein in gastroduodenal diseases is possible.

Site-specific monoclonal antibodies against peanut agglutinin (PNA) from Arachis hypogaea. Immunohistochemical study of tissue-cultured cells and of 27 cases of Hodgkin's disease.

The purpose of this study was to increase the sensitivity of the staining reaction for the T antigen on the surface of neoplastic cells grown in vitro with the use of site-specific monoclonal antibodies (MAbs). The authors describe anti-peanut agglutinin (PNA) MAbs selected by screening the hybridomas with PNA and PNA bound to bovine serum albumin conjugated with the T antigen. The selected hybridomas (F2C8, F3D12, F3A5) were then grown in pristane-sensitized mice or in the Amicon Hollow Fiber System (F2C8). The affinity constant values for PNA were measured, and all the purified MAbs were tested on both native and denatured PNA, wheat germ agglutinin, concanavalin A, and ricin by using the immunoassay dot test and immunoblotting methods. Eleven different cell lines were stained with the three MAbs; similar results were obtained with F2C8 and F3D12. In each case the fluorescence, if present, was associated with the cell membrane, and the intensity of the staining was always stronger when the cells were incubated with the MAbs than when stained with fluorescein-labeled PNA. On the other hand, F3A5 failed to stain unfixed cells preincubated with PNA but stained the same cells after fixation, independently of the presence of PNA. One of the antibodies, F2C8, was used to stain histologic preparations from 27 cases of Hodgkin's disease and was compared with the anti-granulocyte antibody, Leu-M1, which has been used by numerous authors to identify the characteristic Reed-Sternberg cells. The results obtained were qualitatively similar; ie, F2C8 was at least as efficient as anti-Leu-M1 in its ability to stain the typical diagnostic cells in Hodgkin's disease.

Correlation between differentiation and lung colonization by retinoic acid-treated F9 cells as revealed by the expression pattern of extracellular matrix and cell surface antigens.

For study of the correlation between differentiation and organ colonization properties of tumor cells, F9 embryonal carcinoma (EC) cells were treated with retinoic acid, an inducer of differentiation; and their organ colonization pattern was assessed by the experimental metastasis assay. Untreated cells were found to colonize the liver, whereas treated cells colonized the lungs. This pattern held true when metastases were scored after spontaneous death or after a careful microscopic search for micrometastases. Histologic examination revealed that both the tumor nodules produced by the untreated and the treated cells had the characteristics of EC devoid of any evidence of differentiation. The immunohistochemical study of the expression of markers typical of embryonal carcinoma cells or of the extracellular matrix components laminin and collagen type IV, typical of differentiated cells, confirmed these results. However, the lack of expression of stage-specific embryonal antigen 1 (SSEA-1), a marker generally associated with the undifferentiated state, observed only in the tumors obtained after injection of treated cells, indicates that the lung nodules probably derive from cells that have responded to the induction in vitro but have dedifferentiated in vivo.

Expression of myelin components in mouse Schwann cells in culture.

Mouse Schwann cells cultured in vitro are capable of expressing basal levels of the major myelin components P1, P2, P0, and galactocerebroside. Numerical counts of immunostained cultures indicated that between 22 and 40% of the cells are positive up to 21 days for all of the components indicated. Electrophoretic analysis of Schwann cells labeled with a 14C-amino acid mixture revealed the presence of proteins with relative mobilities identical to those of P0 and P1. Positive identification of the two proteins was indicated by immunoprecipitation of P1 and immunoblotting of P0. These data show that in the absence of neurites, Schwann cells in culture can express low levels of myelin characteristic components even in the absence of myelin assembly.

Lipid interaction of the 37-kDa and 58-kDa fragments of the Helicobacter pylori cytotoxin.

Helicobacter pylori cytotoxin vacA (95 kDa) causes a vacuolar degeneration of epithelial cells. There is evidence that this protein toxin acts inside cells, and hence has to cross a cell membrane. This cytotoxin is frequently obtained as two fragments of 58 kDa (p58) and 37 kDa (p37) and it is available only in minute amounts. Here, its membrane interaction was studied with the two fragments, produced in Escherichia coli. Light scattering and energy transfer experiments show that p37 and p58 cause aggregation and fusion of small unilamellar lipid vesicles; only a reversible aggregation is induced at neutral pH, whereas at acid pH fusion also takes place. p58, but not p37, causes potassium efflux from liposomes and this occurs only at acid pH. Hydrophobic photolabelling with photoactivatable phosphatidylcholines inserted into liposomes shows that both fragments are labelled at neutral pH. The amount of labelling of the two fragments is much higher at acid pH, consistent with a further penetration into the hydrophobic core of the lipid bilayer. Tryptophan fluorescence measurements indicate that the two fragments undergo a pH-driven conformational change. These data are consistent with cytotoxin entry in the cell cytosol via an intracellular acidic compartment.

Identification of the Helicobacter pylori VacA toxin domain active in the cell cytosol.

Cells exposed to Helicobacter pylori toxin VacA develop large vacuoles which originate from massive swelling of membranous compartments at late stages of the endocytic pathway. When expressed in the cytosol, VacA induces vacuolization as it does when added from outside. This and other evidence indicate that VacA is a toxin capable of entering the cell cytosol, where it displays its activity. In this study, we have used cytosolic expression to identify the portion of the toxin molecule responsible for the vacuolating activity. VacA mutants with deletions at the C and N termini were generated, and their activity was analyzed upon expression in HeLa cells. We found that the vacuolating activity of VacA resides in the amino-terminal region, the whole of which is required for its intracellular activity.

Pharmacokinetic variability and strategy for therapeutic drug monitoring of saquinavir (SQV) in HIV-1 infected individuals.

AIMS: To investigate the pharmacokinetic profile of the protease inhibitor saquinavir (SQV) after multiple doses in HIV-positive patients and to evaluate the possibility of predicting total body exposure of SQV from concentrations determined at single time points. METHODS: Twenty HIV-positive patients on steady-state treatment with SQV (Hard-Gel-Capsule, Invirase(R)) were enrolled in this study. Serial blood samples were obtained during a dosing interval. SQV plasma concentrations were determined by high performance liquid chromatography (h.p.l.c.) and pharmacokinetic parameters were determined by noncompartmental techniques. RESULTS: There was a marked interindividual variability in SQV pharmacokinetic parameters with a 11-fold variability in total systemic exposure to SQV, as expressed by AUC(0,8h) values (range: 268-3009 ng ml-1 h, CV: 69%). The oral clearance shows an interindividual CV of 75%. A strong correlation (r=0.94) was found between SQV plasma concentration at 3 h (C3 h ) and AUC(0,8h). CONCLUSIONS: This study shows that C3 h is a good predictor for total body exposure of SQV and might be useful to predict SQV disposition in HIV-positive patients on steady-state treatment.

High-performance liquid chromatography method for analyzing the antiretroviral agent efavirenz in human plasma.

Efavirenz (EFV, DMP-266) is a new antiretroviral agent belonging to the class of nonnucleoside reverse transcriptase inhibitors. It has recently been approved by the Food and Drug Administration in management of human immunodeficiency virus (HIV). Preliminary pharmacokinetic studies on EFV in healthy volunteers show that the drug may influence the metabolism of protease inhibitors. For the determination of EFV in human plasma, a validated and specific reverse-phase high-performance liquid chromatography (HPLC) method, with UV detection, was developed. We used 100 microL plasma sample for a liquid-liquid extraction with diethyl ether after basification. The mobile phase was a mixture of acetonitrile and water, pumped at a flow rate of 1.2 mL/min. Ultraviolet detection was carried out at a wavelength of 247 nm. Retention times for EFV and internal standard (IS) were 5.3 and 4.5 minutes, respectively, and there was no chromatographic interference from other commonly administered drugs. The limit of detection was 100 ng/mL. The described assay is a rapid and accurate method for measurement of EFV in plasma: the easy preparation and small sample size makes this assay highly suitable for pharmacokinetic studies and routine clinical analysis in patients with HIV. In addition, the reproducibility of the method is only moderately increased by including IS, so analyzing without IS may be an alternative.

Deletion of the major proteolytic site of the Helicobacter pylori cytotoxin does not influence toxin activity but favors assembly of the toxin into hexameric structures.

The Helicobacter pylori cytotoxin is proteolytically cleaved at a flexible hydrophilic loop into two subunits. Deletion of the loop sequences had no effect on biological activity of the toxin in the HeLa cell vacuolation assay but favored the organization of the protein into hexameric rather than heptameric structures.

Binding of the Helicobacter pylori vacuolating cytotoxin to target cells.

The vacuolating cytotoxin of Helicobacter pylori, VacA, enters the cytoplasm of target cells and causes vacuolar degeneration by interfering with late stages of endocytosis. By using indirect immunofluorescence and flow cytometry, we have demonstrated that VacA binds to specific high-affinity cell surface receptors and that this interaction is necessary for cell intoxication.

Molecular characterization of the 128-kDa immunodominant antigen of Helicobacter pylori associated with cytotoxicity and duodenal ulcer.

Helicobacter pylori has been associated with gastritis, peptic ulcer, and gastric adenocarcinoma. We report the nucleotide sequence and expression of an immunodominant antigen of H. pylori and the immune response to the antigen during disease. The antigen, named CagA (cytotoxin-associated gene A), is a hydrophilic, surface-exposed protein of 128 kDa produced by most clinical isolates. The size of the cagA gene and its protein varies in different strains by a mechanism that involves duplication of regions within the gene. Clinical isolates that do not produce the antigen do not have the gene and are unable to produce an active vacuolating cytotoxin. An ELISA to detect the immune response against a recombinant fragment of this protein detects 75.3% of patients with gastroduodenal diseases and 100% of patients with duodenal ulcer (P < 0.0005), suggesting that only bacteria harboring this protein are associated with disease.

Helicobacter pylori cytotoxin: importance of native conformation for induction of neutralizing antibodies.

We have attempted to express the Helicobacter pylori vacuolating cytotoxin in Escherichia coli. Although the 95-kDa VacA polypeptide was expressed abundantly, it completely lacked any biological activity. In addition, this material failed to induce neutralizing antibodies after immunization of rabbits. In contrast, highly purified high-molecular-mass cytotoxin from the supernatant of H. pylori cultures was active in a HeLa cell assay and effectively induced a neutralizing response in rabbits. Neutralizing sera were shown to contain a high proportion of antibodies which recognized conformational epitopes found only on the native toxin. The data indicate that toxin-neutralizing epitopes are conformational and that potential vaccines based on the cytotoxin may benefit from the use of the intact molecule.