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Biomedical subjects

C Montecucco

Publications and source records attributed to C Montecucco.

At least 37 records · Page 2Linked to original sources
PubMed2001

Neutrophil-activating protein (HP-NAP) versus ferritin (Pfr): comparison of synthesis in Helicobacter pylori.

We recently reported that the neutrophil-activating protein (HP-NAP) of Helicobacter pylori is capable of binding iron in vitro. To more fully understand the relationship between iron and HP-NAP the synthesis of HP-NAP was compared to that of Pfr, another iron-binding protein of H. pylori. Synthesis of HP-NAP and Pfr in growing cultures of H. pylori was analysed under iron depletion and iron, copper, nickel and zinc overload. The synthesis of HP-NAP and Pfr in H. pylori was also analysed under conditions of varying pH and oxidative stress. In addition, recombinant HP-NAP and Pfr were produced in Escherichia coli to assess the contribution of the two proteins to increased survival of E. coli under heavy metal overload. Our data reveal that both HP-NAP and Pfr accumulate in the stationary phase of growth. HP-NAP synthesis is not regulated by iron depletion or overload or by the presence of copper, nickel or zinc in liquid medium and it does not confer resistance to these metals when produced in E. coli. Except for an increase in the synthesis of Pfr at pH 5.7 neither the pH or oxidative stress conditions investigated had an affect on the synthesis of either protein. An increase in Pfr synthesis was observed under iron overload and a decrease was observed under conditions of copper, nickel and zinc overload confirming previous reports. Recombinant Pfr, as well as conferring resistance to iron and copper as previously reported, also conferred resistance to zinc overload when produced in E. coli.

Bacterial Proteins↗
PubMed2001

Helicobacter pylori neutrophil-activating protein stimulates tissue factor and plasminogen activator inhibitor-2 production by human blood mononuclear cells.

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a virulence factor that activates phagocytic NADPH-oxidase. The effect of HP-NAP on the production of tissue factor (TF), plasminogen activator inhibitor-2 (PAI-2), and urokinase-type plasminogen activator (u-PA) by human blood mononuclear cells (MNC) was evaluated by using functional and immunological assays and mRNA analysis. HP-NAP induced time- and dose-dependent increases in TF and PAI-2, with a maximal effect at 300 nmol/L (>15-fold increase in antigens). No changes in u-PA were observed. When whole bacteria were used, an H. pylori mutant lacking HP-NAP was significantly less active than the wild-type strain. MNC from a patient with chronic granulomatous disease behaved as do normal cells, which indicates that HP-NAP effects can occur independently of NADPH-oxidase. HP-NAP, by inducing the coordinate expression of cell procoagulant and antifibrinolytic activities, might favor fibrin deposition and contribute to the inflammatory reaction of gastric mucosa elicited by H. pylori.

Bacterial Proteins↗
PubMed2001

How the loop and middle regions influence the properties of Helicobacter pylori VacA channels.

VacA is a pore-forming cytotoxin produced by Helicobacter pylori in several strain-specific isoforms, which have been classified in two main families, m1 and m2, according to the sequence of a variable "midregion." Both forms are associated with gastric pathologies and can induce vacuolation of cultured cells. The comparison of two representative toxins, m1 17874 and m2 9554, has indicated that the m2 form is less powerful in vacuolation assays and that its effects are more strongly cell type dependent. To rationalize these differences and to investigate structure-function relationships in this toxin, we have compared the properties of the channels formed by these two variants and by a construct derived from 17874 by deleting a loop that connects the two toxin domains, which is shorter in 9554 than in 17874. Although the channels formed by all three proteins are similar, m2 9554 channels have, on average, a lower conductance and are less anion-selective and more voltage-dependent than the m1 pores. Furthermore, the rate of incorporation of 9554 VacA into planar bilayers depends on lipid composition much more strongly than that of 17874. The comparison with the behavior of the loop deletion mutant indicates that this latter property, as well as a portion of the conductance decrease, may be attributed to the reduction in loop length. The differences in pore properties are proposed to account in part for the different cytotoxicity exhibited by the two toxin isoforms. We furthermore present evidence suggesting that the conformation of the membrane-embedded toxin may be influenced by the lipid composition of the membrane itself.

Amino Acid Sequence↗
PubMed2001

Tetanus and botulinum neurotoxins: turning bad guys into good by research.

The neuroparalytic syndromes of tetanus and botulism are caused by neurotoxins produced by bacteria of the genus Clostridium. They are 150 kDa proteins consisting of three-domains, endowed with different functions: neurospecific binding, membrane translocation and specific proteolysis of three key components of the neuroexocytosis apparatus. After binding to the presynaptic membrane of motoneurons, tetanus neurotoxin (TeNT) is internalized and transported retroaxonally to the spinal cord, where it blocks neurotransmitter release from spinal inhibitory interneurons. In contrast, the seven botulinum neurotoxins (BoNT) act at the periphery and inhibit acetylcholine release from peripheral cholinergic nerve terminals. TeNT and BoNT-B, -D, -F and -G cleave specifically at single but different peptide bonds, VAMP/synaptobrevin, a membrane protein of small synaptic vesicles. BoNT types -A, -C and -E cleave SNAP-25 at different sites within the COOH-terminus, whereas BoNT-C also cleaves syntaxin. BoNTs are increasingly used in medicine for the treatment of human diseases characterized by hyperfunction of cholinergic terminals.

Animals↗
PubMed2001

Active-site mutagenesis of tetanus neurotoxin implicates TYR-375 and GLU-271 in metalloproteolytic activity.

Tetanus neurotoxin (TeNT) blocks neurotransmitter release by cleaving VAMP/synaptobrevin, a membrane associated protein involved in synaptic vesicle fusion. Such activity is exerted by the N-terminal 50kDa domain of TeNT which is a zinc-dependent endopeptidase (TeNT-L-chain). Based on the three-dimensional structure of botulinum neurotoxin serotype A (BoNT/A) and serotype B (BoNT/B), two proteins closely related to TeNT, and on X-ray scattering studies of TeNT, we have designed mutations at two active site residues to probe their involvement in activity. The active site of metalloproteases is composed of a primary sphere of residues co-ordinating the zinc atom, and a secondary sphere of residues that determines proteolytic specificity and activity. Glu-261 and Glu-267 directly co-ordinates the zinc atom in BoNT/A and BoNT/B respectively and the corresponding residue of TeNT was replaced by Asp or by the non conservative residue Ala. Tyr-365 is 4.3A away from zinc in BoNT/A, and the corresponding residue of TeNT was replaced by Phe or by Ala. The purified mutants had CD, fluorescence and UV spectra closely similar to those of the wild-type molecule. The proteolytic activity of TeNT-Asp-271 (E271D) is similar to that of the native molecule, whereas that of TeNT-Phe-375 (Y375F) is lower than the control. Interestingly, the two Ala mutants are completely devoid of enzymatic activity. These results demonstrate that both Glu-271 and Tyr-375 are essential for the proteolytic activity of TeNT.

Amino Acid Sequence↗
PubMed2001

Virulence factors of Helicobacter pylori.

To date a number of virulence factors have been identified and characterised from the gastric pathogen Helicobacter pylori. The vacuolating toxin (VacA) is a major determinant of H. pylori-associated gastric disease. In non-polarised cells, VacA alters the endocytic pathway, resulting in the release of acid hydrolases and the reduction of both extracellular ligand degradation and antigen processing. The toxin forms trans-membrane anion-specific channels and reduces the transepithelial electrical resistance of polarized monolayers. Localization of the VacA channels in acidic intracellular compartments causes osmotic swelling which, together with membrane fusion, leads to vacuole formation. The neutrophil-activating protein of H. pylori (HP-NAP) induces the production of oxygen radicals in human neutrophils via a cascade of intracellular activation events which may contribute to the damage of the stomach mucosa. This protein has recently been shown to be an important antigen in the human immune response to H. pylori infection. In addition, mice vaccinated with recombinant HP-NAP were protected against H. pylori challenge. H. pylori strains that are associated with severe tissue damage and inflammation possess the cag pathogenicity island that contains several genes encoding factors involved in the induction of proinflammatory cytokines/chemokines and of a type IV secretion system involved in the delivery of a highly immunogenic protein, CagA, into eukaryotic cells. Recent advances in our understanding of the involvement of VacA, HP-NAP and the CagA/Type IV secretion system in the H. pylori-associated disease process are discussed in this review.

Animals↗
PubMed2001

Living dangerously: how Helicobacter pylori survives in the human stomach.

Helicobacter pylori was already present in the stomach of primitive humans as they left Africa and spread through the world. Today, it still chronically infects more than 50% of the human population, causing, in some cases, severe diseases such as peptic ulcers and stomach cancer. To succeed in these long-term associations, H. pylori has developed a unique set of virulence factors, which allow survival in a unique and hostile ecological niche--the human stomach.

Helicobacter Infections↗
PubMed2001

WHO Collaborating Centre consensus meeting on anti-cytokine therapy in rheumatoid arthritis.

Severe adult rheumatoid arthritis is a cause of progressive disability and increased mortality across Europe. A cure for the disease remains elusive, but control of symptoms and maintenance of individual independence is possible. Anti-cytokine therapies offer a new approach to disease management. They are effective after the failure of full doses of methotrexate, and are at least as effective as methotrexate in retarding the progression of radiological changes. Until more is known about the long-term safety and efficacy of these drugs they should be reserved for patients with severe disease who are progressing despite adequate doses of methotrexate or other disease-modifying anti-rheumatic drugs. They should be continued until therapeutic failure or intolerance. A comprehensive health economic evaluation is needed to optimally direct the use of these drugs. This should be undertaken when long-term safety and efficacy studies are completed.

Antibodies, Monoclonal↗
PubMed2001

Presenting features of polymyalgia rheumatica (PMR) and rheumatoid arthritis with PMR-like onset: a prospective study.

OBJECTIVE: To evaluate in a prospective study whether patients with polymyalgia rheumatica (PMR) and patients with rheumatoid arthritis (RA) with PMR-like onset show distinctive clinical and laboratory features. METHODS: A cohort of 116 consecutive patients with bilateral girdle pain for at least one month and raised erythrocyte sedimentation rate (ESR) was studied and followed up for 12 months. Laboratory tests included determination of ESR, IgM rheumatoid factor, haemoglobin, white blood cell count, platelet count, percentage of CD8 lymphocytes, serum aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and glutamyltransferase concentrations. RESULTS: At first examination, RA was diagnosed in 22/116 (19%) patients and PMR in 94 (81%) patients. During the follow up period, 19 additional patients developed RA, and the diagnosis of PMR was confirmed in 65 (56%) patients at the end of the study. Of the clinical and laboratory features, only the presence of peripheral synovitis could differentiate patients who will develop RA from those with "true" PMR, but the positive predictive value of this feature was poor. CONCLUSION: At present, there are no clinical or routine laboratory features allowing early differentiation between PMR and RA with PMR-like onset.

Aged↗
PubMed2001

The design of vaccines against Helicobacter pylori and their development.

Helicobacter pylori is a gram negative, spiral, microaerophylic bacterium that infects the stomach of more than 50% of the human population worldwide. It is mostly acquired during childhood and, if not treated, persists chronically, causing chronic gastritis, peptic ulcer disease, and in some individuals, gastric adenocarcinoma and gastric B cell lymphoma. The current therapy, based on the use of a proton-pump inhibitor and antibiotics, is efficacious but faces problems such as patient compliance, antibiotic resistance, and possible recurrence of infection. The development of an efficacious vaccine against H. pylori would thus offer several advantages. Various approaches have been followed in the development of vaccines against H. pylori, most of which have been based on the use of selected antigens known to be involved in the pathogenesis of the infection, such as urease, the vacuolating cytotoxin (VacA), the cytotoxin-associated antigen (CagA), the neutrophil-activating protein (NAP), and others, and intended to confer protection prophylactically and/or therapeutically in animal models of infection. However, very little is known of the natural history of H. pylori infection and of the kinetics of the induced immune responses. Several lines of evidence suggest that H. pylori infection is accompanied by a pronounced Th1-type CD4(+) T cell response. It appears, however, that after immunization, the antigen-specific response is predominantly polarized toward a Th2-type response, with production of cytokines that can inhibit the activation of Th1 cells and of macrophages, and the production of proinflammatory cytokines. The exact effector mechanisms of protection induced after immunization are still poorly understood. The next couple of years will be crucial for the development of vaccines against H. pylori. Several trials are foreseen in humans, and expectations are that most of the questions being asked now on the host-microbe interactions will be answered.

Animals↗
PubMed2001

Helicobacter pylori cytotoxin VacA increases alkaline secretion in gastric epithelial cells.

Human infection by the bacterium Helicobacter pylori (Hp) may lead to severe gastric diseases by an ill-understood process involving several virulence factors. Among these, the cytotoxin VacA is associated with higher tissue damage. In this study, the isolated frog stomach model was used to characterize the acute effects of VacA on the gastric epithelium. Our results show that VacA partially inhibits gastric acid output by increasing HCO(3)(-) efflux. Experiments conducted with double-barrelled pH or Cl(-)-selective microelectrodes on surface epithelial gastric cells (SECs) and single gastric glands show that VacA does not impair the activity of the oxyntic cells but renders the apical membrane of SECs more permeable to HCO(3)(-) and Cl(-). Inhibition of this permeation by 5-nitro-2-(3-phenylpropylamino) benzoic acid indicates that this may be due to the formation of anion-selective pores by the toxin. We suggest that VacA-dependent HCO(3)(-) efflux from SECs improves the environmental conditions (pH, CO(2) concentration) of the niche parasitized by Hp, that is the gastric surface. This may favor Hp persistence in the tissue and the secondary development of a chronic inflammation.

Animals↗
PubMed2000

Susceptibility of mitogen-activated protein kinase kinase family members to proteolysis by anthrax lethal factor.

The lethal factor (LF) produced by toxigenic strains of Bacillus anthracis is a Zn(2+)-endopeptidase that cleaves the mitogen-activated protein kinase kinases (MAPKKs) MEK1, MEK2 and MKK3. Using genetic and biochemical approaches, we have extended the study of LF proteolytic specificity to all known MAPKK family members and found that LF also cleaves MKK4, MKK6 and MKK7, but not MEK5. The peptide bonds hydrolysed by LF within all MAPKKs were identified. Cleavage invariably occurs within the N-terminal proline-rich region preceding the kinase domain, thus disrupting a sequence involved in directing specific protein-protein interactions necessary for the assembly of signalling complexes. Alignment of the sequences flanking the site of cleavage reveals the occurrence of some consensus motifs: position P2 and P1' are occupied by hydrophobic residues and at least one basic residue is present between P4 and P7. The implications of these findings for the biochemical activity and functional specificity of LF are discussed.

Amino Acid Sequence↗
PubMed2000

The neutrophil-activating protein (HP-NAP) of Helicobacter pylori is a protective antigen and a major virulence factor.

Helicobacter pylori infection induces the appearance of inflammatory infiltrates, consisting mainly of neutrophils and monocytes, in the human gastric mucosa. A bacterial protein with neutrophil activating activity (HP-NAP) has been previously identified, but its role in infection and immune response is still largely unknown. Here, we show that vaccination of mice with HP-NAP induces protection against H. pylori challenge, and that the majority of infected patients produce antibodies specific for HP-NAP, suggesting an important role of this factor in immunity. We also show that HP-NAP is chemotactic for human leukocytes and that it activates their NADPH oxidase to produce reactive oxygen intermediates, as demonstrated by the translocation of its cytosolic subunits to the plasma membrane, and by the lack of activity on chronic granulomatous disease leukocytes. This stimulating effect is strongly potentiated by tumor necrosis factor alpha and interferon gamma and is mediated by a rapid increase of the cytosolic calcium concentration. The activation of leukocytes induced by HP-NAP is completely inhibited by pertussis toxin, wortmannin, and PP1. On the basis of these results, we conclude that HP-NAP is a virulence factor important for the H. pylori pathogenic effects at the site of infection and a candidate antigen for vaccine development.

Androstadienes↗