A polyphasic approach to the identification of ochratoxin A-producing black Aspergillus isolates from vineyards in Sicily.

Aspergillus strains belonging to section Nigri isolated during a two year survey in eight Sicilian vineyards located on the slopes of Mount Etna (Sicily, Italy) were analysed analyzed in order to characterize species responsible for ochratoxin A (OTA) contamination of grapes. The polyphasic approach permitted analysis of biodiversity of Aspergillus isolates in relation to their morphology, ochratoxigenicity and genetic variability. We assessed OTA production by A. carbonarius, A. niger, A. tubingensis and A. japonicus using an enzyme-linked immunosorbent assay. A. carbonarius isolates were the strongest OTA producers. A subset of 66 representative strains was selected for further DNA-based characterization. PCR assays using species-specific primers discriminated between A. niger, A. carbonarius and A. japonicus on the basis of the target sequences for each species. The PCR-based methods matched morphological characterization in identifying all the black aspergilli (BA) isolates tested, whereas RFLP analysis with RsaI of isolates positive to PCRs with A. niger specific primers identified three A. tubingensis isolates. The identification of thirteen isolates was further confirmed by ITS analysis. By this method, each of the isolates was identified and assigned to an Aspergillus species. The fAFLP analysis of 40 isolates highlighted the power of this technique to discriminate different species and single strains, to verify the presence of mixed populations in the same vineyard, through homogeneous species clusters. No correlation was observed between the clusters and OTA production level or origin.

Molecular determinants of insulin resistance, cell apoptosis and lipid accumulation in non-alcoholic steatohepatitis.

BACKGROUNDS AND AIMS: Non-alcoholic-steatohepatitis (NASH) is closely related to insulin resistance, but it is unknown whether insulin resistance may be localized in hepatocytes. This study investigates insulin signalling in liver tissue from NASH, and the molecular mechanisms by which insulin-resistance could lead to liver damage (apoptosis). Moreover, to investigate the mechanisms of lipid overload we studied key enzymes in hepatocytes lipid metabolism. METHODS AND RESULTS: In liver specimens from 11 patients with NASH and 7 histological normal livers, we measured total and phosphorylated Akt (active form), Bax and Bcl-2 by Western-blot analysis. In addition, we studied AMP-activated protein Kinase and Carnitine-Palmitoyl-Transferase-1 gene expression, key regulators of non-esterified fatty acid synthesis and oxidation, by reverse transcription polymerase chain reaction. In NASH, phosphorylated Akt was impaired (104.3+/-10.6 vs 152.6+/-22.4 AU, p<0.002) and correlated with necroinflammatory score (r=-0.62; p<0.05). Bax/Bcl-2 ratio was increased in NASH. Moreover, we observed a decrease of AMP-activated protein Kinase (10.74+/-6 vs 144.7+/-41.6 AU, p<0.0001) and Carnitine-Palmitoyl-Transferase-1 gene expression (38.7+/-14.6 vs 192.1+/-26.2 AU, p<0.0001), and both were correlated with steatosis score (r=-0.56, p<0.05, r=-0.87, p<0.05 respectively). CONCLUSIONS: Akt, a key molecule of insulin signalling and cell apoptosis is impaired in NASH, suggesting an important role of hepatic insulin resistance in liver failure. Moreover, decreased non-esterified fatty acid oxidation may cause hepatic lipid overload.

Effects of n-3 polyunsaturated fatty acids in subjects with nonalcoholic fatty liver disease.

BACKGROUND: Long-chain polyunsaturated fatty acid omega-3 levels are decreased in the hepatic tissue of patients with nonalcoholic fatty liver disease. Polyunsaturated fatty acids are negative regulators of hepatic lipogenesis and attenuate the inflammatory response in mice. AIM: To investigate whether polyunsaturated fatty acid may be effective in the treatment of nonalcoholic fatty liver disease. METHODS: Forty patients with nonalcoholic fatty liver disease were randomized into two groups for treatment of 6 months duration. Group DP (n=20) received an AHA recommended diet and polyunsaturated fatty acid 2g/day; Group D (n=20) received only the AHA regular diet. Outcome measurements were fatty liver assessed by abdominal ultrasound, liver aminotransferase and tumour necrosis factor-alpha serum levels, and insulin resistance assessed by HOMA(IR). RESULTS: After 6 months of treatment, the DP group displayed a decrease in alanine aminotransferase levels (p<0.01), as well as in triglyceride levels (p<0.01), serum tumour necrosis factor-alpha levels (p<0.05) and in HOMA(IR) (p<0.05). In the D group, no significant modification was observed. In the DP group, complete fatty liver regression was observed in 33.4% of the patients, and an overall reduction in 50%. In contrast, no patient achieved complete regression in the D group, whereas some amount of reduction occurred in 27.7% of the patients; the remaining 72.2% did not change. CONCLUSION: Our results indicate that alanine aminotransferase, triglyceride and serum tumour necrosis factor-alpha levels, as well as fatty liver improved after polyunsaturated fatty acid administration.

Prevalence of the antiphospholipid syndrome in primary systemic vasculitis.

BACKGROUND: The antiphospholipid (APS or Hughes') syndrome, anticardiolipin antibodies (aCL), and the lupus anticoagulant (LA) are associated with systemic lupus erythematosus, malignancy, infection, and drugs. It has been described in patients with primary systemic vasculitis (PSV). OBJECTIVE: To determine the prevalence of APS in patients with PSV attending a vasculitis clinic and the prevalence of patients with positive aCL and/or the LA who do not fulfil the classification criteria for APS. METHODS: All case notes of patients attending the vasculitis clinic over a 12 month period were reviewed. Outpatients and inpatients were both included and were assessed for features of the APS and presence of aPL. Patients with positive aCL or LA tests were classified according to the significance of these results. RESULTS: Of 144 patients with PSV, 25 had positive aCL or LA on at least one occasion, representing a point prevalence of 17%. Of these, nine had definite APS (classified by the Sapporo criteria) and a further four patients had clinical and serological features of APS, although insufficient to satisfy the Sapporo criteria. Twelve had only positive aPL. CONCLUSION: The antiphospholipid syndrome, aCL, and the LA may occur in association with PSV.

MALToma and lupus.

We present two patients with low-grade non-Hodgkin MALT lymphoma (mucosa-associated lymphoid tissue) occurring years before clinical evidence of autoimmune disorders. While the strong association between autoimmune disorders, in particular Sjögren's syndrome and MALToma, is well described, the developement of MALToma years before the onset of an autoimmune disease is very unusual.

Neutral endopeptidase and angiotensin I converting enzyme insertion/deletion gene polymorphism in humans.

Neutral endopeptidase (NEP) hydrolyses angiotensins (Ang) I and II and generates angiotensin-(1-7) [Ang-(1-7)]. In humans, the insertion/deletion (I/D) angiotensin-I converting enzyme (ACE) gene polymorphism determined plasma ACE levels by 40%. In rats, a similar polymorphism determines ACE levels which are inversely associated to NEP activity. The objective of this study is to evaluate the relationship between ACE expression and plasma NEP activity in normotensive subjects and in hypertensive patients. In total, 58 consecutive patients with hypertension, evaluated in our Hypertension Clinic, were compared according to their ACE I/D genotypes with 54 control subjects in terms of both plasma ACE activity and NEP activities. Plasma ACE activity was elevated 51 and 70% in both DD ACE groups (normotensives and hypertensives) compared with their respective ID and II ACE groups (P<0.001). A significant effect of the ACE polymorphism and of the hypertensive status on ACE activity was observed (P<0.001). In normotensive DD ACE subjects, NEP activity was 0.30+/-0.02 U/ml, whereas in the normotensive II ACE and in the normotensive ID ACE subjects NEP activity was increased 65 and 48%, respectively (P<0.001). In the hypertensive DD ACE patients, NEP activity was 0.47+/-0.03 U/mg. An effect of the I/D ACE genotypes on NEP activity (P<0.04) and an interaction effect between the I/D ACE genotype and the hypertensive status were also observed (P<0.001). These results are consistent with a normal and inverse relationship between the ACE polymorphism and NEP activity in normotensive humans (as is also observed in rats). This normal relationship is not observed in hypertensive patients.

Angiotensin I-converting enzyme modulates neutral endopeptidase activity in the rat.

Angiotensin I is a substrate for both ACE and for neutral endopeptidase 24.11 (NEP). We hypothesized that high ACE expression is related to low NEP activity. Accordingly, circulating and tissue NEP and ACE activities were measured by fluorometry in homozygous rats (F(0) and F(2)) for the Lewis microsatellite allele (LL, low ACE) and for the Brown Norway microsatellite allele (BB, high ACE). Plasma, lung, and aortic ACE activities in F(0) and F(2) were higher in BB rats than in LL rats (P<0.01), whereas left ventricular ACE activity was similar in both genotypes. In contrast, NEP activity in the LL group was higher in the serum, aorta, and lungs in F(0) and F(2) homozygous (P<0.05). Plasma ACE activity was inversely correlated with serum (r=-0.6 and -0.598 in F(0) and F(2), respectively; P<0.03) and lung NEP activities (r=-0.77 in F(0) and r=-0.59 in F(2), P<0.01). Aortic ACE and NEP activities were also correlated (r=-0.696 and -0.584 in F(0) and F(2), respectively; P<0.03). In conclusion, genetically determined high ACE expression in rats is inversely related to tissue NEP activity, which could determine lower angiotensin-(1-7) tissue levels.

Role of cardiopulmonary mechanoreceptors in the postural regulation of renin.

To change the stretch on cardiopulmonary mechanoreceptors, large shifts of blood in the capacity space were elicited by tilting and by exerting positive lower body pressure in the tilted position. Twelve volunteers underwent invasive hemodynamic studies and in 10 other subjects cardiac size was determined by radionuclide cardiography. In all 22 subjects tilting caused the expected increase of renin, which was abolished by lower body compression. Decompression caused renin to increase again. Right atrial pressure in invasive studies and end-systolic and end-diastolic counts in noninvasive studies showed a significant and strong negative correlation with renin and norepinephrine levels. Thus, the degree of stretch of the cardiopulmonary mechanoreceptors is a major determinant of reflex regulation of renin release in humans.