RESUMEN
There are two alleles of the vacuolating cytotoxin gene from Helicobacter pylori, which code for toxins with different cell specificities. By analyzing the phenotypes of natural and artificial chimeras between the two forms of the protein, we have delimited a short stretch of amino acids which determine the cell specificity.
Asunto(s)
Proteínas Bacterianas/toxicidad , Toxinas Bacterianas/toxicidad , Citotoxinas/toxicidad , Helicobacter pylori/patogenicidad , Polimorfismo Genético , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Citotoxinas/genética , Células Epiteliales , Células HeLa , Helicobacter pylori/genética , Humanos , Riñón/citología , Datos de Secuencia Molecular , Conejos , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , VacuolasRESUMEN
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.
Asunto(s)
Fármacos Anti-VIH/sangre , Cromatografía Líquida de Alta Presión/métodos , Oxazinas/sangre , Inhibidores de la Transcriptasa Inversa/sangre , Administración Oral , Alquinos , Benzoxazinas , Ciclopropanos , Humanos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Factores de Tiempo , Rayos UltravioletaRESUMEN
Helicobacter pylori has been widely recognized as an important human pathogen responsible for chronic gastritis, peptic ulcers, gastric cancer, and mucosa-associated lymphoid tissue (MALT) lymphoma. Little is known about the natural history of this infection since patients are usually recognized as having the infection only after years or decades of chronic disease. Several animal models of H. pylori infection, including those with different species of rodents, nonhuman primates, and germ-free animals, have been developed. Here we describe a new animal model in which the clinical, pathological, microbiological, and immunological aspects of human acute and chronic infection are mimicked and which allows us to monitor these aspects of infection within the same individuals. Conventional Beagle dogs were infected orally with a mouse-adapted strain of H. pylori and monitored for up to 24 weeks. Acute infection caused vomiting and diarrhea. The acute phase was followed by polymorphonuclear cell infiltration, interleukin 8 induction, mononuclear cell recruitment, and the appearance of a specific antibody response against H. pylori. The chronic phase was characterized by gastritis, epithelial alterations, superficial erosions, and the appearance of the typical macroscopic follicles that in humans are considered possible precursors of MALT lymphoma. In conclusion, infection in this model mimics closely human infection and allows us to study those phases that cannot be studied in humans. This new model can be a unique tool for learning more about the disease and for developing strategies for treatment and prevention.
Asunto(s)
Modelos Animales de Enfermedad , Perros , Infecciones por Helicobacter , Helicobacter pylori , Enfermedad Aguda , Animales , Anticuerpos Antibacterianos/inmunología , Enfermedad Crónica , Endoscopía Gastrointestinal , Femenino , Helicobacter pylori/inmunología , Helicobacter pylori/aislamiento & purificación , Interleucina-8/metabolismo , Masculino , RatonesRESUMEN
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.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Monitoreo de Drogas , VIH-1 , Saquinavir/farmacocinética , Síndrome de Inmunodeficiencia Adquirida/metabolismo , Adolescente , Adulto , Anciano , Humanos , Persona de Mediana EdadRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/toxicidad , Toxinas Bacterianas/toxicidad , Helicobacter pylori/patogenicidad , Vacuolas , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Citosol , Células HeLa/efectos de los fármacos , Células HeLa/patología , Humanos , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/toxicidad , Proteínas Recombinantes/toxicidad , Eliminación de SecuenciaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Citotoxinas/genética , Helicobacter pylori/inmunología , Eliminación de Secuencia/genética , Proteínas Bacterianas/química , Toxinas Bacterianas/química , Sitios de Unión/genética , Sitios de Unión/inmunología , Citotoxinas/química , Células HeLa , Humanos , Mutagénesis Sitio-Dirigida , Eliminación de Secuencia/inmunologíaRESUMEN
BACKGROUND/AIMS: Infection with Helicobacter pylori strains harbouring the cagA gene (cagA+) is associated with an increased risk of developing peptic ulcer and gastric cancer. The aim of this study was to assess whether H pylori isolates with different cagA status were present in patients with non-ulcer dyspepsia, and whether a variable cagA status is relevant to histological gastric mucosal damage and glandular cell proliferation. METHODS: Well separated H pylori colonies (between 2 and 25) from primary plates, per gastric area, for each of 19 patients with non-ulcer dyspepsia were examined for cagA by hybridisation. Western blotting was used to examine both representative colonies for CagA expression and the patients' sera for antibody response to CagA. Glandular gastric cell proliferation was assessed immunohistochemically. RESULTS: Of the 747 colonies examined, 45.3% were cagA+. All colonies from four patients were cagA+, and all colonies from two patients were cagA-. In 13 patients (68%) both cagA+ and cagA- colonies were found. CagA expression of isolates corresponded to their cagA status. H pylori strains with different CagA molecular masses were present in three patients. Results based on all 19 patients studied showed that the prevalence of cagA+ colonies in areas with mucosal atrophy associated or not with intestinal metaplasia (67.9%) was significantly higher than in normal mucosa (44.7%) and mucosa from patients with chronic gastritis (44.0%) (p < 0.001). High levels of cell proliferation were associated with histological atrophy with or without intestinal metaplasia, but not with the possession of cagA by organisms colonising the same mucosal sites. CONCLUSIONS: Most patients with nonulcer dyspepsia are infected by both cagA+ and cagA- H pylori colonies. The cagA status of infecting organisms may play a role in the development of atrophy and intestinal metaplasia.
Asunto(s)
Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Dispepsia/microbiología , Helicobacter pylori/genética , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Antibacterianos/sangre , Western Blotting , División Celular , Dispepsia/inmunología , Dispepsia/patología , Mucosa Gástrica/patología , Helicobacter pylori/inmunología , Humanos , Inmunohistoquímica , Hibridación in Situ , Persona de Mediana EdadRESUMEN
The Helicobacter pylori toxin VacA causes vacuolar degeneration in mammalian cell lines in vitro and plays a key role in peptic ulcer disease. Two alleles, m1 and m2, of the mid-region of the vacA gene have been described, and the m2 cytotoxin always has been described as inactive in the in vitro HeLa cell assay. However, the m2 allele is associated with peptic ulcer and is prevalent in populations in which peptic ulcer and gastric cancer have high incidence. In this paper, we show that, despite the absence of toxicity on HeLa cells, the m2 cytotoxin is able to induce vacuolization in primary gastric cells and in other cell lines such as RK-13. The absence of Hela cell activity is due to an inability to interact with the cell surface, suggesting a receptor-mediated interaction. This result is consistent with the observation that the m2 allele is found in a population that has a high prevalence of peptic ulcer disease and gastric cancer. VacA is the first bacterial toxin described for which the same active subunit can be delivered by different receptor binding domains.
Asunto(s)
Proteínas Bacterianas/toxicidad , Toxinas Bacterianas/toxicidad , Citotoxinas/toxicidad , Helicobacter pylori/patogenicidad , Alelos , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Secuencia de Bases , Citotoxinas/genética , Citotoxinas/fisiología , Cartilla de ADN/genética , ADN Bacteriano/genética , Genes Bacterianos , Células HeLa , Helicobacter pylori/genética , Helicobacter pylori/fisiología , Humanos , Úlcera Péptica/etiología , Úlcera Péptica/microbiología , Reacción en Cadena de la Polimerasa , Neoplasias Gástricas/etiología , Neoplasias Gástricas/microbiología , Transfección , Vacuolas/efectos de los fármacos , VirulenciaRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/aislamiento & purificación , Helicobacter pylori/química , Animales , Anticuerpos Antibacterianos/biosíntesis , Anticuerpos Antibacterianos/aislamiento & purificación , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/aislamiento & purificación , Cromatografía de Afinidad , Ensayo de Inmunoadsorción Enzimática , Células HeLa/metabolismo , Helicobacter pylori/inmunología , Humanos , Immunoblotting , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica , Microscopía FluorescenteRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Citotoxinas/metabolismo , Helicobacter pylori/metabolismo , Vacuolas/metabolismo , Células 3T3 , Animales , Unión Competitiva , Línea Celular , Células HeLa , Humanos , Células Jurkat , Ratones , Receptores de Superficie Celular/metabolismoRESUMEN
Helicobacter pylori (Hp) infection almost invariably results in chronic antral gastritis, but only a proportion of patients develop peptic ulcer. Some Hp strains may be more ulcerogenic than others, but some ulcerogenic mechanisms may also depend on the type of the host immune response. In this study, the antigen specificity and the cytokine profile of 53 Hp-specific CD4+ T cell clones derived from the antral mucosa of five patients with Hp-induced uncomplicated chronic gastritis (CG) were assessed and compared with those of 34 Hp-specific CD4+ T cell clones derived from six Hp-infected patients with chronic gastritis and peptic ulcer (CG-PU). The majority (28/34; 82%) of gastric Hp-specific T cell clones from CG-PU patients expressed the Th1 profile and 17 (all Th1) of the 34 clones were specific for cytotoxin-associated protein (CagA). In contrast, 34 (64%) of the 53 Hp-specific gastric T cell clones derived from CG patients were able to secrete both Th1 and Th2 cytokines (Th0 profile) and only 36% expressed a polarized Th1 profile. The majority (85%) of Hp-specific clones from CG patients recognized Hp antigens other than CagA, since 13/53 (25%) were specific for urease, 6 (11%) for VacA, 6 (11%) for HSP and 20 (38%) for other undefined Hp antigens. Results provide evidence that the type of T helper cell response against Hp may vary according to the antigen involved and suggest that a polarized Th1 response may play a role in the genesis of peptic ulcer, whereas a local Th0 response, including interleukin-4 production, may represent an individual host factor which contributes to lower the degree of gastric inflammation and prevent ulcer complication.
Asunto(s)
Antígenos Bacterianos , Citocinas/análisis , Epítopos/análisis , Gastritis/inmunología , Helicobacter pylori/inmunología , Úlcera Péptica/inmunología , Antro Pilórico/inmunología , Linfocitos T/inmunología , Adulto , Proteínas Bacterianas/análisis , Chaperonina 60/análisis , Enfermedad Crónica , Células Clonales , Femenino , Gastritis/microbiología , Humanos , Masculino , Persona de Mediana Edad , Úlcera Péptica/microbiología , Antro Pilórico/microbiología , Linfocitos T/microbiologíaRESUMEN
Chronic antral gastritis following Helicobacter pylori (Hp) infection is characterized by a cellular inflammatory infiltrate whose cytokines may represent a host-dependent factor influencing the outcome of the infection. The pattern of cytokines produced by the immunologically active cells in the gastric antrum was analyzed at the mRNA level in antral biopsies from five Hp-infected patients with duodenal ulcer and three Hp-negative dyspeptic controls. T cell clones were generated from parallel antral biopsies of the same Hp-infected patients and assessed for reactivity to Hp Ags, cytokine profile, and effector functions. Antral biopsies from all Hp-infected patients showed IFN-gamma, TNF-alpha, and IL-12, but not IL-4, mRNA expression, whereas no cytokine mRNA signal was found in the mucosa of controls. A total of 24 out of the 163 CD4+ T cell clones (15%) derived from Hp-infected patients proliferated in response to a Hp lysate; 11 clones (46%) also reacted with Cag-A, 2 with Vac-A, and 1 with urease. Upon Ag stimulation, 20 out of the 24 Hp-reactive clones (83%) produced IFN-gamma, but not IL-4 or IL-5 (Th1-like), whereas 4 produced IFN-gamma, IL-4, and IL-5 (Th0-like). All Hp-specific clones secreted high levels of TNF-alpha. At low T:B cell ratio, Hp-specific clones expressed Ag-dependent helper function for B cell proliferation and Ig production, whereas at higher T:B cell ratios, 15 Th1 and 2 Th0 clones lysed Ag-pulsed autologous EBV-transformed B cells. Results provide evidence for Hp-specific Th1 effectors in the gastric antrum of Hp-infected patients, where they may play a role in the genesis of either peptic ulcer or Hp-associated gastric B cell lymphoma.
Asunto(s)
Helicobacter pylori/inmunología , Úlcera Péptica/inmunología , Antro Pilórico/inmunología , Células TH1/inmunología , Adulto , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , ADN Bacteriano/genética , ADN Bacteriano/inmunología , Helicobacter pylori/genética , Helicobacter pylori/inmunología , Animales , Toxinas Bacterianas/genética , Toxinas Bacterianas/inmunología , Citotoxinas/genética , Citotoxinas/inmunología , Hibridomas , Inmunización , Ratones , Ratones Endogámicos BALB C , Plásmidos/genéticaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Citotoxinas/metabolismo , Helicobacter pylori/química , Liposomas/metabolismo , Fragmentos de Péptidos/metabolismo , Proteínas Bacterianas/química , Toxinas Bacterianas/química , Citotoxinas/química , Escherichia coli/genética , Fluorescencia , Gramicidina/farmacología , Concentración de Iones de Hidrógeno , Fusión de Membrana , Peso Molecular , Fotoquímica , Potasio/metabolismo , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Dispersión de Radiación , TriptófanoRESUMEN
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.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Proteínas Bacterianas/química , Toxinas Bacterianas/química , Citotoxinas/química , Helicobacter pylori/inmunología , Animales , Reacciones Antígeno-Anticuerpo , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/toxicidad , Toxinas Bacterianas/inmunología , Toxinas Bacterianas/toxicidad , Citotoxinas/inmunología , Citotoxinas/toxicidad , Escherichia coli , Células HeLa , Helicobacter pylori/química , Humanos , Conformación Proteica , Conejos , Proteínas Recombinantes/inmunologíaRESUMEN
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.
Asunto(s)
Modelos Animales de Enfermedad , Gastritis/microbiología , Infecciones por Helicobacter , Helicobacter pylori , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Toxinas Bacterianas/inmunología , Vacunas Bacterianas , Mucosa Gástrica/microbiología , Mucosa Gástrica/patología , Gastritis/inmunología , Gastritis/patología , Gastritis/prevención & control , Infecciones por Helicobacter/inmunología , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/patología , Infecciones por Helicobacter/prevención & control , Helicobacter pylori/enzimología , Helicobacter pylori/crecimiento & desarrollo , Helicobacter pylori/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Organismos Libres de Patógenos Específicos , Ureasa/inmunologíaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Citotoxinas/genética , Infecciones por Helicobacter/microbiología , Helicobacter pylori/genética , Gastropatías/microbiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Bacteriano , Modelos Animales de Enfermedad , Helicobacter pylori/patogenicidad , Humanos , Immunoblotting , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Úlcera Gástrica/microbiologíaRESUMEN
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.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Gastritis/inmunología , Proteínas de Choque Térmico/genética , Infecciones por Helicobacter/inmunología , Helicobacter pylori/genética , Úlcera Péptica/inmunología , Secuencia de Aminoácidos , Secuencia de Bases , Chaperonina 60 , Clonación Molecular , Gastritis/microbiología , Genes Bacterianos/genética , Humanos , Datos de Secuencia Molecular , Úlcera Péptica/microbiología , Proteínas Recombinantes/biosíntesis , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
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.
Asunto(s)
Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Úlcera Duodenal/microbiología , Genes Bacterianos , Helicobacter pylori/genética , Secuencia de Aminoácidos , Antígenos Bacterianos/biosíntesis , Proteínas Bacterianas/biosíntesis , Secuencia de Bases , Donantes de Sangre , Southern Blotting , Cromosomas Bacterianos , Clonación Molecular , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Biblioteca Genómica , Helicobacter pylori/aislamiento & purificación , Helicobacter pylori/patogenicidad , Humanos , Mucosa Intestinal/microbiología , Datos de Secuencia Molecular , Peso Molecular , Oligodesoxirribonucleótidos , Plásmidos , Valores de Referencia , Mapeo Restrictivo , Úlcera Gástrica/microbiología , Virulencia/genéticaRESUMEN
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.