In this study we assessed the seroprevalence of hepatitis E virus (HEV) infection in both the Italian population and immigrants from developing countries in Foggia (Apulia, Southern Italy). The seroprevalence of HEV was determined in 1217 subjects [412 (34%) immigrants and 805 Italian subjects (blood donors, general population, HIV-positive, haemodialysis patients)]. Serum samples were tested for anti-HEV and confirmed by Western blot assay; in positive patients HEV RNA and genotype were also determined. There were 8·8% of patients that were positive to anti-HEV, confirmed by Western blot. The prevalence in immigrants was 19·7%, and in Italians 3·9% (blood donors 1·3%, general population 2·7%, HIV-positive patients 2·0%, haemodialysis patients 9·6%). Anti-HEV IgM was found in 38/107 (35·5%) of the anti-HEV-positive serum samples (34 immigrants, four Italians). This study indicates a higher circulation of HEV in immigrants and Italian haemodialysis patients, whereas a low prevalence of HEV antibodies was seen in the remaining Italian population.

Background: Hepatitis E virus is the etiologic agent of an enterically transmitted viral hepatitis associated with inadequate hygienic conditions.HEV is endemic in developing countries where it occurs in sporadic and endemic form, but autochthonous sporadic cases of hepatitis E have been reported in North America and in Europe, including Italy.The aim of the present study is to assess the seroprevalence of antibodies to HEV in autochthonous Italian population and in immigrants from developing countries in the province of Foggia.Methods: The seroprevalence of HEV was determined in a cohort of 1217 subjects, 412 (34%) were asymptomatic immigrants (mostly from countries in Sub-Saharan Africa) who had recently arrived in Italy and 805 were four different Italian populations (volunteer blood donors, normal healthy individuals, HIV-positive patients, hemodialysis patients).Serum samples were tested for anti-HEV by a commercial enzyme immunoassay (EIA) based on recombinant proteins; repeatedly positive obtained results were confirmed with a Western Blot assay (Recomblot HEV).Results: A total of 107 (8.8%) of the 1217 serum samples examined were reactive to anti-HEV IgG and confirmed by Western Blot.The prevalence in immigrants was 19.6%, in Italians was 4.0% (blood donors 1.3%, normal healthy individuals 3.3%, HIV-positive patients 3.0%, hemodialysis patients 11.5%) Anti-HEV IgM was found in 20/107 subjects (18.7%) of the anti-HEV IgG positive serum samples, 17 in immigrants and 3 in Italians.All anti-HEV positive subjects were asymptomatic and alanine transferase values were elevated only in 8/20 patients with anti-HEV IgM-positive. Conclusions:The results of this study indicate a high circulation of HEV in immigrant population and hemodialysis patients, whereas the prevalence of HEV antibodies was low in the other Italian population, compared to that of other European countries.

We have recently demonstrated that endothelial cells cultured on Gelfoam blocks, but not monolayer matrices can phagocytose and kill Staphyloccus aureus. Experiments determined that penicillin G, included in the endothelial cell growth medium, induces these cells to exhibit the observed bactericidal activity. In this communication, we report on studies aimed at elucidating the mechanism by which penicillin G-induced endothelial cells, cultured on Gelfoam blocks, kill S. aureus. Despite the fact that there is a substantial literature that demonstrates neutrophilic killing of bacteria can be mediated through free radical-dependent and free radical-independent mechanisms, considerably less is known about pathways by which endothelial cells can catalyze similar microbicidal activities. Studies described herein point to the fact that superoxide and products derived from this free radical were not responsible for endothelial killing of S. aureus. Likewise, a possible role for nitric oxide in bacterial killing was explored. As part of this inquiry, we stably transduced a NOS-2 encoding retrovirus into endothelial cells cultured on Gelfoam blocks in the absence of penicillin G. Even though these cells secreted nitric oxide at a rate of 0.5 μM/h per 1 × 106 cells, similar to what has been reported for murine macrophages induced with γ-interferon, in our model, nitric oxide was not found to kill S. aureus. Data presented demonstrate that the microbicidal activity of endothelial cells is mediated through free radical-independent pathways.

A body of evidence has surfaced documenting the ability of endothelial cells cultured on mono-layers to phagocytose but not kill bacteria. Several years ago, a new three-dimensional endothelial cell culturing model was developed, which simulated the morphology of the endothelium in small vessels and capillaries. Given that endothelial cellsmay be derived from the same pluripotent stem cells as macrophages, the question of whether endothelial cells might phagocytose and kill bacteria was explored. Endothelial cells grown on Gelfoam blocks exhibited bactericidal activity towards Staphylococcus aureus, reaching maximal killing of >90% after 2 h. Evidence documents the involvementof bacterial adherence to the plasma membrane of the endothelial cell. This is followed by phagocytosis of S. aureus, leading to intracellular killing. Penicillin G, included in the endothelial cell growth medium, was found to be a critical factor in the bactericidal activity demonstrated by Gelfoam blocks laden with endothelial cells.

The spin trap 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) alone, as well as DMPO or N-tert-butyl-α-phenylnitrone (PBN) in the presence of excess dimethyl sulfoxide (Me2SO), have been used as spin trapping systems for the detection of hydroxyl radical. However, the instability of DMPO and many of its corresponding spin-trapped adducts has limited the usefulness of this spin trap, particularly in biological systems. Spin trapping of multiple free radicals by the PBN/Me2SO system may undermine the sensitivity of this method to detect small, yet biologically significant amounts of hydroxyl radical. The present study was undertaken to select a spin trapping system with greater sensitivity and selectivity toward ·OH than DMPO, DMPO/Me2SO, or PBN/Me2SO. We report that α-hydroxyethyl radical, resulting from the reaction of photolytically generated ·OH with excess ethanol is spin trapped by 4-pyridyl-1-oxide-N-tert-butylnitrone (4-POBN) with a second-order rate constant nearly 10-fold greater than that for DMPO or PBN. In contrast to DMPO spin-trapped ad-ducts, the α-hydroxyethyl radical adduct of 4-POBN, 4-POBN-CH(CH3)OH, is resistant to reduction by superoxide, even in the presence of cysteine. The efficiency of spin trapping and the marked stability of the resulting spin-trapped adduct confer a high degree of sensitivity and demonstrate the potential application of 4-POBN/ETOH toward the detection of hydroxyl radical in biological systems.

Conventional methods of endothelial cell culture on monolayers and beads require enzymatic digestion, traumatic scraping, or centrifugation to transfer cells to other experimental systems. Gelfoam, a porous gelatin block, not only supports the growth of bovine pulmonary artery endothelial cells but also allows the rapid transfer of cell-laden blocks from one experimental system to another with minimal intervention. This property has been shown to be especially useful for the rapid fixation of endothelial cells for microscopy using standard histologic methods. Histology confirmed that the trabecular nature of the substrate allows endothelial cells to line the interstices of the sponge matrix and grow in a configuration that simulates the appearance of the endothelium in small vessels and capillaries. The inoculation of 1 x 10(5) endothelial cells on 7.5 mg Gelfoam (24 x 8 x 2 mm blocks) was enhanced by fibroblast growth factor and resulted in cell attachment by day 2 with a cell doubling time of 1.7 days. In addition, endothelial cells completely infiltrated 1, 5 and 7.5 mg Gelfoam blocks, as verified by histology. Assays to quantify cell number and protein were easily performed. To facilitate cell counting, the Gelfoam matrix was rapidly removed by the addition of 0.05 mg/ml collagenase, a concentration that interfered minimally with the assay for cellular protein concentration. The data demonstrate that Gelfoam is a suitable support growth matrix for the in vitro culture of bovine pulmonary artery endothelial cells.

Enzymatic production of prostaglandins (PGs) from exogenous arachidonic acid was studied in brain microsomal fractions prepared from mice following pentylenetetrazol (PTZ)-induced convulsions. Prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α) measured either by radioimmunoassay or after incubation with [1-14C]arachidonic acid (AA) was significantly increased in microsomes from the convulsed animals. Pretreatment of the mice with the anticonvulsant ethosuximide prevented the enhanced PG production. The increased PG synthesis could not be attributed to an increased substrate availability nor to an activated phospholipase nor to a direct effect of the convulsant on the fatty acid cyclooxygenase. Evidence that a modification of the cyclooxygenase had occured with seizure activity was obtained from kinetic analysis; the apparent Km for the AA was lowered from 30 ± 3 μM in the controls to 12 ± 1 μM in the PTZ-treated mice. Further evidence for a modification of the fatty acid cyclooxygenase was obtained from incubations of the microsomes with catalase to reduce peroxide formation. Limiting peroxide levels did not decrease the microsomal cyclooxygenase activity in the PTZ-treated mice to control levels. Seizure activity induced by picrotoxin and strychnine also increased the microsomal capacity of the convulsed animals to synthesize PGs. The increased brain fatty acid cyclooxygenase activity may result from a biochemical modification of the enzyme induced by seizure activity.

Rat lens microsomal preparations possess the capability of converting exogenous arachidonic acid into prostaglandins (PG) E2 and F2 alpha. The low, yet measurable prostaglandin biosynthetic capacity of the lens microsomes was demonstrated by radioimmunoassay (RIA) and by separation of radiolabeled products after incubation with high specific activity U-14 [C]-arachidonic acid. Maximal formation of radioimmunoassayable PGE2 and PGF2 alpha was measured after a 15-min incubation at 37 degrees C with approximately 0.5 mg protein. Prostaglandin biosynthesis was inhibited by the nonsteroidal anti-inflammatory agents aspirin (IC50 = 52 microM) and indomethacin (IC50 = 20 microM). These results unequivocally demonstrate prostaglandin biosynthesis in rat lens.

The effect of inhibiting endogenous brain thromboxane (TXB2) on pentylenetetrazole‐induced seizures was studied using the thromboxane synthetase inhibitors OKY‐1581 (20 mg/kg) and UK 38,485 (50 mg/kg). Both compounds selectively decreased (> 90%) TXB2 production in brain measured after 2 min of convulsive activity but had no effect on brain PGE2, PGF2α, or 6‐keto‐PGF1α No effect of these agents on the tonic seizure threshold was observed, whereas 10 mg/kg ip indomethacin, an agent which inhibits both TXB2 and prostaglandin production, reduced the tonic seizure threshold from 78 ± 2.6 mg/kg in controls to 62 ± 3.7 mg/kg. Thus, this study concludes that the availability of TXB2 with convulsant activity is unlikely to be a factor in altering tonic seizure activity observed with indomethacin.