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1.
Transplant Proc ; 48(2): 395-8, 2016 Mar.
Article in English | MEDLINE | ID: mdl-27109964

ABSTRACT

BACKGROUND: Recent studies have challenged the dogma that the adult heart is a postmitotic organ and raise the possibility of the existence of resident cardiac stem cells (CSCs). Our study aimed to explore if these CSCs are present in the "ventricular tip" obtained during left ventricular assist device (LVAD) implantation from patients with end-stage heart failure (HF) and the relationship with LV dysfunctional area extent. METHODS: Four consecutive patients with ischemic cardiomyopathy and end-stage HF submitted to LVAD implantation were studied. The explanted "ventricular tip" was used as a sample of apical myocardial tissue for the pathological examination. Patients underwent clinical and echocardiographic examination, both standard transthoracic echocardiography (TTE) and speckle tracking echocardiography (STE), before LVAD implantation. RESULTS: All patients presented severe apical dysfunction, with apical akinesis/diskinesis and very low levels of apical longitudinal strain (-3.5 ± 2.9%). Despite this, the presence of CSCs was demonstrated in pathological myocardial samples of "ventricular tip" in all 4 of the patients. It was found to be a mean of 6 c-kit cells in 10 fields magnification 40×. CONCLUSIONS: Cardiac stem cells can be identified in the LV apical segment of patients who have undergone LVAD implantation despite LV apical fibrosis.


Subject(s)
Heart Failure/therapy , Heart Ventricles/cytology , Heart-Assist Devices , Myocardial Ischemia/therapy , Myocardium/cytology , Stem Cells/cytology , Biopsy , Cardiac Surgical Procedures , Echocardiography , Fibrosis , Heart Failure/diagnostic imaging , Heart Failure/pathology , Heart Ventricles/diagnostic imaging , Heart Ventricles/surgery , Humans , Myocardial Ischemia/diagnostic imaging , Myocardial Ischemia/pathology , Myocardium/pathology , Prosthesis Implantation
3.
Protein Expr Purif ; 45(1): 142-9, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16146695

ABSTRACT

The IgA1 protease of Streptococcus pneumoniae is a Zn-metalloproteinase of 1964 amino acids that specifically cleaves the hinge region of IgA1, the predominant class of immunoglobulin present on mucosal membranes. This protease is associated to the bacterial cell surface via an N-terminal membrane anchor. Following proteolysis it is released in several forms of different molecular weight. Here, we describe the cloning, expression, and characterization of the enzymatic activity and immunogenicity of three fragments of IgA1 protease, including a large one lacking only the 103 N-terminal amino acids that constitute a typical prokaryotic signal sequence. Further, a proteolytically inactive mutant was generated by replacement of the glutamate residue with an alanine residue in the active site motif HExxH (1605-1609). This is the first report of recombinant active forms of S. pneumoniae IgA1 protease, which open the possibility of identifying specific inhibitors that could interfere with the mucosal colonization by pneumococcus. Moreover the inactive mutant could be considered as a candidate vaccine component.


Subject(s)
Gene Expression Regulation, Enzymologic , Pneumonia, Pneumococcal/genetics , Serine Endopeptidases , Streptococcus pneumoniae/enzymology , Cloning, Molecular , Humans , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Serine Endopeptidases/genetics , Serine Endopeptidases/isolation & purification , Serine Endopeptidases/metabolism
4.
Curr Opin Microbiol ; 4(1): 41-6, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11173032

ABSTRACT

Contact-dependent activation of the cag organelle, a type IV secretion system of Helicobacter pylori, promotes translocation of CagA into the host cell. CagA is an immunodominant antigen of H. pylori, encoded by cag. It is thought to be associated with severe clinical outcomes, but has an unclear role in pathogenesis. Now we know that CagA is injected into the host and is tyrosine-phosphorylated by a membrane-associated eukaryotic tyrosine kinase. After activation, CagA induces morphological changes in the host, as well as actin reorganization, variations in the cell cycle and autocrine effects. Subversion of cell control may ultimately lead to cellular damage and to increased risks for gastric cancer development. cag instability contributes to long-term persistence within the host by attenuating bacterial virulence. We still do not know if additional factors are co-translocated with CagA and we do not know their specific mechanisms of action, but there is a strong experimental evidence that indicates that cag is the major player in the host-pathogen relationship.


Subject(s)
Helicobacter pylori/pathogenicity , Antigens, Bacterial/genetics , Antigens, Bacterial/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Helicobacter pylori/genetics , Helicobacter pylori/metabolism , Humans , Virulence
5.
Dig Liver Dis ; 32(6): 458-67, 2000.
Article in English | MEDLINE | ID: mdl-11057919

ABSTRACT

BACKGROUND: In vitro studies showed that Helicobacter pylori strains carrying the cag pathogenicity island are able to induce epithelial secretion of Interleukin-8. AIMS: To evaluate the assessment of cag pathogenicity island and the expression of Interleukin-8 in the gastric mucosa of Helicobacter pylori-infected patients and correlate these data with the activity of gastritis and Helicobacter pylori density. METHODS: cag status was determined by polymerase chain reaction directly on gastric biopsies from 13 Helicobacter pylori+ patients with non-ulcer dyspepsia and 13 Helicobacter pylori+ with duodenal ulcer. Interleukin-8 gene transcription and protein expression were analysed by in situ hybridization and immunofluorescence, respectively. Gastritis activity and Helicobacter pylori density were also investigated. RESULTS: cag was present in 20/26 of Helicobacter pylori+ patients: in 7/13 non-ulcer dyspepsia (53.8%] and in 13/13 duodenal ulcer patients (100%), (p<0.05). Interleukin-8 mRNA and protein expression in epithelial and inflammatory cells was higher in cag+ than in cag- patients (p<0.005). Gastritis activity significantly correlated with cag (p<0.05) and Interleukin-8 expression (p<0.005]. Helicobacter pylori density was enhanced in cag+ [p<0.005] and correlated with Interleukin-8 expression (p<0.0051. CONCLUSIONS: The present study demonstrates that in Helicobacter pylori-infected human gastric mucosa, cag+ infection is associated with enhanced Interleukin-8 expression, higher levels of active gastritis and bacterial density, and presence of duodenal ulcer.


Subject(s)
Antigens, Bacterial , Bacterial Proteins/genetics , Duodenal Ulcer/microbiology , Helicobacter Infections/microbiology , Helicobacter pylori/genetics , Helicobacter pylori/pathogenicity , Interleukin-8/biosynthesis , Bacterial Proteins/biosynthesis , Gastric Mucosa/microbiology , Gastritis/microbiology , Gene Expression Regulation , Humans , In Situ Hybridization , Polymerase Chain Reaction
6.
J Clin Microbiol ; 38(2): 607-12, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10655354

ABSTRACT

Enterotoxigenic Bacteroides fragilis (ETBF) strains are associated with diarrheal disease in children. These strains produce a zinc metalloprotease enterotoxin, or fragilysin, that can be detected by a cytotoxicity assay with HT-29 cells. Recently, three different isoforms or variants of the enterotoxin gene, designated bft-1, bft-2, and bft-3, have been identified and sequenced. We used restriction fragment length polymorphism analysis of the PCR-amplified enterotoxin gene to detect the isoforms bft-1 and bft-2 or bft-3 borne by ETBF. By sequencing the portion of the bft gene corresponding to the mature toxin in some strains and applying allele-specific PCR for strains categorized as bft-2 or bft-3, we found in our collection two strains harboring bft-3, a variant that had been described for isolates from East Asia. Analysis of 66 ETBF strains from different sources showed that bft-1 is the most frequent allele, being present in 65% of isolates; it is largely predominant in isolates from feces of adults, while bft-2 is present in isolates from feces of children. This association is statistically significant (P, 0.0064). Sixteen strains were examined by Southern hybridization using, as probes, the bft and second metalloprotease genes, both included in a pathogenicity islet. Five strains were found to harbor double copies of both genes, suggesting that the whole islet was duplicated. Four of these strains, harboring bft-1 (three strains) or bft-2 (one strain), were found to produce a large amount of biologically active toxin, as determined by a cytotoxicity assay with HT-29 cells. The strains harboring bft-3, either in a single copy or in double copies, produced the smallest amount of toxin in our collection.


Subject(s)
Alleles , Bacteroides Infections/microbiology , Bacteroides fragilis/genetics , Bacteroides fragilis/isolation & purification , Enterotoxins/genetics , Metalloendopeptidases/genetics , Adult , Animals , Cell Line , Cell Survival/drug effects , Child , Diarrhea/microbiology , Enterotoxins/biosynthesis , Enterotoxins/toxicity , Feces/microbiology , Gene Dosage , Genes, Duplicate , Humans , Metalloendopeptidases/biosynthesis , Metalloendopeptidases/toxicity , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Protein Isoforms , Sequence Analysis, DNA
7.
Folia Microbiol (Praha) ; 43(3): 275-8, 1998.
Article in English | MEDLINE | ID: mdl-9717254

ABSTRACT

In Helicobacter pylori, a pathogenicity island (PAI) of approximately 40 kb, named cag, is present in a subset of strains. The strains containing the PAI are more virulent than those that do not contain it, and are associated with peptic ulcer and gastric cancer. A putative secretory mechanism is encoded by this PAI. This secretory system is thought to be involved in the induction of the proiflammatory lymphokine IL-8 and tyrosine phosphorylation of proteins in the gastric cells. We are currently investigating the potential toxic factors exported by this region.


Subject(s)
Gastric Mucosa/cytology , Gastric Mucosa/microbiology , Helicobacter Infections/microbiology , Helicobacter pylori/pathogenicity , Humans , Virulence
8.
Am J Med Genet ; 78(4): 341-4, 1998 Jul 24.
Article in English | MEDLINE | ID: mdl-9714436

ABSTRACT

The main mutation in fragile X patients is the expansion of the CGG repeat in the first exon of the FMR1 gene, associated with hypermethylation of the proximal CpG island. An increasing number of atypical cases have been reported showing the coexistence of full mutation and premutated or normal-sized alleles. These genotypes are more difficult to detect, and if a PCR strategy alone is adopted, they can be incorrectly identified. We report on a fragile X man with severe phenotype and mosaicism for full mutation and a (CGG)7 normal allele, the shortest fragment reported as yet in mosaics. This case of mosaicism, as other similar cases previously reported, suggests that the normal-length allele can derive from a deletion during the same early stage of development in which the full mutation expansion also arose.


Subject(s)
Fragile X Syndrome/genetics , Mosaicism , Mutation , Nerve Tissue Proteins/genetics , RNA-Binding Proteins , Adult , Alleles , Blotting, Southern , CpG Islands/genetics , DNA Methylation , Exons/genetics , Fragile X Mental Retardation Protein , Genetic Testing , Humans , Male , Polymerase Chain Reaction , Sequence Deletion , Trinucleotide Repeat Expansion/genetics , Trinucleotide Repeats/genetics
9.
Vaccine ; 16(1): 33-7, 1998 Jan.
Article in English | MEDLINE | ID: mdl-9607006

ABSTRACT

We have previously shown that infection of mice with H. pylori can be prevented by oral immunization with H. pylori antigens given together with E. coli heat-labile enterotoxin (LT) as adjuvant. Since LT cannot be used in humans because of its unacceptable toxicity, we investigated whether protection of mice could be achieved by co-administration of antigens with non-toxic LT mutants. Here we show that CD1/SPF mice are protected against infection after oral vaccination with either purified H. pylori antigens (native and recombinant VacA, urease and CagA), or whole-cell vaccine formulations, given together with the non-toxic mutant LTK63 as a mucosal adjuvant. Furthermore we show that such protection is antigen-specific since immunization with recombinant or native VacA plus LTK63 conferred protection against infection by an H. pylori Type I strain, which expresses VacA, but not against challenge with a Type II strain which is not able to express this antigen. These results show that: (1) protection against H. pylori can be achieved in the mouse model of infection using subunit recombinant constructs plus non-toxic mucosal adjuvants; and (2) this mouse model is an useful tool in testing H. pylori vaccine formulations for eventual use in humans.


Subject(s)
Adjuvants, Immunologic , Antigens, Bacterial/immunology , Bacterial Toxins , Bacterial Vaccines/therapeutic use , Enterotoxins , Escherichia coli Proteins , Helicobacter Infections/prevention & control , Helicobacter pylori/immunology , Animals , Bacterial Vaccines/immunology , Male , Mice , Vaccines, Synthetic/immunology , Vaccines, Synthetic/therapeutic use
10.
Proc Natl Acad Sci U S A ; 93(25): 14648-53, 1996 Dec 10.
Article in English | MEDLINE | ID: mdl-8962108

ABSTRACT

cagA, a gene that codes for an immunodominant antigen, is present only in Helicobacter pylori strains that are associated with severe forms of gastroduodenal disease (type I strains). We found that the genetic locus that contains cagA (cag) is part of a 40-kb DNA insertion that likely was acquired horizontally and integrated into the chromosomal glutamate racemase gene. This pathogenicity island is flanked by direct repeats of 31 bp. In some strains, cag is split into a right segment (cagI) and a left segment (cagII) by a novel insertion sequence (IS605). In a minority of H. pylori strains, cagI and cagII are separated by an intervening chromosomal sequence. Nucleotide sequencing of the 23,508 base pairs that form the cagI region and the extreme 3' end of the cagII region reveals the presence of 19 ORFs that code for proteins predicted to be mostly membrane associated with one gene (cagE), which is similar to the toxin-secretion gene of Bordetella pertussis, ptlC, and the transport systems required for plasmid transfer, including the virB4 gene of Agrobacterium tumefaciens. Transposon inactivation of several of the cagI genes abolishes induction of IL-8 expression in gastric epithelial cell lines. Thus, we believe the cag region may encode a novel H. pylori secretion system for the export of virulence determinants.


Subject(s)
Antigens, Bacterial/genetics , Bacterial Proteins/genetics , Genes, Bacterial , Helicobacter pylori/genetics , Base Sequence , Chromosome Mapping , Evolution, Molecular , Helicobacter pylori/pathogenicity , Molecular Sequence Data , Sequence Analysis , Virulence/genetics
11.
Clin Infect Dis ; 23(1): 101-6, 1996 Jul.
Article in English | MEDLINE | ID: mdl-8816137

ABSTRACT

A cytotoxin inducing vacuolation in HEp-2 cells was detected in 19 (3.1%) of 618 stool specimens from children with diarrhea but in none of 135 from control children. Common enteric pathogens were found in only two (10.5%) of the 19 cytotoxin-positive stool specimens. The vacuoles induced by stool filtrates resembled those induced by the vacuolating toxin (VacA) of Helicobacter pylori. The vacuolating toxin was heat-labile and protease-sensitive, and it had an apparent molecular weight of > 100,000 but was not neutralized by an antiserum to H. pylori VacA. Although proper prospective case-control studies are needed to definitely assess the etiologic association between the new vacuolating cytotoxin and diarrhea, the present study suggests that microorganisms of the gastrointestinal tract produce a Helicobacter-like vacuolating toxin and may be responsible for cases of childhood diarrhea whose etiology is currently considered unknown.


Subject(s)
Bacterial Proteins/isolation & purification , Bacterial Toxins/isolation & purification , Cytotoxins/isolation & purification , Diarrhea/microbiology , Feces/chemistry , Feces/microbiology , Cell Line , Child , Child, Preschool , Diarrhea/epidemiology , Diarrhea/etiology , Female , Helicobacter Infections/epidemiology , Helicobacter Infections/etiology , Helicobacter Infections/microbiology , Helicobacter pylori/pathogenicity , Humans , Italy/epidemiology , Male , Vacuoles/microbiology , Vacuoles/pathology , Virulence
12.
Infect Immun ; 63(1): 94-8, 1995 Jan.
Article in English | MEDLINE | ID: mdl-7806390

ABSTRACT

Colonization of the mucosa of the stomach and the duodenum by Helicobacter pylori is the major cause of acute and chronic gastroduodenal pathologies in humans. Duodenal ulcer formation strongly correlates with the expression of an antigen (CagA) that is usually coeexpressed with the vacuolating cytotoxin (VacA), a protein that causes ulceration in the stomach of mice. However, the relationship between these two virulence factors is unknown. To define whether CagA and VacA are coexpressed in all clinical isolates and their relationships, we collected 43 clinical isolates of H. pylori and studied their genetic and phenotypic properties. Based on this analysis, most of the strains could be classified into two major types. Type I bacteria had the gene coding for CagA and expressed the CagA protein and the vacuolating cytotoxin. Type II bacteria did not have the gene coding for CagA and did not express either the CagA protein or the vacuolating cytotoxin. Type I and type II bacteria represented 56 and 16%, respectively, of the 43 clinical isolates, while the remaining 28% had an intermediate phenotype, expressing CagA independently of VacA or vice versa. This finding shows that although it is present in most cytotoxic strains, CagA is not necessary for the expression of the vacuolating cytotoxin.


Subject(s)
Antigens, Bacterial , Bacterial Proteins/biosynthesis , Helicobacter Infections/microbiology , Helicobacter pylori/classification , Helicobacter pylori/pathogenicity , Bacterial Proteins/genetics , Bacterial Toxins/biosynthesis , Bacterial Toxins/genetics , Base Sequence , Blotting, Southern , Cytotoxins/biosynthesis , Cytotoxins/genetics , Genes, Bacterial , Helicobacter pylori/genetics , Helicobacter pylori/isolation & purification , Humans , Molecular Sequence Data , Plasmids
13.
J Exp Med ; 179(5): 1653-58, 1994 May 01.
Article in English | MEDLINE | ID: mdl-8163943

ABSTRACT

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.


Subject(s)
Bacterial Proteins/genetics , Bacterial Toxins/genetics , Cytotoxins/genetics , Helicobacter Infections/microbiology , Helicobacter pylori/genetics , Stomach Diseases/microbiology , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA, Bacterial , Disease Models, Animal , Helicobacter pylori/pathogenicity , Humans , Immunoblotting , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Peptide Fragments/genetics , Stomach Ulcer/microbiology
14.
Eur J Clin Microbiol Infect Dis ; 12(10): 739-45, 1993 Oct.
Article in English | MEDLINE | ID: mdl-8307041

ABSTRACT

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.


Subject(s)
Antigens, Bacterial/immunology , Bacterial Proteins/immunology , Helicobacter Infections/diagnosis , Helicobacter pylori/immunology , Adult , Aged , Aged, 80 and over , Antibodies, Bacterial/blood , Antigens, Bacterial/biosynthesis , Bacterial Proteins/biosynthesis , Blotting, Western , Humans , Immunoenzyme Techniques , Immunoglobulin G/blood , Middle Aged , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/immunology
15.
Mol Microbiol ; 9(3): 645-52, 1993 Aug.
Article in English | MEDLINE | ID: mdl-8105364

ABSTRACT

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.


Subject(s)
Antibodies, Bacterial/blood , Gastritis/immunology , Heat-Shock Proteins/genetics , Helicobacter Infections/immunology , Helicobacter pylori/genetics , Peptic Ulcer/immunology , Amino Acid Sequence , Base Sequence , Chaperonin 60 , Cloning, Molecular , Gastritis/microbiology , Genes, Bacterial/genetics , Humans , Molecular Sequence Data , Peptic Ulcer/microbiology , Recombinant Proteins/biosynthesis , Sequence Analysis, DNA , Sequence Homology, Amino Acid
16.
J Exp Med ; 178(1): 17-25, 1993 Jul 01.
Article in English | MEDLINE | ID: mdl-8100267

ABSTRACT

The adult liver is an organ without constitutive lymphoid components. Therefore, any intrahepatic T cell found in chronic hepatitis should have migrated to the liver after infection and inflammation. Because of the little information available on the differences between intrahepatic and peripheral T cells, we used recombinant proteins of the hepatitis C virus (HCV) to establish specific T cell lines and clones from liver biopsies of patients with chronic hepatitis C and compared them with those present in peripheral blood mononuclear cells (PBMC). We found that the protein nonstructural 4 (NS4) was able to stimulate CD4+ T cells isolated from liver biopsies, whereas with all the other HCV proteins we consistently failed to establish liver-derived T cell lines from 16 biopsies. We then compared NS4-specific T cell clones obtained on the same day from PBMC and liver of the same patient. We found that the 22 PBMC-derived T cell clones represent, at least, six distinct clonal populations that differ in major histocompatibility complex restriction and response to superantigens, whereas the 27 liver-derived T cell clones appear all identical, as further confirmed by cloning and sequencing of the T cell receptor (TCR) variable and hypervariable regions. Remarkably, none of the PBMC-derived clones has a TCR identical to the liver-derived clone, and even with polymerase chain reaction oligotyping we did not find the liver-derived clonotypic TCR transcript in the PBMC, indicating a preferential intrahepatic localization of these T cells. Functionally, the liver-derived T cells provided help for polyclonal immunoglobulin (Ig)A production by B cells in vitro that is 10-fold more effective than that provided by the PBMC-derived clones, whereas there is no difference in the help provided for IgM and IgG production. Altogether these results demonstrate that the protein NS4 is highly immunogenic for intrahepatic CD4+ T cells primed by HCV in vivo, and that there can be compartmentalization of some NS4-specific CD4+ T cells to the liver of patients with chronic hepatitis C.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , Hepacivirus/immunology , Hepatitis C/immunology , Liver/immunology , T-Lymphocytes/physiology , Viral Nonstructural Proteins/immunology , Adult , Base Sequence , Cell Line , Chronic Disease , Female , Humans , Leukocytes, Mononuclear/immunology , Male , Middle Aged , Molecular Sequence Data
17.
Proc Natl Acad Sci U S A ; 90(12): 5791-5, 1993 Jun 15.
Article in English | MEDLINE | ID: mdl-8516329

ABSTRACT

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.


Subject(s)
Antigens, Bacterial/genetics , Bacterial Proteins/genetics , Duodenal Ulcer/microbiology , Genes, Bacterial , Helicobacter pylori/genetics , Amino Acid Sequence , Antigens, Bacterial/biosynthesis , Bacterial Proteins/biosynthesis , Base Sequence , Blood Donors , Blotting, Southern , Chromosomes, Bacterial , Cloning, Molecular , DNA, Bacterial/genetics , DNA, Bacterial/isolation & purification , Enzyme-Linked Immunosorbent Assay , Escherichia coli/genetics , Genomic Library , Helicobacter pylori/isolation & purification , Helicobacter pylori/pathogenicity , Humans , Intestinal Mucosa/microbiology , Molecular Sequence Data , Molecular Weight , Oligodeoxyribonucleotides , Plasmids , Reference Values , Restriction Mapping , Stomach Ulcer/microbiology , Virulence/genetics
18.
J Biol Chem ; 268(14): 10490-4, 1993 May 15.
Article in English | MEDLINE | ID: mdl-8387521

ABSTRACT

The type I interleukin-1 receptor (IL-1R) is capable of transducing a signal resulting in promoter activation in T cells. This signal transduction is dependent on the cytoplasmic domain, which consists of 213 amino acids. In contrast to the type I receptor, the type II IL-1R has a small cytoplasmic tail, and it is not clear whether this receptor is a signal-transducing or a regulatory molecule. Here we report that the type II IL-1R does not mediate gene activation in Jurkat cells. However, a hybrid receptor composed of the extracellular and transmembrane regions of the human type II interleukin-1 fused to the cytoplasmic domain of the human type I IL-1R was capable of transducing a signal across the membrane resulting in a pattern of gene activation identical to that mediated by the type I IL-1R. Our results indicated that the extracellular domain of the type II IL-1R was capable of functionally interacting with interleukin-1 and transmitting the resulting signal to a heterologous cytoplasmic domain.


Subject(s)
Gene Expression Regulation, Neoplastic/drug effects , Interleukin-1/pharmacology , Receptors, Interleukin-1/physiology , Recombinant Fusion Proteins/metabolism , Signal Transduction , Base Sequence , Dose-Response Relationship, Drug , Escherichia coli/genetics , Gene Expression/drug effects , Humans , Kinetics , Molecular Sequence Data , NF-kappa B/genetics , Oligodeoxyribonucleotides , Polymerase Chain Reaction/methods , Promoter Regions, Genetic/genetics , Receptors, Interleukin-1/genetics , Recombinant Proteins/pharmacology , Signal Transduction/drug effects , Simian virus 40/genetics , T-Lymphocytes , Transcriptional Activation , Tumor Cells, Cultured
20.
Proc Natl Acad Sci U S A ; 89(7): 2990-4, 1992 Apr 01.
Article in English | MEDLINE | ID: mdl-1313575

ABSTRACT

The interaction of the immunodominant pertussis toxin peptide containing residues 30-42 (p30-42) with soluble DR1 molecules and the T-cell receptor (TCR) of 12 DR1-restricted human T-cell clones has been analyzed. Peptide analogues of p30-42 containing single alanine substitutions were used in DR1-binding and T-cell proliferation assays to identify the major histocompatibility complex and TCR contact residues. Each T-cell clone was found to recognize p30-42 with a different fine specificity. However, a common core comprising amino acids 33-39 was found to be important for stimulation of all T-cell clones. Within this core two residues, Leu33 and Leu36, interact with the DR1 molecule, whereas Asp34, His35, Thr37, and Arg39 are important for TCR recognition in most of the clones. Computer modeling of the structure of p30-42 showed that an alpha-helical conformation is compatible with the experimental data. The analysis of TCR rearrangement revealed that the peptide was recognized by T-cell clones expressing different variable region alpha (V alpha) and variable region beta (V beta) chains, although a preferential use of V alpha 8-V beta 13 and V alpha 11-V beta 18 combinations was found in clones from the same donor. Understanding the details of the interaction of antigenic peptides with the major histocompatibility complex and TCR molecules should provide the theoretical basis to design T-cell epitopes and obtain more immunogenic vaccines.


Subject(s)
HLA-DR1 Antigen/immunology , Pertussis Toxin , Receptors, Antigen, T-Cell, alpha-beta/immunology , Receptors, Antigen, T-Cell/immunology , Virulence Factors, Bordetella/immunology , Amino Acid Sequence , Base Sequence , Binding Sites , Clone Cells , Gene Rearrangement, alpha-Chain T-Cell Antigen Receptor , Gene Rearrangement, beta-Chain T-Cell Antigen Receptor , Humans , In Vitro Techniques , Models, Molecular , Molecular Sequence Data , Oligodeoxyribonucleotides/chemistry , Peptides/chemistry , Peptides/immunology , Protein Binding , Receptors, Antigen, T-Cell, alpha-beta/genetics , Virulence Factors, Bordetella/chemistry
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