Multivariate probability distribution for sewer system vulnerability assessment under data-limited conditions.

The lack of geometrical and hydraulic information about sewer networks often excludes the adoption of in-deep modeling tools to obtain prioritization strategies for funds management. The present paper describes a novel statistical procedure for defining the prioritization scheme for preventive maintenance strategies based on a small sample of failure data collected by the Sewer Office of the Municipality of Naples (IT). Novelty issues involve, among others, considering sewer parameters as continuous statistical variables and accounting for their interdependences. After a statistical analysis of maintenance interventions, the most important available factors affecting the process are selected and their mutual correlations identified. Then, after a Box-Cox transformation of the original variables, a methodology is provided for the evaluation of a vulnerability map of the sewer network by adopting a joint multivariate normal distribution with different parameter sets. The goodness-of-fit is eventually tested for each distribution by means of a multivariate plotting position. The developed methodology is expected to assist municipal engineers in identifying critical sewers, prioritizing sewer inspections in order to fulfill rehabilitation requirements.

An ancestral molecular response to nanomaterial particulates.

The varied transcriptomic response to nanoparticles has hampered the understanding of the mechanism of action. Here, by performing a meta-analysis of a large collection of transcriptomics data from various engineered nanoparticle exposure studies, we identify common patterns of gene regulation that impact the transcriptomic response. Analysis identifies deregulation of immune functions as a prominent response across different exposure studies. Looking at the promoter regions of these genes, a set of binding sites for zinc finger transcription factors C2H2, involved in cell stress responses, protein misfolding and chromatin remodelling and immunomodulation, is identified. The model can be used to explain the outcomes of mechanism of action and is observed across a range of species indicating this is a conserved part of the innate immune system.

Effects of nonylphenol on vitellogenin synthesis in adult males of the spotted ray Torpedo marmorata.

The aim of this investigation was to assess the effects of nonylphenol (NP), an oestrogen-like environmental pollutant, on the vitellogenin (VTG) synthesis in adult males of the aplacental viviparous cartilaginous fish Torpedo marmorata. The VTG recovery in males is considered a biomarker of xeno-oestrogenic pollution as this lipophosphoglycoprotein is physiologically induced by oestrogens only in females of oviparous and ovoviparous vertebrates. Using in situ hybridization and immunohistochemistry, T. marmorata males injected with nonylphenol showed the presence of VTG in the liver and the kidney. In particular, vtg messenger (m)RNA and VTG protein were expressed in the liver, whereas in the kidney cells only the presence of VTG was recorded. By contrast, no expression for VTG was detected in the testis. These results demonstrate that in T. marmorata NP induces the expression of vtg only in the liver; the presence of VTG in the kidney and its absence in the testis are discussed.

Cytokines kill malaria parasites during infection crisis: extracellular complementary factors are essential.

Malaria infection crisis, at which the parasitemia drops precipitously and the parasite loses infectivity to the mosquito vector, occurs in many natural malaria systems, and has not been explained. We demonstrate that in a simian malaria parasite (Plasmodium cynomolgi in its natural host, the toque monkey), the loss of infectivity during crisis is due to the death of circulating intraerythrocytic gametocytes mediated by crisis serum. These parasite-killing effects in crisis serum are due to the presence in the serum of cytokines tumor necrosis factor and interferon gamma, which are produced by the host as a result of the malaria infection. The killing activity of each cytokine is absolutely dependent upon the presence of additional, as yet unidentified factor(s) in the crisis serum.

Mice from a genetically resistant background lacking the interferon gamma receptor are susceptible to infection with Leishmania major but mount a polarized T helper cell 1-type CD4+ T cell response.

Mice with homologous disruption of the gene coding for the ligand-binding chain of the interferon (IFN) gamma receptor and derived from a strain genetically resistant to infection with Leishmania major have been used to study further the role of this cytokine in the differentiation of functional CD4+ T cell subsets in vivo and resistance to infection. Wild-type 129/Sv/Ev mice are resistant to infection with this parasite, developing only small lesions, which resolve spontaneously within 6 wk. In contrast, mice lacking the IFN-gamma receptor develop large, progressing lesions. After infection, lymph nodes (LN) and spleens from both wild-type and knockout mice showed an expansion of CD4+ cells producing IFN-gamma as revealed by measuring IFN-gamma in supernatants of specifically stimulated CD4+ T cells, by enumerating IFN-gamma-producing T cells, and by Northern blot analysis of IFN-gamma transcripts. No biologically active interleukin (IL) 4 was detected in supernatants of in vitro-stimulated LN or spleen cells from infected wild-type or deficient mice. Reverse transcription polymerase chain reaction analysis with primers specific for IL-4 showed similar IL-4 message levels in LN from both types of mice. The IL-4 message levels observed were comparable to those found in similarly infected C57BL/6 mice and significantly lower than the levels found in BALB/c mice. Anti-IFN-gamma treatment of both types of mice failed to alter the pattern of cytokines produced after infection. These data show that even in the absence of IFN-gamma receptors, T helper cell (Th) 1-type responses still develop in genetically resistant mice with no evidence for the expansion of Th2 cells.

Mucosal adjuvanticity and immunogenicity of LTR72, a novel mutant of Escherichia coli heat-labile enterotoxin with partial knockout of ADP-ribosyltransferase activity.

Heat-labile Escherichia coli enterotoxin (LT) has the innate property of being a strong mucosal immunogen and adjuvant. In the attempt to reduce toxicity and maintain the useful immunological properties, several LT mutants have been produced. Some of these are promising mucosal adjuvants. However, so far, only those that were still toxic maintained full adjuvanticity. In this paper we describe a novel LT mutant with greatly reduced toxicity that maintains most of the adjuvanticity. The new mutant (LTR72), that contains a substitution Ala --> Arg in position 72 of the A subunit, showed only 0.6% of the LT enzymatic activity, was 100,000-fold less toxic than wild-type LT in Y1 cells in vitro, and was at least 20 times less effective than wild-type LT in the rabbit ileal loop assay in vivo. At a dose of 1 microg, LTR72 exhibited a mucosal adjuvanticity, similar to that observed with wild-type LT, better than that induced by the nontoxic, enzymatically inactive LTK63 mutant, and much greater than that of the recombinant B subunit. This trend was consistent for both the amounts and kinetics of the antibody induced, and priming of antigen-specific T lymphocytes. The data suggest that the innate high adjuvanticity of LT derives from the independent contribution of the nontoxic AB complex and the enzymatic activity. LTR72 optimizes the use of both properties: the enzymatic activity for which traces are enough, and the nontoxic AB complex, the effect of which is dose dependent. In fact, in dose-response experiments in mice, 20 microg of LTR72 were a stronger mucosal adjuvant than wild-type LT. This suggests that LTR72 may be an excellent candidate to be tested in clinical trials.

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.

Transmission blocking immunity in Plasmodium vivax malaria: antibodies raised against a peptide block parasite development in the mosquito vector.

One approach towards the development of a vaccine against malaria is to immunize against the parasite sexual stages that mediate transmission of the parasite from man to mosquito. Antibodies against these stages, ingested with the blood meal, inhibit the parasite development in the mosquito vector, constituting "transmission blocking immunity." Most epitopes involved in transmission-blocking immunity depend on the tertiary conformational structure of surface antigens. However, one of the transmission-blocking monoclonal antibodies we have raised against Plasmodium vivax reacts with a linear epitope on both asexual stages and gametes. This monoclonal antibody (A12) is capable of totally blocking development of the parasite in the mosquito host when tested in membrane feeding assays with gametocytes from P. vivax-infected patients. Immune screening of a P. vivax lambda gt11 genomic expression library with A12 led to the isolation of a clone to which was mapped the six-amino acid epitope recognized by A12. Antisera raised in mice against a 12-mer synthetic peptide containing this epitope coupled to bovine serum albumin not only had high titers of antipeptide antibodies as measured by enzyme-linked immunosorbent assay, but in addition recognized the same 24- and 57-kD parasite components as A12 on Western blots and reacted with the parasite by immunofluorescence. When tested in membrane feeding assays, these antibodies have significant suppressive effects on parasite development in the mosquito.

Immunological fingerprint of 4CMenB recombinant antigens via protein microarray reveals key immunosignatures correlating with bactericidal activity.

Serogroup B meningococcus (MenB) is a leading cause of meningitis and sepsis across the world and vaccination is the most effective way to protect against this disease. 4CMenB is a multi-component vaccine against MenB, which is now licensed for use in subjects >2 months of age in several countries. In this study, we describe the development and use of an ad hoc protein microarray to study the immune response induced by the three major 4CMenB antigenic components (fHbp, NHBA and NadA) in individual sera from vaccinated infants, adolescents and adults. The resulting 4CMenB protein antigen fingerprinting allowed the identification of specific human antibody repertoire correlating with the bactericidal response elicited in each subject. This work represents an example of epitope mapping of the immune response induced by a multicomponent vaccine in different age groups with the identification of protective signatures. It shows the high flexibility of this microarray based methodology in terms of high-throughput information and minimal volume of biological samples needed.

Helicobacter pylori virulence and genetic geography.

Isolated for the first time in 1982 from human gastric biopsy, Helicobacter pylori is responsible for gastritis, peptic ulcer, and gastric cancer. A pathogenicity island acquired by horizontal transfer, coding for a type IV secretion system, is a major determinant of virulence. The infection is now treated with antibiotics, and vaccines are in preparation. The geographic distribution suggests coevolution of man and Helicobacter pylori.

Cytokine gene polymorphisms and the risk of adenocarcinoma of the stomach in the European prospective investigation into cancer and nutrition (EPIC-EURGAST).

BACKGROUND: The relative contribution to gastric cancer (GC) risk of variants in genes that determine the inflammatory response remains mostly unknown and results from genotyping studies are inconsistent. PATIENTS AND METHODS: A nested case-control study within the prospective European Prospective Investigation into Cancer and Nutrition cohort was carried out, including 248 gastric adenocarcinomas and 770 matched controls. Twenty common polymorphisms at cytokine genes [interleukin (IL)1A, IL1B, IL1RN, IL4, IL4R, IL6, IL8, IL10, IL12A, IL12B, lymphotoxin alpha and tumor necrosis factor (TNF)] were analyzed. Antibodies against Helicobacter pylori (Hp) and CagA were measured. RESULTS: IL1RN 2R/2R genotype [odds ratio (OR) 2.43; 95% confidence interval (CI) 1.19-4.96] and allele IL1RN Ex5-35C were associated with an increased risk of Hp(+) non-cardia GC. IL8 -251AA genotype was associated with a decreased risk of Hp(+) non-cardia GC (OR 0.51; 95% CI 0.32-0.81), mainly of the intestinal type. These associations were not modified by CagA status. Carriers of IL1B -580C and TNF -487A alleles did not associate with an increased risk. A moderately increased risk of Hp(+) non-cardia GC for IL4R -29429T variant was observed (OR 1.74; 95% CI 1.15-2.63). CONCLUSION: This prospective study confirms the association of IL1RN polymorphisms with the risk of non-cardia GC and indicates that IL8 -251T>A may modify the risk for GC.

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.

New carriers and adjuvants in the development of vaccines.

Different approaches are being followed to enhance the immunogenicity of established and newly developed vaccines. Some of them foresee the utilization of carrier molecules recognized by the immune systems of a large proportion of the human population. Others are directed mainly towards the development of improved adjuvants and delivery systems.

Characterization of murine monoclonal antibodies against a repetitive synthetic peptide from the circumsporozoite protein of the human malaria parasite, Plasmodium falciparum.

Monoclonal antibodies (mAbs) were raised in mice against the synthetic peptide (NANP)40, consisting of 40 (NANP) repeats of the circumsporozoite (CS) protein of the human malaria parasite, Plasmodium falciparum, and characterized. (i) Five of these mAbs recognized the P. falciparum CS protein in western blot experiments and in immunofluorescence assays using different preparations of sporozoites. The remaining two mAbs (CT3.2 and CT3.3, both IgG1) gave negative results by both techniques. (ii) When the anti-(NANP)40 peptide mAbs were functionally tested in vitro to assess their ability to inhibit the attachment and penetration of the parasites into cultured human liver cells, six of them exhibited inhibitory activities ranging between 66 and 90%. CT3.2 mAbs, also, inhibited sporozoite attachment and penetration, despite the negative results by immunofluorescence and western blot experiments. However, when immunofluorescence was repeated in the presence of calcium, CT3.2 did reveal a positive recognition of P. falciparum sporozoites, suggesting that this mAb could recognize the (NANP) sequence when calcium was bound to the repetitive peptide. (iii) Furthermore, the binding of an anti-(NANP)40 IgM mAb (CT1) to the solid-phase peptide was not inhibited by preincubation of the peptide with a mAb against the P. falciparum CS protein. (iv) Finally, one anti-(NANP)40 IgG1 mAb (CT3.1) was unable to bind to the shorter (NANP)3 peptide, although it recognized the (NANP)40 peptide and the P. falciparum CS protein. The results presented here suggest that heterogeneous antibody populations are produced upon immunization of mice with (NANP)40 synthetic peptide and that epitopes different from those simply related to the linear (NANP) amino acid sequence are likely to be present in long (NANP)n constructs as well as in the repetitive domain of the P. falciparum CS protein.

Molecular basis of vaccination.

Vaccines represent the most cost-effective means to prevent infectious diseases. Most of the vaccines which are currently available were developed long before the era of molecular biology and biotechnology. They were obtained following empirical approaches leading to the inactivation or to the attenuation of microorganisms, without any knowledge neither of the mechanisms of pathogenesis of the disease they were expected to protect from, nor of the immune responses elicited by the infectious agents or by the vaccine itself. The past two decades have seen an impressive progress in the field of immunology and molecular biology, which have allowed a better understanding of the interactions occurring between microbes and their hosts. This basic knowledge has represented an impetus towards the generation of better vaccines and the development of new vaccines. In this monograph we briefly summarize some of the most important biotechnological approaches that are currently followed in the development of new vaccines, and provide details on an approach to vaccine development: the genetic detoxification of bacterial toxins. Such an approach has been particularly successful in the rational design of a new vaccine against pertussis, which has been shown to be extremely efficacious and safe. It has been applied to the construction of powerful mucosal adjuvants, for administration of vaccines at mucosal surfaces.

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.

Inhibition of the vacuolating and anion channel activities of the VacA toxin of Helicobacter pylori.

VacA, the vacuolating cytotoxin secreted by Helicobacter pylori, is believed to be a major causative factor in the development of gastroduodenal ulcers. This toxin causes vacuolation of cultured cells and it has recently been found to form anion-selective channels upon insertion into planar bilayers as well as in the plasma membrane of HeLa cells. Here, we identify a series of inhibitors of VacA channels and we compare their effectiveness as channel blockers and as inhibitors of VacA-induced vacuolation, confirming that the two phenomena are linked. This characterization opens the way to studies in other experimental systems and to the search for a specific inhibitor of VacA action.

Helicobacter pylori and stomach cancer: a case-control study in Venezuela.

The role of Helicobacter pylori infection in gastric cancer was evaluated in a high-risk population in Venezuela using serological assays in a study of 302 cases and 483 neighborhood controls. To investigate the claim that assays for H. pylori should use antigens derived from local strains, four different assays derived from Venezuelan and European strains were used. Prevalence of IgG H. pylori antibodies in controls was very high, with estimates between 72 and 92%. Prevalence was similar in cases and controls. However, cases had lower antibody titers. This effect was observed only in subjects with low pepsinogen (PG) levels PGI/PGII <3.0), which suggested that extensive atrophy in cases causes a loss of H. pylori infection, with a consequent reduction in antibody titer. In addition, advanced cases (stage II or higher) had lower antibody titers than less advanced cases, which indicated that the lower antibody titers in cases may be attributable partially to a diminished immune response. All of the four assays for anti-H. pylori antibodies gave similar results. No evidence was found for the superiority of the assay based on Venezuelan strains. These results are consistent with other case-control studies in high-risk populations and highlight the difficulties of investigating H. pylori infection in retrospective studies.

Towards vaccine optimisation.

New molecular technologies have accelerated the search for sub-unit candidate vaccines. However, once identified the use of a candidate antigen must be optimised to reap the maximum benefit from the eventual vaccine. This optimisation should take into account both the needs of the target population, and the various ways of potentiating the protective immune response induced. One must be sure that the final product will be used. Hence, vaccine optimisation should strive toward meeting the needs of a specific epidemiological problem within the economic constraints of a given situation. This may be possible using novel delivery systems designed to limit the number of doses needed, improve the stability or facilitate the delivery of a particular vaccine. In meeting the needs of a target population in a field situation, one must also keep in mind certain safety factors that go beyond the usual regulatory constraints. The immune response to vaccine candidates can be potentiated in many ways. The ability to preferentially induce specific protective effector mechanisms: i.e., antibody isotypes, T-cell subsets, and T-cell sub-subsets, is becoming a reality. Carrier molecules designed to avoid the problems of epitope suppression and competition, and perhaps an eventual "carrier jam," are being developed. Adjuvants and immunostimulants may also help, but the critical issue here remains their acceptability for use in man. Finally novel strategies for the induction of the immune response may also potentiate the immune response in the optimisation of vaccines.

Peptide-primed CD4+ cells and malaria sporozoites.

We have mapped a T cell epitope in the circumsporozoite (CS) protein of the murine malaria parasite, Plasmodium yoelii. A 21-mer synthetic peptide corresponding to the amino acid positions 59-79 (referred to as Py1), induced specific proliferation in BALB/c and C57BL/6 mice, and provided help for the production of antibodies to peptides from the repetitive region, (QGPGAP)n, of the same CS protein, when mice were immunized with the Py1 peptide conjugated to the repetitive peptide. Long-term CD3+CD4+CD8-TCR alpha beta+ T cell lines and clones were derived from both strains of mice. These lines and clones, that proliferated in an MHC-restricted fashion, did not recognize peptides from the homologous region of another murine malaria parasite, P. berghei. About 50% of these clones produced detectable amounts of IFN-gamma and IL-2, whereas the remaining produced IL-4, IL-5, and IL-6. In preliminary experiments, some of these clones specifically inhibited P. yoelii sporozoite development in vitro and conferred protection in vivo in passive transfer experiments. These findings show that heterogenous T cell populations are activated in mice upon immunization with a short peptide from the P. yoelii CS protein and that some of these cells could be active in the effector arm of the immune response against malaria sporozoites.