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The possibility of using a recombinant fragment of the CagA (128 kDa protein) for the diagnosis of Helicobacter pylori infection was evaluated. Following cloning of the gene coding for the CagA, a recombinant fragment of it was expressed in Escherichia coli, purified and used in Western blot and an EIA to screen sera from 82 patients with gastroduodenal disease who underwent endoscopic examination. In Western blot, good correlation was found between the serological data obtained with the recombinant antigen and those obtained using non-purified extracts of Helicobacter pylori. The EIA using the antigen showed a sensitivity of 96.2% and a specificity of 96.6% compared with Western blot. These data indicate that the recombinant protein is a reliable antigen for detection of infections with Helicobacter pylori strains that are associated with disease. The EIA assay described may be used in follow-up of the progression of the illness and the results of therapy.

All available bafilomycins (A1, B1, C1 and D) inhibit and revert macroscopic vacuolization induced by Helicobacter pylori cell-free extracts. Bafilomycin A1 displays the highest activity, followed by bafilomycin B1, C1 and D. The different potency of bafilomycins correlates with their ability to inhibit the vacuolar-type ATPase (V-ATPase) and to dissipate the membrane pH gradient of intracellular acidic organelles. These results suggest that bafilomycins should be considered as possible therapeutic agents in the treatment of gastritis.

The E6 open reading frame of the human papillomavirus (HPV) 16 codes for a protein of 158 amino acids with a theoretical molecular weight of 19.1 kD. A peptide corresponding to 147-158 amino acids (NH2-RSSRTRRETQLC-COOH) was synthesized, coupled to haemocyanin and used for immunization of rabbits. The antibodies obtained were used for the immunohistochemical analysis of a series of 41 paraffin-embedded biopsies including 29 cases of HPV 16-associated cervical lesions, five HPV 6, four HPV 11, and three HPV 18-associated genital warts. Expression of a reacting molecule (both intranuclear and cytoplasmic) could be demonstrated in 28/29 (96.6%) of the HPV 16 lesions, in 2/5 (40%) of the HPV 6, in 2/4 (50%) of the HPV 11, and in 3/3 of HPV 18 lesions. The intensity of the staining was weak in the HPV 6 and HPV 11 lesions, whereas it was classified as intense in 21/29 (72%) of the E6-positive HPV 16 lesions. Negative staining was almost invariably (5/6, 83%) confined to HPV-non-cervical intraepithelial neoplasia (HPV-NCIN) lesions, only 5/18 (27%) of which showed an intense staining. In contrast, intense staining was associated with CIN; 11/12 (92%) in HPV-CIN I and 8/11 (73%) in HPV-CIN II lesions. In the HPV 16 group, intense reactivity was more frequent in lesions shown to make clinical progression (8/9, 89%) as compared with those which underwent spontaneous regression (10/15, 66.7%). Topography of the immunostaining paralleled the localization of HPV DNA hybridization signals in 20/21 (95%) evaluable HPV 16 cases, in contrast to only 1/3 in HPV 18 cases and in none of the HPV 6 and HPV 11 lesions. These differences between HPV 16 and HPV 6/11 lesions in the staining with the HPV 16 E6 protein antibody might help explain at least some of the differences in their known biological behaviour.

Bafilomycin A1, a specific inhibitor of the vacuolar-type H(+)-ATPase, responsible for acidification of intracellular compartments, prevents the vacuolization of Hela cells induced by H. pylori, with an inhibitory concentration giving 50% of maximal (ID50) of 4 nM. Bafilomycin A1 is also very efficient in restoring vacuolated cells to a normal appearance. The vacuolating activity of Helicobacter pylori is not inhibited by a series of specific inhibitors of vacuolar H(+)-ATPases. These findings indicate that a transmembrane pH gradient is needed for the formation and growth of vacuoles caused by the bacterium and that this pH gradient is due to the activity of a vacuolar ATPase proton pump of HeLa cells.

Formaldehyde treatment is a method routinely used to detoxify diphtheria, tetanus, and pertussis toxins as well as other molecules suitable for vaccine production. To investigate whether chemical detoxification alters the immunological properties of vaccine components, we have treated the pertussis toxin mutant PT-9K/129G with formaldehyde and tested the properties of the resulting molecules. Very low concentrations of formaldehyde stabilize the molecule without affecting the physicochemical and immunological parameters. Increasing doses of formaldehyde abolish the mitogenic and hemagglutinating activities of PT-9K/129G. At the same time, the molecule loses the ability to be recognized by a monoclonal antibody specific for a major protective epitope on the S1 subunit of pertussis toxin and its affinity for anti-pertussis toxin polyclonal antibodies is also reduced. In marked contrast, the ability of PT-9K/129G to be recognized by human T-cell clones is not affected by Formalin treatment. In vivo, the formaldehyde-treated molecules induce amounts of specific antibodies comparable with those of untreated molecules but significantly lower levels of toxin-neutralizing antibodies. Furthermore, the formaldehyde-treated molecules also show a reduced protective activity in the intracerebral challenge assay.

the introduction of two amino acid substitutions within the enzymatically active subunit S1 of pertussis toxin (PT) abolishes its ADP-ribosyltransferase activity and toxicity on CHO cells (Pizza et al., Science 246:497-500, 1989). These genetically inactivated molecules are also devoid of other in vivo adverse reactions typical of PT, such as induction of leukocytosis, potentiation of anaphylaxis, stimulation of insulin secretion, and histamine sensitivity. However, the mutant PT molecules are indistinguishable from wild-type PT in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and maintain all the physical and chemical properties of PT, including affinity for toxin-neutralizing poly- and monoclonal antibodies. Either alone or stabilized with formaldehyde, PT mutants are able to induce high levels of neutralizing antibodies and to protect mice in a dose-dependent fashion against intracerebral challenge with virulent B. pertussis. These results clearly show that these genetically inactivated PT molecules are nontoxic but still immunogenic and justify their development as a component of a new, safer acellular vaccine against whooping cough.

Filamentous hemagglutinin (FHA), a 220-kDa protein that mediates the adhesion of Bordetella pertussis to eukaryotic cells, is a component of acellular vaccines against whooping cough. To identify the subregions of FHA that are immunogenic for T cells, 16 human T-cell clones were raised against purified FHA and tested for the recognition of recombinant and proteolytic fragments. The clones were found to map either in the carboxy-terminal or the amino-terminal part of the FHA molecule, but none of them recognized the central region, which contains a sequence that is homologous to that of the eukaryotic protein fibronectin. These data suggest that subregions of FHA that do not contain sequences that are potentially cross-reactive with self proteins may be sufficient to induce an immune response against the whole protein.

Colonization of human gastric mucosa with cytotoxic strains of the bacterium Helicobacter pylori is associated with peptic ulcer and with chronic gastritis. Since little is known about the T-cell response to H. pylori, we investigated the CD4+ T-cell response both in peripheral blood mononuclear cells (PBMCs) and at the site of infection. First, we compared the bulk PBMC proliferative response to the bacterium in individuals with and without symptoms of gastroduodenal disease. We found that the PBMCs from virtually all individuals proliferate in response to heat-inactivated bacteria. Second, we cloned H. pylori-specific CD4+ T lymphocytes from the PBMCs of three patients and from both the gastric mucosa and PBMCs of a fourth patient. We have found that CD4+ T-cell clones specific for H. pylori from peripheral blood samples and gastric mucosae of infected patients are major histocompatibility complex class II restricted and discriminate between several cytotoxic and noncytotoxic bacterial strains. Moreover, they are polyclonal in terms of T-cell receptor usage and major histocompatibility complex restriction. Our results demonstrate that the T-cell response to the whole bacterium in PBMCs does not correlate with antibody response, infection, or disease. However, H. pylori-specific CD4+ T cells are detectable, at the clonal level, in both the periphery and gastric mucosa of infected patients. Localization of these cells at the site of disease suggests they are effectors of the immune response to the bacteria.

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.

The study of the virulence characteristics of Helicobacter pylori (HP) seeks to contribute to explaining why only few persons infected with HP develop peptic ulcers. Cytotoxicity plays a crucial role in the pathogenetic mechanisms of most infections of the digestive system. Some HP isolates produce one or more substances which induce intracytoplasmic vacuolization in mammahan cells in culture. Several studies have shown that vacuolating activity or cytotoxicity of these isolates is associated with proteins of apparent molecular weights of 130, 95, and 82 kDa in SDS-PAGE [3,4]. This association is based mainly on the presence of these proteins in concentrated broth culture filtrates of cytotoxic strains and their absence in those of noncytotoxic ones. In addition, by immunoblotting with human sera a serological recognition of these proteins is more prevalent among subjects infected with cytotoxic strain than in persons colonized by non-cytotoxic HP [1]. Sera from patients infected with HP are also able to neutralize the vacuolization suggesting that cytotoxin is produced in vivo [5].

Helicobacter pylori (HP) is the agent of type B gastritis and a cofactor in the development of peptic ulcer and probably also of gastric cancer. Certain HP strains produce a toxin which induces intracellular vacuolization on mammalian cells in vitro. Proteins associated with vacuolating activity (VA) have molecular weights of 130, 95, and 80 kDa and can be detected broth and in concentrated broth culture filtrates (CBCF) of cytotoxic strains [1].

Helicobacter pylori (HP) strains differ considerably in their virulence characteristics. Only certains organisms, for example, produce a toxic substance which causes intracytoplasmic vacuolization on cells in vitro [2, 3]. Cytotoxic HP strains (CTHP) can also be considered ulcerogenic because they have been isolated with greater frequency from patients with peptic ulcers than from patients with chronic gastritis only [2]. Thus the vacuolating toxin most probably plays a crucial role in the pathogenic mechanism of the gastric mucosa damage.

Two cases of Campylobacter mucosalis enteritis in children are reported. The patients recovered without antimicrobial therapy. Strains were isolated only by the feces filtration technique. In one child, bactericidal antibodies to the homologous strain were detected in a convalescent-phase serum sample. C. mucosalis should be considered a primary intestinal pathogen.

ObjectivesTo investigate the effect of the proton pump inhibitor omeprazole on the function of purified urease enzyme of Helicobacter pylori and to examine the in vitro activities of omeprazole against urease-positive and -negative Helicobacter strains.MethodsUrease was purified chromatographically

In 1924 Ramon described the inactivation of diphtheria toxin by formaldehyde treatment. This method allowed the introduction of mass vaccination against diphtheria and tetanus and opened the way to the inactivation of viruses by chemical treatment. In this review we describe the use of genetic manipulations for the inactivation of pertussis toxin. The toxin inactivated by this new method is an antigen superior to those obtained by chemical treatment and has been used to develop a new vaccine against whooping cough.

Polyclonal antibodies able to recognize protein-acetaldehyde conjugates were produced and characterized. The antibodies react with sodium cyanoborohydride-reduced Schiff's bases between acetaldehyde and a protein, independently of the nature of the macromolecule binding the acetaldehyde moiety. Only conjugates between acetaldehyde or propionaldehyde and a protein are recognized; conjugates obtained with other aldehydes are not reactive. Results concerning the formation of acetaldehyde adducts with carrot (Daucus carota L.) proteins are presented as well as the presence of such conjugates in ethanol-treated carrot cell cultures, a system highly sensitive to the presence of ethanol in the culture medium.

The complete E7 protein-encoding open reading frame of human papillomavirus type 16 (HPV-16) was expressed in the fission yeast Schizosaccharomyces pombe, under the control of a cloned yeast promoter. The HPV-16 E7 protein synthesized in S. pombe is a 17-kDa phosphoprotein which is recognized by anti-E7 antibodies (raised in rabbits against E7 fusion protein produced in Escherichia coli). The mobility during sodium dodecyl sulfate-polyacrylamide-gel electrophoresis of native E7 phosphoprotein synthesized in S. pombe is identical to that of the E7 phosphoprotein immunoprecipitated from human CaSki cells. Immunofluorescence staining showed that HPV-16 E7 phosphoprotein is localized in the nuclei of transformed S. pombe. These results indicate that E7 protein synthesized by S. pombe is apparently indistinguishable from HPV-16 protein synthesized in higher eukaryotic cells expressing genes of HPV-16, and also that the phosphorylated, nuclear HPV-16 E7 protein is synthesized in S. pombe in a form compatible with its biological activity. © 1990.

A mouse monoclonal antibody against abscisic acid (ABA) was produced and characterized. It was raised using ABA conjugated to the carrier protein through the carboxyl (Cl) group as immunogen. It did not discriminate between free ABA or its ester derivatives. This antibody, which is the first monoclonal against Cl-conjugated ABA, shows interesting characteristics. It has high affinity (Ka=1.5 × 109 L/mol) and specificity. Compounds structurally similar to ABA, such as phaseic acid, dihydrophaseic acid, and both the 2,trans-isomer and the (R)-enantiomer of ABA, are not reactive. The narrow linear range of the standard curve (0.018–1.8 pmol) ensures great precision of the assay. This monoclonal antibody has been used for the quantification of ABA conjugates in crude aqueous extracts of bean leaves by radioimmuno-assay (RIA). The fractionation of the extracts by high-performance liquid chromatography (HPLC) confirmed the absence of cross-reacting compounds. Because of its affinity and specificity, in combination with antibodies against free ABA, this antibody should be a sound tool for studying the metabolism and immunolocalization of ABA in plant tissues.

Antral biopsy culture supernatants from 14 subjects with chronic gastritis, known to have IgA antibodies to the 120 kilodalton protein, showed positive recognition of this antigen in western blots against a cytotoxin positive strain of Helicobacter pylori but gave negative reactions with two cytotoxin negative strains. Control immunoblots with culture supernatants from 13 non-responders to the protein were all negative. This indicates a direct association between expression of the 120 kilodalton protein in H pylori strains and cytotoxicity.

A series of 74 punch biopsies, derived from 513 women prospectively followed for cervical human papillomavirus (HPV) infections (including HPV-NCIN, HPV-CIN I, HPV-CIN II, and HPV-CIN III lesions), and 43 control cases (consisting of normal epithelia, nonspecific cervicitis, and classical CIN lesions) were analysed for expression of cytokeratin polypeptides using the ABC technique and monoclonal antibodies SK 56-23 (wide-spectrum antibody), SK 60-61 (specific for keratins 8 and 18), and SK 2-27 (detecting keratins 14, 16, and 17). HPV typing was carried out using the in situ hybridization technique with DNA probes for HPV 6, 11, 16, 18, and 31. All layers of the exocervical epithelium were regularly stained with the antibody SK 56-23, and the staining pattern remained unaltered in all cervical lesions studied. In contrast to the normal exocervical epithelium, which remained negative with SK 60-61, positive staining was observed in 3 of 15 cervicitis cases and 6 of 23 classical CIN lesions. Interestingly, the majority (69 of 74, 93.2%) of both HPV-NCIN and HPV-CIN lesions showed positive staining with this antibody either in all layers or in suprabasal cells. Antibody SK 2-27 stained the basal cells of the normal exocervical epithelium with remarkable specificity. In 18 of 19 HPV-NCIN lesions, basal cells could not be stained by SK 2-27 monoclonal, but the suprabasal cells were stained instead. In HPV-CIN, but not in classical CIN, this antibody demonstrated the presence of the epitope typical of the cytokeratins 14, 16, and 17 in all layers of the epithelium, the highest frequency (9 of 12, 75%) being found in HPV 16-induced lesions. These disturbances of cytokeratin patterns in cervical epithelium could be associated with cell transformation by HPV, leading to development of HPV-CIN, and could be specific for this virus. The present data are of interest in assessing the stage of maturation of the squamous cells in progressing cervical HPV infections.

In this study antibodies specific for different intermediate-sized proteins (cytokeratins and neurofilaments) have been tested on a series of neuroendocrine (NE) lung tumors in order to evaluate their diagnostic validity. In particular we used a panel of polyclonal anti-neurofilament 200-kilodalton subunits whose reactivity against phospho-dependent epitopes was known. At least one NF subunit was constantly present and in all cases coexpression of cytokeratins and neurofilaments was confirmed. However, in cases of carcinoid tumor (CT) the results were homogeneous, while the cases of small cell lung carcinoma (SCLC) showed a much wider range of immunostaining. Our investigation confirms the hypothesis that the phosphorylation state is a significant determinant of immunohistochemical properties of neurofilaments. This might explain the large number of negative results obtained in previous investigations on NE tumors. The phosphorylation of neurofilaments may also be considered an indication of the degree of differentiation of the tumor.

Immunization with chemically detoxified pertussis toxin can prevent severe whooping cough with an efficacy similar to that of the cellular pertussis vaccine, which normally gives unwanted side effects. To avoid the reversion to toxicity and the loss of immunogenicity that may follow chemical treatment of pertussis toxin, inactive toxins were constructed by genetic manipulation. A number of genetically engineered alleles of the pertussis toxin genes, constructed by replacing either one or two key amino acids within the enzymatically active S1 subunit, were introduced into the chromosome of strains of Bordetella pertussis, B. parapertussis, and B. bronchiseptica. These strains produce mutant pertussis toxin molecules that are nontoxic and immunogenic and that protect mice from the intracerebral challenge with virulent Bordetella pertussis. Such molecules are ideal for the development of new and safer vaccines against whooping cough.

We have evaluated by means of immunocytochemistry the distribution of various cytoskeletal and contractile proteins (cytokeratins, vimentin, desmin and alpha-smooth muscle actin) in 23 salivary or lacrimal gland primary tumours (15 pleomorphic adenomas and 8 carcinomas in pleomorphic adenoma), one third of which contained areas of normal gland. Normal epithelial luminal cells were stained by cytokeratin antibodies with a general specificity, while myoepithelial cells were selectively stained by a monoclonal antibody (SK2-27) reacting in immunoblots with cytokeratin polypeptides 14, 16 and 17, according to the classification of Moll et al. (1982) and by an antibody directed against alpha-smooth muscle actin (Skalli et al. 1986). In pleomorphic adenomas, both epithelial and myoepithelial cells displayed typical topographic distributions; moreover, myoepithelial cells showed two distinct cytoskeletal phenotypes. These findings could account in part for the heterogeneity of aspects observed in this tumour. In carcinomas, malignant cells were always positive to cytokeratin antibodies with general specificity and myoepithelial cells were absent as judged by anticytokeratin SK2-27 and anti-alpha-smooth muscle actin immunostainings. However, interestingly, there was in all cases a strong positivity for alpha-smooth muscle actin in stromal cells, similarly to what has previously been described for mammary carcinoma (Skalli et al. 1986). Our findings may be useful for the interpretation of the histogenesis of salivary and lacrimal tumour and stromal cells.

Summary A new mouse monoclonal antibody against abscisic acid (ABA) was produced and characterized. This antibody is highly specific for free (S)-ABA and shows a good affinity constant. A newly developed solid phase radioimmunoassay (RIA) for the quantitation of ABA content in plant extracts is described. This assay proved to be more sensitive and precise than the commonly used liquid phase RIA. Using this assay ABA was quantified in tomato and bean leaves and in Sechium endosperm. Results were compared with those obtained using the other two monoclonal antibodies produced up to now against ABA. Results obtained with RIA were validated by physicochemical methods.

ObjectiveTo assess seropositivity to Heiicobacter pylori cytotoxin-associated proteins in a group of children with H. pylori gastritis and to correlate seropositivity to their disease characteristics, in a prospective study.Patients and methodsSera were collected from 68 children with H. pylori gast

Proteins to be used as vaccines are frequently treated with formaldehyde, although little is known about the effects of this treatment on protein antigenicity. To investigate the effect of formaldehyde treatment on antigen recognition by T cells, we compared the in vitro T-cell response to proteins that have been formaldehyde treated with the response to untreated proteins. We found that peripheral blood mononuclear cells from individuals vaccinated with three formaldehyde-treated proteins (pertussis toxin, filamentous hemagglutinin, pertactin) of Bordetella pertussis showed little or no response to the formaldehyde-treated proteins but proliferated very well in response to the corresponding untreated protein. These findings were further confirmed with CD4+ T-cell clones specific for defined epitopes of the bacterial proteins. We found that some epitopes are presented poorly or not at all when formaldehyde-treated proteins are used, whereas other epitopes are equally presented to T-cell clones when either formaldehyde-treated or untreated antigens are used. However, T-cell recognition could be restored by either antigen degradation before formaldehyde treatment or heat denaturation after such treatment. Parallel digestion with trypsin of both formaldehyde-treated and untreated proteins showed that fragments generated from the two forms of the same antigen were different in size. These results demonstrate that formaldehyde treatment can constrain antigen presentation to T cells and that this may be due to an altered proteolytic processing of formaldehyde-treated proteins.

The two published sequences of the pertussis toxin operon differ in 3 bp in the S1 subunit gene. In this report, we provide evidence that Bordetella pertussis strains are able to produce active pertussis toxin only when they contain one of the two possible nucleotide sequences.

Pertussis toxin is a protein containing five noncovalently linked subunits which are assembled into the monomer A (containing the subunit S1) and the oligomer B (containing subunits S2, S3, S4, and S5 in a 1:1:2:1 ratio). Each of the five subunits is synthesized as a precursor containing a secretory leader peptide and is secreted into the periplasm of Bordetella pertussis where the five subunits are assembled into the oligomeric structure and then released into the culture medium. In the absence of subunit S3 the remaining subunits are not secreted into the medium, thus suggesting that the assembled structure is necessary for the release of the toxin into the supernatant. In this study we describe four B. pertussis mutants which secrete into the medium low amounts of the B oligomer of pertussis toxin. These mutants have single or multiple changes in the gene encoding the S1 subunit and synthesize S1 proteins with altered conformation which are not assembled into the holotoxin and are apparently degraded in the periplasm. These data indicate that while the B oligomer alone has the structural information necessary for the extracellular export of pertussis toxin, the S1 subunit is required for its efficient release into the medium.