Endothelial microparticles correlate with erectile dysfunction in diabetic men.

Cell-derived microparticles are supposed to be involved in endothelial dysfunction and atherogenesis. This study aimed to evaluate circulating microparticles in diabetic subjects with erectile dysfunction (ED) and their relation with endothelial dysfunction. Thirty diabetic men with ED and 20 age-matched control subjects without ED were assessed for circulating microparticles and endothelial dysfunction. Flow cytometry was used to assess microparticles by quantification of circulating endothelial (EMP, CD31(+)/CD42b(-)) and platelet (PMP, CD31(+)/CD42b(+)) microparticles in peripheral blood. Endothelium-dependent flow-mediated dilation (FMD) was evaluated in the right brachial artery after reactive hyperemia. Compared with non-diabetic subjects, diabetic men presented significantly higher numbers of EMP (P=0.001), and reduced FMD (P=0.01), with a significant inverse correlation between the number of circulating EMP and the International Index of Erectile Function (IIEF) score (r=-0.457, P=0.01). Multivariate analysis correcting for age, anthropometric indices, glucose and lipid parameters, FMD and PMP identified EMP as the only independent predictor for IIEF score (P=0.03). EMP are elevated in impotent diabetic subjects and independently involved in the pathogenesis of ED.

Neurotoxicity induced by Cefepime in a very old hemodialysis patient.

Neurotoxicity is an unusual complication of cephalosporin therapy. Only few cases of neurotoxicity induced by Cefepime have been described and probably the frequency of Cefepime-induced status epilepticus is underestimated. We report a case of an 82 year-old male, ESRD patient on chronic hemodialysis program affected by pneumonia, who received a treatment with intravenous Cefepime (1 g/day) and developed a seizure 4 days after the starting antibiotic therapy. Cefepime-induced neurotoxicity was suspected and its administration was immediately discontinued. In order to increase Cefepime clearance a hemodialysis session was urgently started and an improvement of his conscious level was observed. On the following day, after a second hemodialysis session his clinical condition and the status of neurotoxicity were completely recovered. The patient was discharged from the hospital in stable clinical condition one week later. At variance with the cases previously reported, the daily dose of Cefepime administrated to our patient was 50% lower and respected drug prescription dosage. Thus, we speculate on the hypothesis that advanced age of our patient and metabolic encephalopathy induced by chronic uremia made him more sensitive to the neurotoxicity induced by the drug. In conclusion, our case suggests that, in very old patients on long-term hemodialysis, it should be considered, to avoid neurotoxicity, to monitor the clinical neurological status, to use Cefepime at lower dosage than that allowed in patients with severe renal impairment (1 g/day) and, when possible, to evaluate Cefepime plasma levels. However, in these patients, other agents of the same class should be considered such as Cefotaxime and Ceftriaxone which are characterized by both an hepatic and renal excretion. In alternative to cephalosporins, antibiotics with the same action spectrum in the absence of neurological toxicity (i.e. Meropenem) should be recommended.

Autonomic dysfunction associates with prolongation of QT intervals and blunted night BP in obese women with visceral obesity.

Prolonged QT intervals and a reduced fall of nocturnal blood pressure (BP) both predict an increased risk of cardiovascular events in obese subjects. We evaluated circadian BP variations (24-h ambulatory BP monitoring), autonomic function (power spectral analysis of RR interval oscillations) and cardiac repolarization times (QTc-dispersion and QTc interval) in 70 obese women, aged 25-44 yr, grouped by WHR into group A (WHR > 0.85, no.=38) and group B (WHR < or = 0.85, no.=32). Compared with non-obese age-matched women (no.=25, BMI=23+/-1.8) and obese women of group B, obese women of group A had higher values of QTc-d (p<0.05) and QTc (p<0.05), an altered sympathovagal balance (ratio of low-frequency/high-frequency power, p<0.01), and a blunted nocturnal drop in BP (p<0.01). In group A, QTc-d and the QTc interval correlated with diastolic night BP (p<0.01) and sympathovagal balance (p<0.01). WHR and plasma insulin levels correlated with QT intervals, reduced nocturnal fall in diastolic BP and sympathovagal balance (p<0.01). Prolongation of cardiac repolarization times and the reduction of nocturnal fall in BP coexist in obese women with visceral obesity, and might contribute to their raised cardiovascular risk. Autonomic dysfunction may be the common mechanism for this association.

Prevalence of TT virus in healthy children and thalassemic pediatric and young adult patients.

BACKGROUND: The recently discovered TT virus (TTV) has been shown to be highly prevalent in patients with cryptogenetic chronic liver disease and fulminant hepatitis. To study the frequency of TTV and to evaluate the possible association with liver disease, 37 pediatric and young adult patients with thalassemia, and 36 healthy children were included in the study. The sera of 100 blood donors selected randomly in the same period were also tested for TTV DNA. METHODS: The TTV amplification by polymerase chain reaction (PCR) was performed using a first set of primers that recognize an internal sequence into N22 and a second set of primers amplifying a sequence within 5;NCR (5; noncoding region). RESULTS: The first set of primers revealed TTV DNA in 73% of thalassemic patients, in 8% of healthy children, and in 5% of healthy blood donors. With the second set of primers, the prevalence of TTV DNA was, respectively, 100% in thalassemic patients, 44.5% in healthy pediatric patients, and 87% in healthy blood donors. All individuals who tested positive for TTV by the first set of primers were also positive by the second primer set. The TTV infection seemed not to be the cause of altered transaminase levels. Sequencing of TTV clones from thalassemic patients showed the presence of different TTV variants in the same serum. CONCLUSION: The prevalence of TTV in polytransfused children is similar to that detected in blood donors. Moreover, TTV can be detected in healthy children of all ages. The presence of TTV seems to have no clinical significance.

Mapping of protein domains of hepatitis A virus 3AB essential for interaction with 3CD and viral RNA.

The small hydrophobic protein 3AB of the picornaviruses, encompassing the replication primer 3B, has been suggested to anchor the viral replication complex to membranes. For hepatitis A virus (HAV) 3AB, we have previously demonstrated its ability to form stable homodimers, to bind to membranes, and to interact specifically with RNA, implicating its multiple involvement in viral replication. In the present report, we show that HAV 3AB additionally interacts with HAV protein 3CD, a feature also described for the corresponding polypeptide of poliovirus. By assessing the interactions of three deletion mutants, distinct domains of HAV 3AB were mapped. The hydrophobic domain and the 3B moiety were found to be essential for the 3AB interaction with 3CD. Both electrostatic and hydrophobic forces are involved in this interaction. The cluster of charged amino acid residues at the C terminus of 3A seems to determine the specificity of 3AB interaction with RNA structures formed at either terminus of the HAV genome. Furthermore, our data implicate that 3A can interact with HAV RNA. Compared with poliovirus 3AB, which by itself is a nonspecific RNA-binding protein, HAV 3AB specifically recognizes HAV RNA structures that might be of relevance for initiation of viral RNA replication.

Prevalence of TT virus in plasma pools and blood products.

A high prevalence of TT virus (TTV), a novel virus recently identified in the serum of a patient with post-transfusion hepatitis of unknown aetiology, has been reported in blood donors worldwide. We investigated the presence of TTV DNA in several lots of blood products and in the corresponding plasma pools. In the process, we determined, from three sets of primers, the one which was most efficient in detecting the viral nucleic acid. This set amplifies the region closest to the 3'-end of the TTV genome which was proved, by sequence analysis, to be more conserved than the other two regions. Whereas all 10 intravenous immunoglobulin and 21 albumin batches were TTV negative, 4/5 factor VIII concentrates and 4/10 intramuscular immunoglobulin batches were TTV positive. A high prevalence of TTV DNA (70%) was found in the plasma pools that were collected from four different countries. These results confirm the worldwide distribution of this virus and show that TTV is removed with a varying efficiency during the manufacture of blood products.

Detection of hepatitis A virus in shellfish by nested reverse transcription-PCR.

A method for the detection of HAV in shellfish, based on the use of guanidinium isothiocyanate-containing solution for RNA extraction and purification steps, followed by nested PCR, is hereby proposed. Tests were carried out on mollusc samples spiked with HAV strain FG. Results showed that in samples subjected only to one round of PCR it was possible to detect HAV at concentrations of 10(3)-10(4) TCID50/10 g of mollusc. The use of the nested PCR renders the system more sensitive and specific enabling the identification of HAV concentrations as low as 1 TCID50/10 g of mollusc. Furthermore thus method, in addition to allowing the avoidance of confirming tests, such as hybridization, proved to be inexpensive and simple to perform.

Polypeptide 3AB of hepatitis A virus is a transmembrane protein.

Hepatitis A virus (HAV) protein 3AB is a membrane-interacting protein containing a stretch of 21 hydrophobic amino acid residues. The nature of its membrane association was studied in detail by analysing various deletion mutants. In vivo and in vitro expression of the wild-type protein and its mutants allowed to demonstrate that the hydrophobic domain interacts with membranes and to define the portions essential for this feature. Furthermore, the results suggest that 3AB behaves as an integral membrane protein. Expression in Escherichia coli showed that 3AB can be isolated, in association with membranes, both in monomeric and in dimeric form. This finding was confirmed in vitro after post-translational incubation of the protein with microsomal membranes. Analysis of deletion mutants demonstrated that the dimerization region colocalises with the hydrophobic transmembrane domain, implicating that HAV 3AB could form oligomers mediated by the interaction of transmembrane alpha-helices.

Application of reverse transcriptase-nested-PCR for detection of poliovirus in mussels.

In order to identify polioviruses in molluscs, we hereby propose a method based on precipitation with PEG 6000 followed by the use of a commercial kit (RNAfast II-Molecular System-San Diego) for the extraction and purification of viral RNA. The RT-PCR phase is followed by a second amplification using nested primers to increase the sensitivity and specificity of the method. Tests were carried out on mollusc samples spiked with Poliovirus 1. Results showed that in samples subjected only to one round of PCR it was possible to detect Poliovirus concentrations as small as 10(3)TCID50/ml. The use of nested-PCR makes the system more sensitive and specific enabling the identification of Poliovirus concentrations as small as 1 TCID50/ml.

Site-directed mutagenesis of hepatitis A virus protein 3A: effects on membrane interaction.

Due to a stretch of hydrophobic amino acids, protein 3A of hepatitis A virus (HAV) has been suggested to act as a membrane anchor or a carrier of the genome-linked protein 3B (VPg) during viral RNA synthesis. Mutagenesis analysis was performed in order to elucidate the role of the N- and C-terminal tracts of protein 3A in cell membrane interaction. Expression of the mutated proteins in E. coli cells demonstrated that the presence of positively charged residues at the C-terminus is not required for membrane anchoring. Changes in the primary sequence involving charged amino acids at the N- and C-termini critically influenced the ability of the protein 3A of a cytopathic strain of HAV to change bacterial membrane permeability. This result demonstrates the strict correlation between the structure and pore-forming potential of HAV protein 3A.

Focal gastric blood perfusion in relation with the endoscopic signs and liver function in cirrhotic patients.

Focal blood perfusion was measured, by means of a laser-Doppler technique, in the gastric upper body of 70 patients with liver cirrhosis and of 33 noncirrhotic controls. Perfusion was found to be lower in patients with pink mosaic-like pattern as compared to controls (p < 0.001). On the contrary, patients with red spots showed a marked increase of the focal gastric blood flow (p < 0.001). No different blood flow values were found between patients with red mosaic-like pattern and controls. Multiple regression showed that focal gastric blood flow perfusion was independently related to the Child score (p < 0.003), suggesting that gastric hemodynamic abnormalities can be favored by functional decompensation of cirrhosis, whereas there was no independent correlation with esophageal varices size, as assessed by their lumen occupancy percentage. Such observations may contribute to the understanding of the pathophysiology of gastric wall lesions in liver cirrhosis

Age affects glutathione content and glutathione-transferase activity in human gastric mucosa.

This study was undertaken to evaluate the influence of age on the content of glutathione, and its amino-acid precursor cysteine and on the activity of glutathione-S-transferase of gastric mucosa in man. We examined 44 gastric mucosal samples taken from the body and the antrum of the stomach of 22 healthy subjects, aged between 19 and 65 years. The results were examined in relationship to their distribution in the stomach, to the sex and to the age of the subjects. Glutathione and glutathione-S-transferase were higher in the gastric body than in the antrum, without differences between males and females. The activity of glutathione-S-transferase was directly related to glutathione content and both decreased with age. Cysteine was not influenced by any of the factors considered. These data indicate that the antioxidative and detoxifying capability of gastric mucosa decreases with age in man.

Effect of liver cirrhosis and age on the glutathione concentration in the plasma, erythrocytes, and gastric mucosa of man.

GSH and its related enzymes are one of the protective mechanisms vs. the oxidative damage, both in the circulation and in various tissues, including gastric mucosa. Patients with liver cirrhosis frequently suffer from a gastropathy caused by portal hypertension and they present low circulating levels of GSH. Aging processes cause an increase of gastric damage, of lipoperoxidative phenomenons, and a decrease of GSH in animals. The aim of this study was the evaluation, in humans, of the effect of both these factors, age and liver cirrhosis, on the global pool of GSH and on the antioxidant capability of the cells of gastric mucosa. Therefore, we evaluated the effect of liver cirrhosis and age on the circulating levels of GSH, both in the plasma and in the erythrocytes, and the GSH concentration and the activity of the total GSH-transferase (GSH-T) in gastric mucosa of healthy subjects and in patients affected by liver cirrhosis. Age, but not liver cirrhosis, induced a significant decrease of GSH and GSH-T activity in gastric mucosa; on the contrary, the plasma levels of GSH decreased in cirrhotics but not in elderly healthy subjects. In the erythrocytes, GSH was affected by both these factors (age and liver cirrhosis). These findings indicate that both in patients with liver disease and in elderly healthy subjects the GSH-related cellular defensive mechanisms are depressed and therefore susceptibility to oxidative damage may increase.

Recombinant expression of hepatitis A virus protein 3A: interaction with membranes.

The function of hepatitis A virus (HAV) protein 3A and its structural requirements were studied in vitro and in a bacterial expression system by comparing the polypeptide precursor 3AB derived from a cytopathogenic strain with that of an attenuated strain. Although the precursor polypeptides 3AB of both HAV strains bind to microsomal membranes after translation in vitro they differ in inducing membrane permeability when expression is induced in bacteria. Intake and release of macromolecules was dramatically increased when 3AB of the cytopathogenic strain was expressed. Amino acid sequence alignments suggest that membrane binding might be due to a hydrophobic stretch near the C-terminus of 3A found in all picornaviruses whereas the ability to induce permeability of E. coli membranes is determined by an amphipathic helix formed at the N-terminus of 3A of HAVFG.

Complete nucleotide sequence of a cytopathic hepatitis A virus strain isolated in Italy.

The molecular basis of the cytopathic effect induced in cell culture by some hepatitis A virus (HAV) strains and variants has not been determined. In order to assess the molecular mechanism(s) underlying this particular phenotype the genome of an Italian cytopathic isolate (strain FG) was sequenced from cDNAs obtained by RT-PCR. Sequence analysis revealed the presence of mutations common to either adapted or cytopathic variants of HAV. In particular, amino acid deletions in proteins VP1 and 3A were detected. Expression of protein 3A in E. coli showed that the N-terminal deletion renders this protein toxic to bacteria.

Chemical sclerosing cholangitis after injection of scolicidal solution.

Sclerosing cholangitis may be due to developmental immunological, infective, vascular or chemical factors (1). Hydatid cysts of the liver may communicate with the biliary tree. This is the reason why intracystic injection of scolicidal solution before surgery may cause spreading of the solution into the bile ducts. This complication has already been described in literature (2,3). We present a new well-documented case of sclerosing cholangitis after the injection of formaldehyde into a hydatid cyst of the liver to kill it, and give some suggestions how to avoid this complication.

Mutations in the 3A genomic region of two cytopathic strains of hepatitis A virus isolated in Italy.

Two strains of hepatitis A virus (HAV) were isolated in cell culture and found to induce a cytopathic effect at early passages. The nucleotide sequences of the 5' non-translated region (5'NTR) and of genes 2B, 2C, 3A and 3B were determined for these strains and found to contain mutations similar to those detected in cell-culture adapted variants of HAV strain HM175. In addition, gene 3A shows a deletion of three aspartic acid residues near the N-terminus of the polypeptide. In combination with variations in the 5'NTR and in genes 2B and 2C, the absence of an aspartic acid residue in position 4 of gene 3A of three cytopathic clones of HM175 suggests a possible role of the 3A protein in determining the cytopathic phenotype.

Glutathione prevents ethanol induced gastric mucosal damage and depletion of sulfhydryl compounds in humans.

Whether parenteral administration of reduced glutathione prevented ethanol induced damage to and depletion of sulfhydryl compounds in the human gastric mucosa was investigated. Ten healthy volunteers underwent endoscopy on three separate occasions. Gastric mucosal damage was induced by spraying 80% ethanol on to the gastric mucosa through the biopsy channel of the endoscope. The gastric mucosal score, total sulfhydryls, glutathione, and cysteine were evaluated in basal conditions and after ethanol administration with and without pretreatment with parenteral glutathione. Glutathione significantly decreased the extent of ethanol induced macroscopic injury to the mucosa of the gastric body and antrum. Glutathione's protective effect is associated with appreciable inhibition of ethanol induced depletion of gastric sulfhydryl compounds. This is the first report of protection against ethanol induced gastric mucosal damage by a sulfhydryl containing agent in humans.