A case of inflammatory ascites.

UNLABELLED: CASE BACKGROUND: Ascites appears mainly as a consequence of portal hypertension in patients with liver cirrhosis, or can be caused by several other causes, such us congestive heart failure, peritoneal malignancy, or tuberculosis. In some cases, ascites can pose a diagnostic challenge for clinicians and in some patients, despite thorough and extensive work-up, the origin of this ascites remains unknown. CASE REPORT: In the unusual case hereby reported, a 52-year-old man developed severe ascites in a few weeks, in the absence of known liver disease or congestive hearth failure. We performed laboratory analysis, endoscopic, and imaging investigations, including abdominal contrast-enhanced computed tomography and 18-fluorodeoxyglucose-positron emission tomography. Peritoneal fluid analysis showed exudative fluid without neoplastic cells. A diagnostic laparoscopy with multiple diagnostic biopsies was carried out, but no macroscopic cause of the ascites was found; histopathological examination showed minimal aspects of diffuse and non-specific chronic inflammation. CONCLUSIONS: We decided to empirically treat the patient with steroid therapy (methylprednisolone: 0·5 mg/kg/day). Over a period of 6 weeks, his ascites resolved and at 2 months, he was in remission on low-dose methylprednisolone. Our final hypothesis was reactive inflammatory ascites. The literature on ascites and its management has also been reviewed.

Development of a haemolytic-enzymatic assay with mediated amperometric detection for palytoxin analysis: application to mussels.

An electrochemical sensor for palytoxin (PlTX) detection, based on a strip of eight screen-printed electrodes connected to a cost-effective and portable apparatus, is reported. Sheep erythrocytes were used to test the palytoxin detector and degree of haemolysis was evaluated by measuring release of the cytosolic lactate dehydrogenase (LDH). Percentage haemolysis and, therefore, the amount of LDH measured, by use of NADH/pyruvate and appropriate electrochemical mediators, was correlated with the concentration of the toxin. Two different electrochemical approaches were investigated for evaluation of LDH release, but only one based on the use of a binary redox mediator sequence (phenazine methosulfate in conjugation with hexacyanoferrate(III)) proved useful for our purpose. After analytical and biochemical characterization, the sensor strip was used to measure palytoxin. Sheep blood and standard solutions of PlTX were left to react for two different incubation times (24 h or 4 h), resulting in working ranges of 7 × 10(-3)-0.02 ng mL(-1) and 0.16-1.3 ng mL(-1), respectively. The specificity of the test for palytoxin was evaluated by use of ouabain, which acts in the same way as PlTX on the Na(+)/K(+)-ATPase pump. A cross-reactivity study, using high concentrations of other marine biotoxins was also conducted. Experiments to evaluate the matrix effect and recovery from mussels are discussed.

Norovirus contamination in different shellfish species harvested in the same production areas.

AIMS: To investigate Norovirus (NoV) contamination of mussels, clams and oysters harvested in two class B harvesting areas of the delta of the Po river, to choose a species as an indicator. METHODS AND RESULTS: Environmental parameters (temperature and salinity) and hydrometric levels of the tributary river were measured. Seventy shellfish samples (35 samples per area) were examined for Escherichia coli and NoV (GI and GII). NoV contamination was found in 51.4% of samples, of which, 2.9% contained only NoV GI, 14.3% only NoV GII, while the majority of the samples (34.3%) contained both genogroups. Most of the positive results (90.0%) were obtained in the period between November 2008 and April 2009. CONCLUSIONS: No significant differences were found between the results from the two harvesting areas and the three shellfish species. However, on the basis of the average C(t) values, the recovery rate (from 0.46 to 1.15%) and the distribution of positive results in the samplings, mussels seem to be a suitable indicator species to monitor viral contamination in these areas. SIGNIFICANCE AND IMPACT OF THE STUDY: The data allow the optimization of monitoring plans to improve the prevention strategies in terms of money and time, by the intensification of controls in the cold season and the use of one species as indicator.

Pulsed-field gel electrophoresis and PCR characterization of environmental Vibrio parahaemolyticus strains of different origins.

The present study used pulsed-field gel electrophoresis (PFGE) characterization to examine the intraspecies variability and genetic relationships among environmental isolates of Vibrio parahaemolyticus from different European countries. This is first study performed on environmental V. parahaemolyticus that included more than one European country.

Norovirus detection in shellfish using two Real-Time RT-PCR methods.

Shellfish are recognized as a potential vehicle of viral diseases. The aim of the present study was to determine the ability of two real-time RT-PCR methods (an in-house method and a commercial kit) for detecting Norovirus (NoV) belonging to genogroups GI and GII in shellfish. The analyses were performed both on a Norovirus Reference Panel (NRP), consisting of synthetic RNA, and on naturally contaminated mussels. For the experiments carried out on the NRP a statistically significant difference (?2=8.03) was shown between the results obtained by the two methods. The in-house real-time RT-PCR allowed the detection of all genotypes belonging to GI and GII, while the commercial kit was not suitable for the detection of the majority of the GI sequences constituting the panel. No significant difference was instead detected in the experiments carried out on shellfish, where the presence of GI was always concomitant with GII. Both methods were suitable for detection of NoV in shellfish, however the in-house real-time RT-PCR method had the advantage of differentiating GI and GII contamination. As regards the shellfish analysed, a considerable frequency of NoV contamination (34.4% of the samples) was detected, with a predominance of NoV GII.

Local insulin-like growth factor I expression is essential for Purkinje neuron survival at birth.

IGF1, an anabolic and neuroprotective factor, promotes neuronal survival by blocking apoptosis. It is released into the bloodstream by the liver, or synthesized locally by muscles and neural cells, acting in an autocrine or paracrine fashion. Intriguingly, genetic studies conducted in invertebrate and murine models also suggest that an excess of IGF1 signaling may trigger neurodegeneration. This emphasizes the importance of gaining a better understanding of the mechanisms controlling IGF1 regulation and gene transcription. In the cerebellum, Igf1 expression is activated just before birth in a subset of Purkinje cells (PCs). Mice carrying a null mutation for HLH transcription factor EBF2 feature PC apoptosis at birth. We show that Igf1 is sharply downregulated in Ebf2 null PCs starting before the onset of PC death. In vitro, EBF2 binds a conserved distal Igf1 promoter region. The pro-survival PI3K signaling pathway is strongly inhibited in mutant cerebella. Finally, Ebf2 null organotypic cultures respond to IGF1 treatment by inhibiting PC apoptosis. Consistently, wild type slices treated with an IGF1 competitor feature a sharp increase in PC death. Our findings reveal that IGF1 is required for PC survival in the neonatal cerebellum, and identify a new mechanism regulating its local production in the CNS.

Influenza A H1N1 and severe asthma exacerbation.

The pandemic influenza A H1N1 will affect millions of subjects. This influenza can cause respiratory complications with possible death. We have described two case reports of acute severe asthma exacerbation combined to influenza A H1N1, caracterized by severe respiratory failure. The diagnosis of influenza A H1N1 was confirmed with the multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay. These patients, apart from asthma, do not have other diseases; but they did not take adequate therapy. In addition to conventional therapy (corticosteroids, bronchodilator and antibiotics) oseltamivir 75 mg bid was immediately added. After few days the patients improved and therefore in a short time they were discharged. During this period, in the case of severe asthma exacerbations, one must always think of influenza A H1N1 as the possible cause. It is necessary to use oseltamivir precociously to avoid severe complications. All asthmatic patients must regularly take their therapy especially during pandemic influenza A H1N1.

Purkinje cell phenotype restricts the distribution of unipolar brush cells.

Cerebellar unipolar brush cells (UBCs) are glutamatergic interneurons of the granular layer. Previous studies have identified three distinct UBC subsets in the mouse cerebellar cortex: one expressing the calcium-binding protein calretinin (CR), a second expressing both the metabotropic glutamate receptor (mGluR)1alpha and phospholipase C(PLC)beta4, and a third expressing PLCbeta4 but not mGluR1alpha. We have investigated UBC topography in two strains of mutant mice: early B-cell factor 2 (Ebf2) null and scrambler. In Ebf2 null mice Purkinje cell topography is disrupted due to Purkinje cell death and ectopic gene expression. The topography of all three classes of UBCs is also abnormal: the CR(+) UBCs, which are normally aligned with zebrin II stripes, become homogeneously distributed; the numerical density of mGluRlalpha(+) UBCs is increased; and many PLCbeta4(+) UBCs are located ectopically. The UBC ectopia is not a cell-intrinsic action of the Ebf2 gene-analysis of the constitutive expression of a beta-galactoside reporter under the control of the Ebf2 promoter reveals no Ebf2 expression in UBCs at any stage of cerebellar development. In scrambler (Dab1(scm)), most Purkinje cells are ectopic but nevertheless have normal adult gene expression patterns. In scrambler, UBCs associate with specific ectopic Purkinje cell clusters. Finally, similar associations with specific Purkinje cell clusters are seen during normal cerebellar development. These data suggest that UBCs become regionally restricted during development through a non-cell-autonomous mechanism involving embryonic interactions with different Purkinje cell subtypes.

Climate change and food safety: an emerging issue with special focus on Europe.

According to general consensus, the global climate is changing, which may also affect agricultural and livestock production. The potential impact of climate change on food security is a widely debated and investigated issue. Nonetheless, the specific impact on safety of food and feed for consumers has remained a less studied topic. This review therefore identifies the various food safety issues that are likely to be affected by changes in climate, particularly in Europe. Amongst the issues identified are mycotoxins formed on plant products in the field or during storage; residues of pesticides in plant products affected by changes in pest pressure; trace elements and/or heavy metals in plant products depending on changes in their abundance and availability in soils; polycyclic aromatic hydrocarbons in foods following changes in long-range atmospheric transport and deposition into the environment; marine biotoxins in seafood following production of phycotoxins by harmful algal blooms; and the presence of pathogenic bacteria in foods following more frequent extreme weather conditions, such as flooding and heat waves. Research topics that are amenable to further research are highlighted.

Erythromycin resistance in Streptococcus pyogenes and macrolide consumption in a central Italian region.

BACKGROUND: The study investigated macrolide resistance in Streptococcus pyogenes in a central Italian area from 2001 to 2006 and the possible correlation between antibiotic consumption and fluctuations of resistance percentages. MATERIALS AND METHODS: Macrolide and lincosamide susceptibility of 1,419 S. pyogenes isolates was tested by Kirby Bauer method. Macrolide consumption was evaluated as defined daily dose/1,000 inhabitants per day (DID), according to the World Health Organization anatomic therapeutic chemical classification. Spearman's correlation coefficient was used to assess the association between resistance and use of (1) all macrolides pooled, (2) once daily, (3) twice daily, and (4) three times daily dosage regimens. RESULTS: : In total, 320 strains (22.6%) were erythromycin-resistant, 11.4% with the M phenotype and 11.2% with the MLS phenotype. There was a significant decrease in erythromycin resistance during the study period-from 28.1% in 2001 to 15.6% in 2006 (p < 0.01). No significant correlation was found between erythromycin resistance and local overall macrolide consumption, neither during the same year nor during the previous year. In contrast, a significant correlation was found between resistance rates and once-daily macrolide use during the preceding 6 months in Siena r = 0.747, p = 0.008). CONCLUSION: The known greater selective effect of long-acting agents could establish a pressure outcome, resulting in a specific local epidemiology during a relatively short time gap.

A bienzyme electrochemical probe for flow injection analysis of okadaic acid based on protein phosphatase-2A inhibition: an optimization study.

A bienzyme electrochemical probe has been assembled and used to monitor the inhibition of the enzyme protein phosphatase-2A (PP2A) by okadaic acid (OA), taking advantage of the particular characteristics of a biochemical pathway in which PP2A is involved. This enzyme has significant activity toward glycogen phosphorylase a (PHOS a), which in turn catalyzes the conversion of glycogen to glucose-1-phosphate (G-1-P). In addition, PP2A is strongly inhibited by OA and its derivatives. Due to this combination of properties, PP2A was employed to develop an assay system involving a preliminary phase of off-line enzymatic incubations (OA/PP2A, PP2A/PHOS a, PHOS a/glycogen+phosphate). This off-line step was followed by the electrochemical detection of H2O2, which is the final product of two sequential enzymatic reactions: G-1-P with alkaline phosphatase (AP) producing glucose, then glucose with glucose oxidase (GOD) producing hydrogen peroxide. These two enzymes were coimmobilized on a nylon net membrane that was placed over an H2O2 platinum probe inserted into a flow injection analysis (FIA) system. During a first phase of the study, all analytical parameters were optimized. During a subsequent phase, the inhibition of PP2A enzyme by OA was evaluated. The calibration of the system shows a working range for detection of OA between 30 and 250 pg ml(-1). The total analysis time is the sum of 50 min for the off-line enzymatic incubations and 4 min for the biosensor response.

Occurrence of enteric viruses in shellfish and relation to climatic-environmental factors.

AIMS: To investigate the presence of enteric viruses [hepatitis A (HAV) and norovirus (NoV)] in shellfish harvested from the deltaic area of the Po river in relation to environmental factors. METHODS AND RESULTS: Fortnightly sampling of shellfish was carried out in two lagoon areas (category B production areas) and one sea area (category A). Environmental parameters in the lagoon and hydrometric level of the tributary river were monitored throughout the sampling period. Samples (n = 120) were analysed for bacterial (E. coli and Salmonella) and viral (HAV and NoV) contamination; samples from category B areas were analysed before and after purification treatment. All the samples were negative for HAV whereas 10 samples (8.3%), all harvested in the lagoon areas, were positive for NoV. Sequencing identified the strains as genotypes II.4 and II.b. None of the samples was found to be contaminated after depuration. CONCLUSIONS: The monitoring showed a low frequency of NoV presence; viral contamination, detected exclusively in shellfish collected from the deltaic area (category B), could be influenced by the flow of the tributary river. The data collected are useful for the design of targeted prevention strategies and for the modulation of control plans after meteorological events.

Purkinje cell subtype specification in the cerebellar cortex: early B-cell factor 2 acts to repress the zebrin II-positive Purkinje cell phenotype.

The mammalian cerebellar cortex is highly compartmentalized. First, it is subdivided into four transverse expression domains: the anterior zone (AZ), the central zone (CZ), the posterior zone (PZ), and the nodular zone (NZ). Within each zone, the cortex is further subdivided into a symmetrical array of parasagittal stripes. The most extensively studied compartmentation antigen is zebrin II/aldolase c, which is expressed by a subset of Purkinje cells forming parasagittal stripes. Stripe phenotypes are specified early in cerebellar development, in part through the action of early B-cell factor 2 (Ebf2), a member of the atypical helix-loop-helix transcription factor family Collier/Olf1/EBF. In the murine cerebellum, Ebf2 expression is restricted to the zebrin II-immunonegative (zebrin II-) Purkinje cell population. We have identified multiple cerebellar defects in the Ebf2 null mouse involving a combination of selective Purkinje cell death and ectopic expression of multiple genes normally restricted to the zebrin II- subset. The nature of the cerebellar defect in the Ebf2 null is different in each transverse zone. In contrast to the ectopic expression of genes characteristic of the zebrin II+ Purkinje cell phenotype, phospholipase Cbeta4 expression, restricted to zebrin II- Purkinje cells in control mice, is well maintained, and the normal number of stripes is present. Taken together, these data suggest that Ebf2 regulates the expression of genes associated with the zebrin II+ Purkinje cell phenotype and that the zebrin II- Purkinje cell subtype is specified independently.

Epidemiological and virological characterization of a large community-wide outbreak of hepatitis A in southern Italy.

A large outbreak of hepatitis A virus (HAV) infection occurred in 2004 in Campania, a region of southern Italy, with 882 cases reported between 1 January and 1 August. The local public health authorities and the Italian National Institute of Health carried out investigations in order to characterize the agent, identify the source of infection and the route of transmission, and implement appropriate control measures. A web-based reporting system enhanced the flow of information between public health authorities, providing real-time epidemic curves and frequency distributions. The same 1B HAV genotype was found in 90% of sera from a subset of patients with acute disease, suggesting a local common source. A case-control study in the municipality with the highest attack rate showed that raw seafood consumption, in particular if illegally sold in water, was strongly associated with HAV illness. Samples of seafood systematically collected from retailers were found contaminated by HAV.

Comparison of different biochemical and molecular methods for the identification of Vibrio parahaemolyticus.

AIMS: Multicentre evaluation of biochemical and molecular methods for the identification of Vibrio parahaemolyticus. METHODS AND RESULTS: For the biochemical identification methods, API 20E and API 20NE and Alsina's scheme were evaluated in intra- and interlaboratory tests in order to determine the accuracy and concordance of each method. Both in intra- and interlaboratory tests, the Alsina's scheme showed the highest sensitivity (86% of correct identifications in the interlaboratory test). False-positive results were obtained by all methods (specificity was 95% for API 20E, 73% for API 20NE and 84% for Alsina's scheme) and concordance varied from 65% of API 20NE to 84% of API 20E. For the molecular identifications, polymerase chain reaction (PCR) for the detection of toxR gene, tl gene and pR72H fragment were tested on 30 strains by two laboratories. The PCR for toxR showed the highest inclusivity (96%), exclusivity (100%) and concordance (97%). CONCLUSIONS: Among the biochemical identification methods tested, the Alsina's scheme gave more reliable results; however, in order to avoid false-positive results, all the biochemical identifications should be confirmed by means of molecular methods. SIGNIFICANCE AND IMPACT OF THE STUDY: Availability of an efficient identification method of Vibrio parahaemolyticus to use in official control of fisheries products.

Behavior of aeromonas hydrophila in bottled mineral waters.

The growth and survival of Aeromonas hydrophila in three types of natural mineral waters were investigated. Mineral waters with different levels of mineral content (low, medium, and high) were experimentally contaminated with A. hydrophila, stored at different temperatures (10 degrees C and 20 degrees C), and analyzed at intervals over a 60-day period. Water samples that were not experimentally contaminated were investigated for indigenous A. hydrophila. The results confirmed that A. hydrophila may occur naturally in mineral waters and showed that the level of mineral content, temperature, length of storage, and, in some cases, the type of container used may favor the growth of A. hydrophila. The greatest proliferation was observed in water with a low mineral content stored in PET bottles at 10 degrees C, in which A. hydrophila peaked at day 28 (4.47 +/- 0.01 log CFU/100 ml). At 20 degrees C, the same load was observed at day 60. The presence of high densities of A. hydrophila in bottled mineral water can constitute a risk for some groups of consumers, such as elderly and immunocompromised persons.

Detecting the presence of infectious hepatitis A virus in molluscs positive to RT-nested-PCR.

AIMS: The objective of this study was to determine the presence of infectious hepatitis A virus (HAV) in molluscs naturally contaminated with viral HAV-RNA. METHODS AND RESULTS: One hundred and forty-two mollusc samples were analysed for the presence of viral HAV-RNA using RT-nested-PCR; positive samples were then analysed with an integrated method, cell-culture RT-PCR, to detect infectious virus. Viral HAV-RNA was detected in 34.5% of the samples while 12.7% of the total samples were positive for the presence of infectious virus. CONCLUSIONS: The results demonstrate the validity of the screening method (RT-nested-PCR) and the necessity of applying a method that is capable of detecting the presence of infectious HAV. SIGNIFICANCE AND IMPACT OF THE STUDY: The study demonstrates that in any case, to determine the safety for human consumption, the results of RT-nested-PCR must be confirmed with an integrated cell-culture PCR method.

Closed-circuit system for the depuration of mussels experimentally contaminated with hepatitis A virus.

In Italy, the consumption of raw or slightly cooked mussels represents the most important risk factor for the transmission of hepatitis A virus (HAV). Although there exist effective methods for the bacterial depuration of contaminated mussels, these methods are poorly effective on enteric viruses. The objective of the present study was to evaluate the effectiveness of a closed-circuit depuration system that uses both ozone and UV light for disinfecting water and that allows salinity and temperature, important parameters for the metabolism of mussels (Mytilus galloprovincialis), to be maintained at constant levels. The results showed that this depuration method decreased the viral load (from 1.72 log 50% tissue culture infective dose [TCID50] ml(-1) to <1 log TCID50 ml(-1) within 24 h and from 3.82 log TCID50 ml(-1) to <1 log TCID50 ml(-1) within 48 h). However, in both cases, after 120 h of depuration, a residual amount of virus capable of replicating in cells was detected. These results show that depuration, even if performed with advanced systems, may not guarantee the absence of virus.

Xebf3 is a regulator of neuronal differentiation during primary neurogenesis in Xenopus.

During primary neurogenesis in Xenopus, a cascade of helix--loop--helix (HLH) transcription factors regulates neuronal determination and differentiation. While XNeuroD functions at a late step in this cascade to regulate neuronal differentiation, the factors that carry out terminal differentiation are still unknown. We have isolated a new Xenopus member of the Ebf/Olf-1 family of HLH transcription factors, Xebf3, and provide evidence that, during primary neurogenesis, it regulates neuronal differentiation downstream of XNeuroD. In developing Xenopus embryos, Xebf3 is turned on in the three stripes of primary neurons at stage 15.5, after XNeuroD. In vitro, XEBF3 binds the EBF/OLF-1 binding site and functions as a transcriptional activator. When overexpressed, Xebf3 is able to induce ectopic neurons at neural plate stages and directly convert ectodermal cells into neurons in animal cap explants. Expression of Xebf3 can be activated by XNeuroD both in whole embryos and in animal caps, indicating that this new HLH factor might be regulated by XNeuroD. Furthermore, in animal caps, XNeuroD can activate Xebf3 in the absence of protein synthesis, suggesting that, in vitro, this regulation is direct. Similar to XNeuroD, but unlike Xebf2/Xcoe2, Xebf3 expression and function are insensitive to Delta/Notch-mediated lateral inhibition. In summary, we conclude that Xebf3 functions downstream of XNeuroD and is a regulator of neuronal differentiation in Xenopus.

Reduced fertility and spermatogenesis defects in mice lacking chromosomal protein Hmgb2.

High mobility group 2 protein (Hmgb2) is a member of the HMGB protein family, which includes the ubiquitous Hmgb1 and the embryo-specific Hmgb3. The three proteins are more than 80% identical at the amino acid level and their biochemical properties are indistinguishable. Hmgb1 is an abundant component of all mammalian nuclei and acts as an architectural factor that bends DNA and promotes protein assembly on specific DNA targets. Cells that lack Hmgb1 can survive, although mutant mice die shortly after birth. As Hmgb2 is present in all cultured cells and is abundant in thymus, the preferred source for HMGB proteins, it was considered a ubiquitous variant of Hmgb1. We show that in adult mice Hmgb2 is restricted mainly to lymphoid organs and testes, although it is widely expressed during embryogenesis. Mice that lack Hmgb2 are viable. However, male Hmgb2(-/-) mice have reduced fertility, that correlates with Sertoli and germ cell degeneration in seminiferous tubules and immotile spermatozoa. Significantly, Hmgb2 is expressed at very high levels in primary spermatocytes, while it is barely detectable in spermatogonia and elongated spermatids. This peculiar pattern of expression and the phenotype of mutants indicate that Hmgb2 has a specialised role in germ cell differentiation.