Pathogenic mechanisms and current epidemiological status of HEV infection in asymptomatic blood donors and patients with chronic diseases.

In recent years, the seroprevalence of anti-hepatitis E virus immunoglobulins (HEV) has increased in European countries with significant variability among the different geographical areas. HEV infection is spread in a wide range of animal species of which domestic pigs and wild boar represent the main reservoirs of genotype 3 and 4 (the genotypes present also in Europe). European citizens are incidental hosts, mainly infected by direct contact or consumption of foods derived from undercooked or insufficient hygiene handling infected pork products or wild boar meat. Epidemiologically, the HEV incidence is low in humans but serological data show a high proportion of subclinical infection caused by genotypes 3 or 4. In the general population, asymptomatic infection represents a high potential risk in particular subjects such as blood component recipients or occupationally exposed workers. This review offers a landscape of the current epidemiological status of HEV infection (genotypes 1, 2, 3, 4, 7) both in European asymptomatic subjects, patients with chronic diseases, and domestic pig impact on humans. We also underline advantages/disadvantages of high sensitivity and specificity tests using for detecting viral RNA or anti-HEV antibodies.

Phospholamban ablation rescues the enhanced propensity to arrhythmias of mice with CaMKII-constitutive phosphorylation of RyR2 at site S2814.

KEY POINTS: Mice with Ca(2+) -calmodulin-dependent protein kinase (CaMKII) constitutive pseudo-phosphorylation of the ryanodine receptor RyR2 at Ser2814 (S2814D(+/+) mice) exhibit a higher open probability of RyR2, higher sarcoplasmic reticulum (SR) Ca(2+) leak in diastole and increased propensity to arrhythmias under stress conditions. We generated phospholamban (PLN)-deficient S2814D(+/+) knock-in mice by crossing two colonies, S2814D(+/+) and PLNKO mice, to test the hypothesis that PLN ablation can prevent the propensity to arrhythmias of S2814D(+/+) mice. PLN ablation partially rescues the altered intracellular Ca(2+) dynamics of S2814D(+/+) hearts and myocytes, but enhances SR Ca(2+) sparks and leak on confocal microscopy. PLN ablation diminishes ventricular arrhythmias promoted by CaMKII phosphorylation of S2814 on RyR2. PLN ablation aborts the arrhythmogenic SR Ca(2+) waves of S2814D(+/+) and transforms them into non-propagating events. A mathematical human myocyte model replicates these results and predicts the increase in SR Ca(2+) uptake required to prevent the arrhythmias induced by a CaMKII-dependent leaky RyR2. ABSTRACT: Mice with constitutive pseudo-phosphorylation at Ser2814-RyR2 (S2814D(+/+) ) have increased propensity to arrhythmias under ß-adrenergic stress conditions. Although abnormal Ca(2+) release from the sarcoplasmic reticulum (SR) has been linked to arrhythmogenesis, the role played by SR Ca(2+) uptake remains controversial. We tested the hypothesis that an increase in SR Ca(2+) uptake is able to rescue the increased arrhythmia propensity of S2814D(+/+) mice. We generated phospholamban (PLN)-deficient/S2814D(+/+) knock-in mice by crossing two colonies, S2814D(+/+) and PLNKO mice (SD(+/+) /KO). SD(+/+) /KO myocytes exhibited both increased SR Ca(2+) uptake seen in PLN knock-out (PLNKO) myocytes and diminished SR Ca(2+) load (relative to PLNKO), a characteristic of S2814D(+/+) myocytes. Ventricular arrhythmias evoked by catecholaminergic challenge (caffeine/adrenaline) in S2814D(+/+) mice in vivo or programmed electric stimulation and high extracellular Ca(2+) in S2814D(+) /(-) hearts ex vivo were significantly diminished by PLN ablation. At the myocyte level, PLN ablation converted the arrhythmogenic Ca(2+) waves evoked by high extracellular Ca(2+) provocation in S2814D(+/+) mice into non-propagated Ca(2+) mini-waves on confocal microscopy. Myocyte Ca(2+) waves, typical of S2814D(+/+) mice, could be evoked in SD(+/+) /KO cells by partially inhibiting SERCA2a. A mathematical human myocyte model replicated these results and allowed for predicting the increase in SR Ca(2+) uptake required to prevent the arrhythmias induced by a Ca(2+) -calmodulin-dependent protein kinase (CaMKII)-dependent leaky RyR2. Our results demonstrate that increasing SR Ca(2+) uptake by PLN ablation can prevent the arrhythmic events triggered by SR Ca(2+) leak due to CaMKII-dependent phosphorylation of the RyR2-S2814 site and underscore the benefits of increasing SERCA2a activity on SR Ca(2+) -triggered arrhythmias.

Toxic effect on human spermatozoa by Chlamydia trachomatis purified lipopolysaccharide.

We have investigated the effect that lipopolysaccharide extracted from Chlamydia trachomatis has on human spermatozoa. A lipopolysaccharide of 0.1 microgram ml-1 caused a spermatozoa mortality rate of 65 +/- 4% evaluated by eosin exclusion test. The toxic activity occurred rapidly even after brief incubation times, reaching the maximum (100% mortality) within 60 min.

Growth hormone modulates IL-alpha and IFN-gamma release by murine splenocytes activated by LPS or porins of Salmonella typhimurium.

The effect of growth hormone (GH) on the release of IL-1alpha and IFN-gamma from murine splenocytes was investigated. Their release from splenocytes activated by Salmonella enterica serovar Typhimurium lipopolysaccharide (LPS) 0.5 microg/ml was increased by c. 65% in the presence of GH 100 pg/ml. With splenocytes activated by S. Typhimurium porins 5 microg/ml, GH increased the production of both IL-1alpha and IFN-gamma by c. 56%. Polymyxin treatment abolished the cytokine-releasing activity of LPS but had no effect on the activity of the porin preparation.

Growth hormone release of interleukin-1 alpha, interferon-gamma and interleukin-4 from murine splenocytes stimulated with staphylococcal protein A, toxic shock syndrome toxin-1 and streptococcal lysin S.

We investigated changes in the IL-1 alpha, IFN-gamma and IL-4 release from splenocytes in the presence of growth hormone (GH). Splenocytes were stimulated with Protein A (PA), Toxic Shock Syndrome Toxin-1 (TSST-1) and Streptolysin S (SLS). In the presence of GH, splenocytes stimulated with PA, induced a 40% and 50% drop in IL-1 alpha and IFN-gamma release respectively, compared to controls, while no changes were shown in IL-4 release. The release of IFN-gamma by TSST-1-stimulated splenocytes fell by 30%, while no changes were shown in IL-1 alpha and IL-4 release after GH. The release of IL-1 alpha by SLS-stimulated splenocytes increased by 50% in the presence of GH. No changes were shown in IFN-gamma and IL-4 release. The results are discussed in terms of the possibility of an expanding function for these endocrine peptides within the immune system.

Effect of growth hormone, prolactin and insulin on the release of IL-1 alpha, IFN-gamma and IL-4 by staphylococcal enterotoxin A-stimulated splenocytes.

The regulation by peptide hormones (Growth Hormone, Prolactin, Insulin) of cytokine secretion by splenocytes stimulated with Staphylococcal Enterotoxin A was studied. Growth hormone increases the release of IFN-gamma from splenocytes stimulated with Enterotoxin A by 50% but considerably decreases IL-1 alpha release by 93%. Prolactin decreases the release of IL-1 alpha by 80%, but has no significant effects on IFN-gamma release. Insulin causes a 50% decrease in IFN-gamma and 95% decrease in IL-1 alpha. IL-4 release was not changed. The results are discussed in terms of the possibility of an interesting function for these endocrine peptides which expands their range of biologic activities within the immune system.

Prolactin regulates IL-1 alpha, IFN-gamma and IL-4 release from mouse splenocytes stimulated with some staphylococcal and streptococcal toxins.

This study investigates changes in IL-1 alpha, IFN-gamma and IL-4 from mouse splenocytes stimulated with Staphylococcal Protein A (PA), or Toxic Shock Syndrome Toxin-1 (TSST-1), or Streptococcal lysin S (SLS) after exposure to Prolactin (PRL). In the presence of PRL, IL-1 alpha and IFN-gamma induction by PA-stimulated splenocytes was reduced by 74% and 25% respectively. On the other hand, IL-4 release was enormously increased. The ability of TSST-1 to induce IFN-gamma release was decreased by 32% after PRL. IL-1 alpha and IL-4 was unchanged compared to controls. In the presence of PRL, IFN-gamma release from splenocytes stimulated with SLS, was increased by 60%, while no changes were shown in IL-1 alpha and IL-4 release.

Presence of Legionella spp. in thermal springs of the Campania region of south Italy.

Water samples from 66 thermal springs in the Campania region of South Italy were cultured for Legionella spp., Pseudomonas aeruginosa, and indicators of faecal pollution. The temperature of the sources ranged from 21 degrees C to 59.5 degrees C. Legionella pneumophila, serogroup 7-10, was isolated from two out of 60 sources on the Island of Ischia and Legionella dumoffii from one mainland source. The temperatures of these sources were 35.2 degrees C, 48.2 degrees C, and 52.0 degrees C respectively. Twelve sources were positive for P. aeruginosa and 6 for Escherichia coli. Our results found that Legionella spp. were present in only three thermal springs, indicating that in the hydrothermal area of the Campania region the presence of this microbial species is very scarce.

Protection of Escherichia coli K12 structural integrity by Ca2+ and Mg2+ in bactericidal and bacteriolytic tests.

Escherichia coli K12 suspended in different media showed a loss of phospholipids. Mg2+ and Ca2+ 0.01 M prevented phospholipid loss and stabilized Escherichia coli K12 for bactericidal and bacteriolytic assays.

Some biological activities of Eikenella corrodens major outer membrane proteins.

Major outer membrane proteins of Eikenella corrodens, an organism frequently isolated from patients with periodontal disease, were tested for some biological activities. Mouse peritoneal macrophages, exposed at low concentrations of the above-mentioned proteins (between 0.05 and 5 micrograms/ml), showed evident and marked morphological modifications consisting of increases in the size and vacuolation of the cells. Higher concentrations showed a toxic effect. Low concentrations resulted in a selective release of lysosomal enzymes without any significant release of lactatedehydrogenase, and cytoplasmic marker; while concentrations of 25-50 micrograms/ml, which were toxic in trypan-blue exclusion test, increased LDH release. Eikenella corrodens major proteins increased the platelet aggregation of ADP and thrombin. The residual complement activity of serum samples incubated with various amounts of proteins at 37 degrees C for 30 minutes appeared strongly reduced with respect to controls, thus showing a consumption of the complement components. These results suggested that Eikenella corrodens major proteins may play a role in the development of periodontal lesions.

Biological activities--lethality, Shwartzman reaction and pyrogenicity--of Salmonella typhimurium porins.

Porins isolated from Salmonella typhimurium and found to contain less than 0.1% w/w of LPS, were found to be lethal at a dose of 100 ng to both LPS-responder (BALB/cByJ) and non-responder (C3H/HeJ) mice sensitized with D-galactosamine. This lethal action could be prevented by anti-TNF-alpha serum given intravenously 10 min before the porin injection but not by polymyxin-B mixed with the porins in a ratio of approximately 300 moles polymyxin-B per mole of porin. The porin preparation was also pyrogenic to rabbits at a dose of 1 microgram/kg and elicited a local Shwartzman reaction when used as the sensitizing and eliciting agent; these reactions were also present when the porins were mixed with polymyxin-B.

Comparative study of some biological activities of porins extracted from various microorganisms.

The outer membrane of Gram-negative bacteria contains some major proteins, called porins, with particular chemical and physical characteristics which reflect some specialized functions peculiar to the outer membrane. In recent years the biological properties of Salmonella typhimurium SH5014 porins have been the focus of our experimental studies. The aim of the present research was to make a comparative investigation of the biological activities of porins extracted from other microorganisms classified in the Enterobacteriaceae, together with a taxonomically different one, Eikenella corrodens frequently isolated from gum pockets of patients with periodontal disease. Porins from E. coli K12, Proteus mirabilis and Eikenella corrodens were extracted, purified and separated from LPS by means of phenol extraction. The purity of preparation was checked by sodium dodecyl sulphate polyacrylamide gel electrophoresis in slabs. We studied the interactions of these proteins with some cellular and humoral systems within the host. Mouse peritoneal macrophages in the presence of these proteins show morphological and functional modifications, depending upon the type of porins and the concentrations used. The ability of porins to interact with the complement system was investigated in vitro. A pool of sera from 20 blood donors served as a source of human complement. The results obtained with porins of various sources were shown comparatively.

Activation of complement system by porins extracted from Salmonella typhimurium.

The effect of porins purified from Salmonella typhimurium on the complement system was investigated both in vitro and in vivo. Incubation of porins with either human or guinea pig serum resulted in the consumption of the total complement activity when an amount of porins ranging from 8 to 10 micrograms per 100 microliters of serum was used. The activation of the complement system was temperature dependent, suggesting an active process rather than passive adsorption of the complement components by porins. In addition, the activation had a fast kinetic and proceeded mainly through the classical pathway. This conclusion is supported by the consumption of C1s and C4 in normal human serum treated with porins and also by the depletion of C3 activity in the C1s-deficient serum which was marked only when purified C1s was added to the serum before incubation with porins. Injection of 100 micrograms of porins into guinea pigs induced profound complement consumption at 6 h postinjection that persisted up to 12 h. We conclude from this study that porins can effectively contribute to complement activation and to subsequent biological events induced by gram-negative bacteria.

Immune response in mice and effects on cells by outer membrane porins from Salmonella typhimurium.

In this study, we describe the role of porins extracted from the outer membrane of S. typhimurium on the interaction with the host organism. The tests were performed on CD-1 mice immunized both with whole S. typhimurium cells and with purified porins. The antibody titres obtained in mice treated with purified porins and in those treated with whole Salmonella are 1/320 and 1/80 respectively. Cell-mediated response was found to be stimulated in both mice groups: our results show that T lymphocytes determine an increase in H3-thymidine incorporation and MIF production. An increase in lymphocytes with membrane immunoglobulins was observed in the lymphocyte population from the spleens of the treated mice. Purified porins added to macrophages cultures exerted a toxic effect at concentrations between 10 and 20 micrograms/ml. Subinhibiting concentrations (5 micrograms/ml) led to modifications in the surface tension of macrophages layers adhering to the slides. Moreover, subinhibiting porins concentrations were found to modify macrophage functionality as shown by evaluation of the phagocytic index and of intracellular killing, both of which were found to have diminished with respect to controls. When purified porins were added in vitro to populations enriched with T and B lymphocytes, at concentrations between 1 and 5 micrograms/ml they were found to be mitogenic with respect to B lymphocytes. The results we obtained showed that lymphocytes with IgM increase in porins tests. Eventually, purified porins added to Hela and Hep-2 cells were shown to exert a toxic effect which became detectable in the final concentration of 5 micrograms/ml and 1 micrograms/ml for Hela and Hep-2 cells respectively.

Modifications of surface properties in some enteropathogenic serogroups of Escherichia coli.

Studies have been carried out on protein and fatty acid patterns present in the outer layers of E. coli strains which are considered pathogenic in relation to their serogroup. The patterns were related to surface properties of hydrophobicity of the same cells and to their resistance to phagocytosis. The results obtained demonstrate the presence in the pathogenic serogroups of a proteic band of high molecular weight as well as differences in the percentages of fatty acids with respect to the non-pathogenic serogroups. Furthermore, these serogroups are more resistant to phagocytosis; this resistance seems to be correlated to a greater surface hydrophilia of the pathogenic serogroups, leading to different relations of the lipidic and protein fraction of the outer layers.

Porin content modifies resistance of Salmonella typhimurium to phagocytosis.

Mutants of Salmonella typhimurium, which contain different quantities of outer membrane proteins, show different susceptibility to phagocytosis. We correlate susceptibility to phagocytosis with molecular surface characteristics which are responsible for invasiveness and virulence of bacteria.

Serum-mediated killing of Salmonella typhimurium and Escherichia coli mutants which share a different content of major proteins.

Salmonella typhimurium and Escherichia coli K12 mutants of R chemotype, with varying contents of major proteins, were studied with respect to serum-mediated killing. The mutants demonstrated a different susceptibility to serum lytic action. These results were related to phospholipid and fatty acid content, as well as different physico-chemical surface properties, such as outer membrane fluidity. Tests were carried out on all parameters considered in the literature to demonstrate the resistance to complement. Our results showed that in sensitive strains such as Salmonella strains SH6261, SH6378, SH5551, SH6017 and E. coli PC0479 tests taken alone were not sufficient to explain the resistance to complement. Therefore, complement susceptibility is probably determined by many factors influencing the microheterogeneity of the membrane system.

Prolactin and insulin regulate the release of IL-1-alpha and IFN-gamma from murine splenocytes activated with porins or LPS of Salmonella typhimurium.

Murine splenocytes treated with prolactin (PRL) or insulin were stimulated in vitro with porins or lipopolysaccharide (LPS) of Salmonella typhimurium. It was seen that PRL inhibits the release of IFN-gamma from splenocytes treated with porins by about 20% while having no effect on the release of IL-1-alpha. Splenocytes porin-stimulated splenocytes exhibited a remarkable increase in IL-1-alpha release (100%) and a diminished release of IFN-gamma (about 50%) in the presence of insulin. The splenocytes stimulated with LPS had a reduced release of IL-1-alpha (75%) and IFN-gamma (about 50%) when insulin was added. The data suggest that classical endocrine system participates in a bioregulatory feedback loop that may prevent unwanted toxicity from cytokine excess. However, some bacterial products sometimes enormously unbalance this regulatory network.

Distribution of mecA among methicillin-resistant clinical staphylococcal strains isolated at hospitals in Naples, Italy.

Two hundred and twenty strains of Staphylococcus isolated in Naples, Italy, were surveyed for the distribution of the mecA, the structural gene for penicillin-binding protein 2a, which is the genetic determinant for methicillin-resistance in staphylococci. Screening by a cloned mecA, revealed that of 220 strains, 43 were methicillin-resistant (19.5%) and 177 were methicillin-susceptible (80.5%). Among the 43 resistant strains 23 (53.5%) carried mecA in their genome and 20 (46.5%) did not carry mecA, in spite of their resistance to methicillin. Every group was submitted to the AP-PCR profiling. A quantitative analysis of the patterns divided strains into four different clusters for methicillin-resistant mecA-negative and two different clusters for methicillin-resistant mecA-positive with primer 1, while no clusters were noted with primer 7. We conclude that these clinical isolates from our area, were not found to belong to a single clone, although the predominance of four methicillin-resistant mecA-negative genotypes were noted.

Exchange of phospholipids between Escherichia coli cells and environment.

We studied the exchange of phospholipids between Escherichia coli K-12 cells and the suspension medium containing inactivated guinea-pig serum. In this medium, the release of 3H-labelled phospholipids was proportional both to the quantity of serum and to the temperature of incubation. No phospholipids were released when no guinea-pig serum was added to the medium, or the incubation temperature was 4 degrees C. The release of phospholipids into the medium was accompanied by an uptake of serum phospholipids by the cells, as demonstrated by incorporation of labelled phospholipids from the suspension medium. We conclude that an exchange occurs between the cellular phospholipids and those of the medium. Control tests with 3H-thymidine showed that cellular lysis was not involved.