RESUMO
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.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Mucosa Gástrica/imunologia , Helicobacter pylori/imunologia , Adulto , Células Produtoras de Anticorpos/imunologia , Linfócitos B/imunologia , Biópsia , Células Clonais/imunologia , Feminino , Mucosa Gástrica/citologia , Antígenos HLA/imunologia , Humanos , Imunoglobulinas/biossíntese , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
Pathogenic strains of Helicobacter pylori cause progressive vacuolation and death of epithelial cells. To identify the nature of vacuoles, the distribution of markers of various membrane traffic compartments was studied. Vacuoles derive from the endocytic pathway since they include the fluid-phase marker Lucifer yellow. Early endosome markers such as rab5, transferrin, and transferrin receptor, as well as the lysosomal hydrolase cathepsin D, are excluded from these structures. In contrast, the vacuolar membrane is specifically stained by affinity-purified antibodies against rab7, a small GTPase, localized to late endosomal compartments. The labeling of rab7 on vacuolar membranes increases as vacuolation progresses, without a concomitant increase of cellular rab7. Cell vacuolation is inhibited by the microtubule-depolymerizing agents nocodazole and colchicine. Taken together, these findings indicate that the vacuoles specifically originate from late endosomal compartments.
Assuntos
Helicobacter pylori/patogenicidade , Vacúolos/ultraestrutura , Adenina/análogos & derivados , Adenina/farmacologia , Biomarcadores/análise , Catepsina D/análise , Colchicina/farmacologia , Imunofluorescência , Proteínas de Ligação ao GTP/análise , Células HeLa , Humanos , Immunoblotting , Isoquinolinas , Cinética , Nocodazol/farmacologia , Receptores da Transferrina/análise , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismoRESUMO
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.
Assuntos
Apresentação de Antígeno/efeitos dos fármacos , Formaldeído/farmacologia , Linfócitos T/imunologia , Adulto , Proteínas da Membrana Bacteriana Externa/imunologia , Células Cultivadas , Epitopos , Hemaglutininas/imunologia , Humanos , Toxina Pertussis , Fatores de Virulência de Bordetella/imunologiaRESUMO
AIMS: To see whether the activity of omeprazole on Helicobacter pylori is associated with toxicity of strains; to determine whether omeprazole inhibited vacuolisation of cells in culture induced by H pylori cytotoxin and by ureas, and if omeprazole prevented H pylori motility. METHODS: Minimal inhibitory concentrations (MICs) of omeprazole were determined for seven cytotoxic and five non-cytotoxic H pylori strains. Omeprazole at different concentrations was incubated with cytotoxic and non-cytotoxic extracts of H pylori, or with purified H pylori urease, and added to cells in culture. Inhibition of motility by omeprazole was tested in semi-solid medium. RESULTS: MIC90 of omeprazole was 40 micrograms/ml. MICs for cytotoxic and noncytotoxic organisms were similar. Omeprazole did not prevent vacuolisation induced by the cytotoxic extract, but at high concentrations it inhibited the formation of vacuoles induced by urease. Motility was not inhibited by the drug. CONCLUSIONS: H pylori cytotoxin is not the target of the antimicrobial activity of omeprazole. Should the drug reach clinically effective concentrations in vivo, it could potentially prevent the mucosal damage caused by the vacuolising activity of urease.
Assuntos
Citotoxinas/biossíntese , Helicobacter pylori/efeitos dos fármacos , Omeprazol/farmacologia , Citotoxinas/antagonistas & inibidores , Relação Dose-Resposta a Droga , Células HeLa , Helicobacter pylori/metabolismo , Humanos , Testes de Sensibilidade Microbiana , Vermelho Neutro/metabolismo , Urease/antagonistas & inibidores , Vacúolos/efeitos dos fármacosRESUMO
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.
Assuntos
Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Citotoxinas/genética , Infecções por Helicobacter/microbiologia , Helicobacter pylori/genética , Gastropatias/microbiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Bacteriano , Modelos Animais de Doenças , Helicobacter pylori/patogenicidade , Humanos , Immunoblotting , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Úlcera Gástrica/microbiologiaRESUMO
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.
Assuntos
Antibacterianos/farmacologia , Helicobacter pylori/efeitos dos fármacos , Vacúolos , Adenosina Trifosfatases/antagonistas & inibidores , Animais , Antibacterianos/química , Células HeLa , Helicobacter pylori/patogenicidade , Humanos , Concentração de Íons de Hidrogênio , Lisossomos/enzimologia , Macrolídeos , Estrutura Molecular , Ratos , Vacúolos/enzimologiaRESUMO
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.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Infecções por Helicobacter/diagnóstico , Helicobacter pylori/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/biossíntese , Proteínas de Bactérias/biossíntese , Western Blotting , Humanos , Técnicas Imunoenzimáticas , Imunoglobulina G/sangue , Pessoa de Meia-Idade , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/imunologiaRESUMO
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.
Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Úlcera Duodenal/microbiologia , Genes Bacterianos , Helicobacter pylori/genética , Sequência de Aminoácidos , Antígenos de Bactérias/biossíntese , Proteínas de Bactérias/biossíntese , Sequência de Bases , Doadores de Sangue , Southern Blotting , Cromossomos Bacterianos , Clonagem Molecular , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Biblioteca Genômica , Helicobacter pylori/isolamento & purificação , Helicobacter pylori/patogenicidade , Humanos , Mucosa Intestinal/microbiologia , Dados de Sequência Molecular , Peso Molecular , Oligodesoxirribonucleotídeos , Plasmídeos , Valores de Referência , Mapeamento por Restrição , Úlcera Gástrica/microbiologia , Virulência/genéticaRESUMO
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.
Assuntos
Infecções por Campylobacter/microbiologia , Enterite/microbiologia , Anticorpos Antibacterianos/sangue , Campylobacter/isolamento & purificação , Convalescença , Fezes/microbiologia , Humanos , Lactente , Masculino , Resultado do TratamentoRESUMO
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.
Assuntos
Antibacterianos/farmacologia , Células HeLa/microbiologia , Helicobacter pylori/fisiologia , Macrolídeos , Bombas de Próton/efeitos dos fármacos , ATPases Translocadoras de Prótons/antagonistas & inibidores , Vacúolos/enzimologia , Células HeLa/efeitos dos fármacos , Células HeLa/ultraestrutura , Helicobacter pylori/patogenicidade , Humanos , Concentração de Íons de Hidrogênio , Líquido Intracelular/química , VirulênciaRESUMO
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.
Assuntos
Anticorpos Antivirais/imunologia , Carcinoma in Situ/microbiologia , Proteínas Oncogênicas Virais/imunologia , Papillomaviridae/imunologia , Fragmentos de Peptídeos/imunologia , Proteínas Repressoras , Infecções Tumorais por Vírus/diagnóstico , Neoplasias do Colo do Útero/microbiologia , Cervicite Uterina/microbiologia , Sequência de Aminoácidos , Carcinoma in Situ/diagnóstico , Sondas de DNA de HPV , DNA Viral/análise , Feminino , Humanos , Dados de Sequência Molecular , Fases de Leitura Aberta , Papillomaviridae/classificação , Papillomaviridae/isolamento & purificação , Fatores de Risco , Infecções Tumorais por Vírus/microbiologia , Neoplasias do Colo do Útero/diagnóstico , Cervicite Uterina/diagnósticoRESUMO
We show that solid and liquid media, supplemented only with cyclodextrins and free of blood and its derivatives, support the growth of Helicobacter pylori. These media can be used for primary isolation of the bacteria from biopsy samples, routine laboratory growth, and large-scale industrial fermentation.
Assuntos
Ciclodextrinas/metabolismo , Helicobacter pylori/crescimento & desenvolvimento , Proteínas de Bactérias/análise , Contagem de Colônia Microbiana , Meios de Cultura/química , Eletroforese em Gel de Poliacrilamida , Helicobacter pylori/enzimologia , Helicobacter pylori/metabolismoRESUMO
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.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Gastrite/imunologia , Helicobacter pylori/imunologia , Idoso , Antígenos de Bactérias/química , Proteínas de Bactérias/química , Western Blotting , Citotoxinas/imunologia , Humanos , Imunoglobulina A/imunologia , Pessoa de Meia-Idade , Peso MolecularRESUMO
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.
RESUMO
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.
Assuntos
Toxina Pertussis , Vacina contra Coqueluche/imunologia , Fatores de Virulência de Bordetella/imunologia , Animais , Bordetella pertussis , Humanos , Camundongos , Vacina contra Coqueluche/efeitos adversos , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/uso terapêutico , Fatores de Virulência de Bordetella/uso terapêutico , Coqueluche/prevenção & controleRESUMO
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.
Assuntos
Adesinas Bacterianas , Epitopos/imunologia , Hemaglutininas/imunologia , Ativação Linfocitária , Linfócitos T/imunologia , Fatores de Virulência de Bordetella , Sequência de Aminoácidos , Reações Cruzadas/imunologia , Humanos , Dados de Sequência Molecular , Oligopeptídeos/imunologia , Fragmentos de Peptídeos/imunologia , Proteínas Recombinantes/imunologiaRESUMO
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.
Assuntos
Toxina Pertussis , Fatores de Virulência de Bordetella/genética , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Bordetella pertussis/genética , DNA Bacteriano , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Espectrometria de Massas de Bombardeamento Rápido de ÁtomosRESUMO
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.