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Biomedical subjects

G Del Giudice

Publications and source records attributed to G Del Giudice.

At least 19 recordsLinked to original sources
PubMed2006

Plasma and dietary carotenoid, retinol and tocopherol levels and the risk of gastric adenocarcinomas in the European prospective investigation into cancer and nutrition.

Despite declining incidence rates, gastric cancer (GC) is a major cause of death worldwide. Its aetiology may involve dietary antioxidant micronutrients such as carotenoids and tocopherols. The objective of this study was to determine the association of plasma levels of seven common carotenoids, their total plasma concentration, retinol and alpha- and gamma-tocopherol, with the risk of gastric adenocarcinoma in a case-control study nested within the European Prospective Investigation into Cancer and Nutrition (EPIC), a large cohort involving 10 countries. A secondary objective was to determine the association of total sum of carotenoids, retinol and alpha-tocopherol on GCs by anatomical subsite (cardia/noncardia) and histological subtype (diffuse/intestinal). Analytes were measured by high-performance liquid chromatography in prediagnostic plasma from 244 GC cases and 645 controls matched by age, gender, study centre and date of blood donation. Conditional logistic regression models adjusted by body mass index, total energy intake, smoking and Helicobacter pylori infection status were used to estimate relative cancer risks. After an average 3.2 years of follow-up, a negative association with GC risk was observed in the highest vs the lowest quartiles of plasma beta-cryptoxanthin (odds ratio (OR) = 0.53, 95% confidence intervals (CI) = 0.30-0.94, P(trend) = 0.006), zeaxanthin (OR = 0.39, 95% CI = 0.22-0.69, P(trend) = 0.005), retinol (OR = 0.55, 95% CI = 0.33-0.93, P(trend) = 0.005) and lipid-unadjusted alpha-tocopherol (OR = 0.59, 95% CI = 0.37-0.94, P(trend) = 0.022). For all analytes, no heterogeneity of risk estimates or significant associations were observed by anatomical subsite. In the diffuse histological subtype, an inverse association was observed with the highest vs lowest quartile of lipid-unadjusted alpha-tocopherol (OR = 0.26, 95% CI = 0.11-0.65, P(trend) = 0.003). These results show that higher plasma concentrations of some carotenoids, retinol and alpha-tocopherol are associated with reduced risk of GC.

Adenocarcinoma↗
PubMed2003

Occult hepatitis C virus infection in type II mixed cryoglobulinaemia.

Mixed cryoglobulinaemia, when not secondary to other well-defined immunological disorders, is commonly associated with hepatitis C virus (HCV) infection. However, a minority of cases lack evidence of HCV infection and are, therefore, defined as 'true essential' mixed cryoglobulinaemias. We thoroughly investigated three such patients to determine the aetiology of this disorder. Antibodies to HCV (anti-HCV) and HCV RNA, detected by sensitive enzyme-linked immunosorbent and polymerase chain reaction assays in serum and in concentrated cryoglobulins, were repeatedly negative in the three patients. Despite the lack of evidence for HCV infection, two of them were still treated with interferon alpha-2a assuming unrecognized viral infection. Both patients demonstrated excellent clinical and laboratory responses, but cryoglobulinaemia relapsed after the withdrawal of therapy. At the time of relapse, HCV RNA genomic sequences were detected for the first time in the cryoprecipitates of both patients. In the third case, HCV RNA was demonstrated for the first time during a flare of cryoglobulinaemia coincident with varicella infection. In all three patients anti-HCV antibodies remained negative throughout follow-up. We conclude that some apparently 'essential' forms of mixed cryoglobulinaemia can be caused by occult HCV infection. Interferon therapy can be taken into consideration in such HCV-negative cases.

Adult↗
PubMed2002

Enhancement of protective efficacy following intranasal immunization with vaccine plus a nontoxic LTK63 mutant delivered with nanoparticles.

Most vaccines are still given parenterally. Mucosal vaccination would offer different advantages over parenteral immunization, including blocking of the pathogens at the portal of entry. In this paper, nontoxic Escherichia coli heat-labile enterotoxin (LT) mutants and Supramolecular Biovector systems (SMBV) were evaluated in mice as mucosal adjuvants and delivery systems, respectively, for intranasal immunization with the conjugated group C meningococcal vaccine. The conjugated vaccine formulated together with the LT mutants and the SMBV induced very high titers of serum and mucosal antibodies specific for the group C meningococcal polysaccharide. This vaccination strategy also induced high titers of antibodies with bactericidal activity, which is known to correlate with efficacy. Importantly, the mucosal vaccination, but not the conventional parenteral vaccination, induced bactericidal antibodies at the mucosal level. These data strongly support the feasibility of development of intranasal vaccines with an enhanced protective efficacy against meningococci and possibly against other encapsulated bacteria.

Adjuvants, Immunologic↗
PubMed2001

Vacuolation induced by VacA toxin of Helicobacter pylori requires the intracellular accumulation of membrane permeant bases, Cl(-) and water.

The protein vacuolating toxin A (VacA) of Helicobacter pylori converts late endosomes into large vacuoles in the presence of permeant bases. Here it is shown that this phenomenon corresponds to an accumulation of permeant bases and Cl(-) in HeLa cells and requires the presence of extracellular Cl(-). The net influx of Cl(-) is due to electroneutral, Na(+), K(+), 2Cl(-) cotransporter-mediated transport. Cell vacuolation leads to cell volume increase, consistent with water flux into the cell, while hyper-osmotic media decreased vacuole formation. These data represent the first evidence that VacA-treated cells undergo an osmotic unbalance, reinforcing the hypothesis that the VacA chloride channel is responsible for cell vacuolation.

Ammonium Chloride↗
PubMed2001

What are the limits of adjuvanticity?

Vaccines developed traditionally following empirical approaches have often limited problems of immunogenicity, probably due to the low level of purity of the active component(s) they contain. The application of new technologies to vaccine development is leading to the production of purer (e.g. recombinant) antigens which, however, tend to have a poorer immunogenicity as compared to vaccines of the previous generation. The search for new vaccine adjuvants involves issues related to their potential limits. Since the introduction of aluminium salts as vaccine adjuvants more than 70 years ago, only one adjuvant has been licensed for human use. The development of some of these new vaccine adjuvants has been hampered by their inacceptable reactogenicity. In addition, some adjuvants work strongly with some antigens but not with others, thus, limiting their potentially widespread use. The need to deliver vaccines via alternative routes of administration (e.g. the mucosal routes) in order to enhance their efficacy and compliance has set new requirements in basic and applied research to evaluate their efficacy and safety. Cholera toxin (CT) and labile enterotoxin (LT) mutants given along with intranasal or oral vaccines are strong candidates as mucosal adjuvants. Their potential reactogenicity is still matter of discussions, although available data support the notion that the effects due to their binding to the cells and those due to the enzymatic activity can be kept separated. Finally, adjuvanticity is more often evaluated in terms of antigen-specific antibody titers induced after parenteral immunization. It is known that, in many instances, antigen-specific antibody titers do not correlate with protection. In addition, very little is known on parameters of cell-mediated immunity which could be considered as surrogates of protection. Tailoring of new adjuvants for the development of vaccines with improved immunogenicity/efficacy and reduced reactogenicity will represent one of the major challenges of the ongoing vaccine-oriented research.

Adjuvants, Immunologic↗
PubMed2001

Mucosal vaccines: non toxic derivatives of LT and CT as mucosal adjuvants.

Most vaccines are still delivered by injection. Mucosal vaccination would increase compliance and decrease the risk of spread of infectious diseases due to contaminated syringes. However, most vaccines are unable to induce immune responses when administered mucosally, and require the use of strong adjuvant on effective delivery systems. Cholera toxin (CT) and Escherichia coli enterotoxin (LT) are powerful mucosal adjuvants when co-administered with soluble antigens. However, their use in humans is hampered by their extremely high toxicity. During the past few years, site-directed mutagenesis has permitted the generation of LT and CT mutants fully non toxic or with dramatically reduced toxicity, which still retain their strong adjuvanticity at the mucosal level. Among these mutants, are LTK63 (serine-to-lysine substitution at position 63 in the A subunit) and LTR72 (alanine-to-arginine substitution at position 72 in the A subunit). The first is fully non toxic, whereas the latter retains some residual enzymatic activity. Both of them are extremely active as mucosal adjuvants, being able to induce very high titers of antibodies specific for the antigen with which they are co-administered. Both mutants have now been tested as mucosal adjuvants in different animal species using a wide variety of antigens. Interestingly, mucosal delivery (nasal or oral) of antigens together with LTK63 or LTR72 mutants also conferred protection against challenge in appropriate animal models (e.g. tetanus, Helicobacter pylori, pertussis, pneumococci, influenza, etc). In conclusion, these LTK63 and LTR72 mutants are safe adjuvants to enhance the immunogenicity of vaccines at the mucosal level, and will be tested soon in humans.

Adjuvants, Immunologic↗
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

Rationally designed strings of promiscuous CD4(+) T cell epitopes provide help to Haemophilus influenzae type b oligosaccharide: a model for new conjugate vaccines.

The age-related and T cell-independent immunological properties of most capsular polysaccharides limit their use as vaccines, especially in children under 2 years of age. To overcome these limitations, polysaccharide antigens have been successfully conjugated to a variety of carrier proteins, such as diphtheria toxoid or tetanus toxoid (TT) and the diphtheria mutant (CRM197) to produce very successful glycoconjugate vaccines. The increasing demand for new conjugate vaccines requires the availability of additional carriers providing high and long-lasting T helper cell immunity. Here we describe the design and construction of three recombinant carrier proteins (N6, N10, N19) constituted by strings of 6, 10 or 19 human CD4(+) T cell epitopes from various pathogen-derived antigens, including TT and proteins from Plasmodium falciparum, influenza virus and hepatitis B virus. Each of these epitopes is defined as universal in that it binds to many human MHC class II molecules. When conjugated to Haemophilus influenzae type b (Hib) oligosaccharide, these carriers elicit a potent anti-Hib antibody response in mice. In the case of the N19-Hib conjugate, this response is at least as good as that observed with CRM197-Hib, a conjugate vaccine currently used for mass immunization. We also show that some of the universal epitopes constituting the recombinant carriers are specifically recognized by two human in vitro systems, suggesting that T cell memory is provided by the selected epitopes. The data indicate that rationally designed recombinant polyepitope proteins represent excellent candidates for the development and clinical testing of new conjugate vaccines.

Amino Acid Sequence↗
PubMed2001

Intranasal immunization with SAG1 and nontoxic mutant heat-labile enterotoxins protects mice against Toxoplasma gondii.

Effective protection against intestinal pathogens requires both mucosal and systemic immune responses. Intranasal administration of antigens induces these responses but generally fails to trigger a strong protective immunity. Mucosal adjuvants can significantly enhance the immunogenicities of intranasally administered antigens. Cholera toxin (CT) and heat-labile enterotoxin (LT) are strong mucosal adjuvants with a variety of antigens. Moreover, the toxicities of CT and LT do not permit their use in humans. Two nontoxic mutant LTs, LTR72 and LTK63, were tested with Toxoplasma gondii SAG1 protein in intranasal vaccination of CBA/J mice. Vaccination with SAG1 plus LTR72 or LTK63 induced strong systemic (immunoglobulin G [IgG]) and mucosal (IgA) humoral responses. Splenocytes and mesenteric lymph node cells from mice immunized with LTR72 plus SAG1, but not those from mice immunized with LTK63 plus SAG1, responded to restimulation with a T. gondii lysate antigen in vitro. Gamma interferon and interleukin 2 (IL-2) production by splenocytes and IL-2 production by mesenteric lymph node cells were observed in vitro after antigen restimulation, underlying a Th1-like response. High-level protection as assessed by the decreased load of cerebral cysts after a challenge with the 76K strain of T. gondii was obtained in the group immunized with LTR72 plus SAG1 and LTK63 plus SAG1. They were as well protected as the mice immunized with the antigen plus native toxins. This is the first report showing protection against a parasite by using combinations of nontoxic mutant LTs and SAG1 antigen. These nontoxic mutant LTs are now attractive candidates for the development of mucosally delivered vaccines.

Adjuvants, Immunologic↗
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

The Helicobacter pylori VacA toxin is a urea permease that promotes urea diffusion across epithelia.

Urease and the cytotoxin VacA are two major virulence factors of the human pathogen Helicobacter pylori, which is responsible for severe gastroduodenal diseases. Diffusion of urea, the substrate of urease, into the stomach is critically required for the survival of infecting H. pylori. We now show that VacA increases the transepithelial flux of urea across model epithelia by inducing an unsaturable permeation pathway. This transcellular pathway is selective, as it conducts thiourea, but not glycerol and mannitol, demonstrating that it is not due to a loosening of intercellular junctions. Experiments performed with different cell lines, grown in a nonpolarized state, confirm that VacA permeabilizes the cell plasma membrane to urea. Inhibition studies indicate that transmembrane pores formed by VacA act as passive urea transporters. Thus, their inhibition by the anion channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid significantly decreases toxin-induced urea fluxes in both polarized and nonpolarized cells. Moreover, phloretin, a well-known inhibitor of eukaryotic urea transporters, blocks VacA-mediated urea and ion transport and the toxin's main biologic effects. These data show that VacA behaves as a low-pH activated, passive urea transporter potentially capable of permeabilizing the gastric epithelium to urea. This opens the novel possibility that in vivo VacA may favor H. pylori infectivity by optimizing urease activity.

Animals↗
PubMed2001

O6-alkylguanines, dietary N-nitroso compounds, and their precursors in gastric cancer.

Several N-nitroso compounds, present in foods and beverages or formed in the stomach from their precursors, act as alkylating agents. By using a highly reliable technique (high-resolution gas chromatography-mass spectrometry with negative-ion chemical ionization and selected ion recording), we measured a series of specific O6-alkylguanines in snap-frozen paired stomach tissue samples (tumor and noninvolved mucosa) obtained at surgery from 24 gastric cancer patients identified in Florence, Italy. Samples of noninvolved mucosa had higher levels of total O6-alkylguanines and more frequently detectable levels (54%) than tumor samples (29.2%). O6-propylguanine and O6-methylguanine were the single adducts most frequently detected in noninvolved mucosa and tumor tissue, respectively. Tumor samples showed higher levels of total O6-alkylguanines in female patients (p = 0.03) and among those with a diffuse histological type (p = 0.06) or seronegative for Helicobacter pylori CagA antibodies (p = 0.06). Mean dietary nitrate intake was significantly higher in patients with detectable levels of adducts in tumor samples (p = 0.03). Estimated intakes of dimethylamine and N-nitrosodimethylamine correlated with total levels of O6-alkylguanines in noninvolved gastric mucosa. These findings, although based on a small series of cases, support a role for N-nitroso compounds from dietary sources in the etiology of gastric cancer.

Aged↗
PubMed2001

Towards the development of vaccines against Helicobacter pylori: status and issues.

Helicobacter pylori is one of the most common bacteria worldwide. It infects more than 50% of the human population, and causes serious gastric diseases, such as chronic gastritis, peptic ulcer and, in some individuals, gastric cancer. The current treatment with antibiotics is not without drawbacks. The development of vaccines could help enormously in reducing the burden of H. pylori-associated diseases. A large body of evidence from experimental animal models has shown that vaccine-mediated prevention or vaccine-induced eradication of H. pylori is feasible. Several issues, however, are still unresolved, concerning the quality of the induced immune response necessary to achieve strong and sustained protective immunity in man. Several trials in humans are planned for the next few years. These studies will tell whether vaccination against H. pylori is feasible in man, and will offer the great opportunity to understand better the relationship between this ancient bacterium and humans.

Animals↗
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↗
PubMed2000

Blockers of VacA provide insights into the structure of the pore.

The cytotoxic effects of the Helicobacter pylori toxin VacA, an important etiogenic factor in human gastric diseases, are due to its ability to form anion-selective pores in target cell membranes. We have studied the inhibition of channel activity by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), representatives of two popular classes of chloride channel blockers, to gain information on the mechanism of blocking and on the unknown structure of the VacA pore. The data indicate that both compounds produce a fast block by binding to separate but mutually exclusive sites within the channel lumen. DIDS binds close to the pore opening on the side of protein insertion, whereas NPPB blocks at a position in the opposite half of the channel. Although DIDS reaches the blocking site by traveling along the lumen, inhibition by NPPB appears to involve mainly partition of the compound into the membrane, voltage-independent diffusion from it to the inhibitory position, and voltage-dependent exit. The data are consistent with a pore that can be more easily entered from the side of protein insertion than from the opposite end.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2000

Phase variation in H type I and Lewis a epitopes of Helicobacter pylori lipopolysaccharide.

Helicobacter pylori NCTC 11637 lipopolysaccharide (LPS) expresses the human blood group antigens Lewis x (Le(x)), Le(y), and H type I. In this report, we demonstrate that the H type I epitope displays high-frequency phase variation. One variant expressed Le(x) and Le(y) and no H type I as determined by serology; this switch was reversible. Insertional mutagenesis in NCTC 11637 of JHP563 (a poly(C) tract containing an open reading frame homologous to glycosyltransferases) yielded a transformant with a serotype similar to the phase variant. Structural analysis of the NCTC 11637 LPS confirmed the loss of the H type I epitope. Sequencing of JHP563 in strains NCTC 11637, an H type I-negative variant, and an H type I-positive switchback variant showed a C14 (gene on), C13 (gene off), and C14 tract, respectively. Inactivation of strain G27, which expresses Le(x), Le(y), H type I, and Le(a), yielded a transformant that expressed Le(x) and Le(y). We conclude that JHP563 encodes a beta3-galactosyltransferase involved in the biosynthesis of H type I and Le(a) and that phase variation in H type I is due to C-tract changes in this gene. A second H type I-negative variant (variant 3a) expressed Le(x) and Le(a) and had lost both H type I and Le(y) expression. Inactivation of HP093-HP094 resulted in a transformant expressing Le(x) and lacking Le(y) and H type I. Structural analysis of a mutant LPS confirmed the serological data. We conclude that the HP093-HP094 alpha2-fucosyltransferase (alpha2-FucT) gene product is involved in the biosynthesis of both Le(y) and Le(x). Finally, we inactivated HP0379 in strain 3a. The transformant had lost both Le(x) and Le(a) expression, which demonstrates that the HP0379 gene product is both an alpha3- and an alpha4-FucT. Our data provide understanding at the molecular level of how H. pylori is able to diversify in the host, a requirement likely essential for successful colonization and transmission.

Antibodies, Monoclonal↗