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Background: Tuber aestivum Vittad., known as black summer truffle, represents high-value food especially used as garnishment in nouvelle cuisine. The aim of this study was to investigate on the viable microbial populations associated with T. aestivum ascomata collected in different sites of Sicily and one locality of Umbria (Italy). Methods: The ripe ascomata of black summer truffles were collected from Central Italy. Cell densities of spoilage bacteria, fecal indicators, potential pathogens, yeasts, and molds were analyzed. Statistical analysis was conducted with XLSTAT software. Results: The microbiological counts of truffles ranged between 6.00 and 9.63 log Colony Forming Unit (CFU)/g for total mesophilic count and between 6.18 and 8.55 log CFU/g for total psychrotrophic count; pseudomonads were in the range 6.98-9.28 log CFU/g. Listeria spp. and coagulase-positive streptococci detected in no samples. Coagulase-negative streptococci were found in some samples with 2.11-4.76 log CFU/g levels. Yeasts and filamentous fungi were detected at consistent levels of 3.60-7.81 log CFU/g. Significant differences (p

Truffles are valuable underground mushrooms with significant economic importance. In recent years, their cultivation has achieved satisfactory results, but not for all species. The harvesting of white truffles ( Tuber magnatum Picco) is still dependent on natural production, which is at risk due to various issues, such as improper forest management. A useful practice to protect natural resources is to promote the expansion of productive forests. In this study, we investigate the dynamics of the microbiome in an old and new truffle forest using an amplicon sequencing approach of the fungal ITS region and the prokaryotic 16S rRNA gene. We monitor the soil biological community's development to compare differences and similarities between the primary productive forest and the expanding area over a two-year sampling period. In particular, we observed the colonization of vacant ecological niches by certain fungi, such as those belonging to the genus Mortierella . Additionally, we examined the competitive interactions between saprotrophs and ectomycorrhizal fungi (ECM). In both study areas, the bacterial community was dominated by Pseudomonadota, Planctomycetota, and Actinomycetota. The behavior of the Tuber genus differed significantly from other ECMs and displayed positive correlations with bacterial taxa such as Ktedonobacter , Zavarzinella , and Sphingomonas . The present work provides an initial overview of expanding white truffle habitats. Further, more specific research is needed to explore potential connections between individual taxa .

Although only a few species of Tuber account for the major truffle sales volume, many species that are not considered delicacies are finding their way to the market, especially in regions where the traditionally appreciated ones do not occur. This is the case for whitish truffles. Specimens of whitish truffles were collected in pecan ( Carya illinoinensis ) orchards in Uruguay in October 2021. Morphological and molecular methods were used to characterize and assess their identity as Tuber maculatum Vittad. An SPME extraction of volatile compounds and GC-MS analyses were performed to characterize the aromatic profile of these specimens and evaluate their potential applications. Among the 60 VOCs detected, 3-octenone (mushroom odor), 3-octanol (moss, nut, mushroom odor), and 2H-pyran-2-one (no odor), followed by octen-1-ol-acetate (no odor) and 2-undecanone (orange, fresh, green odor) were the major compounds in T. maculatum fruiting bodies. The attributes of exotic edible mushrooms of commercial value in the region are highlighted. In particular, this work emphasizes the characteristics of truffles as a byproduct of pecan cultivation.

Non-alcoholic fatty liver disease (NAFLD) is a common chronic liver condition with significant risk of progression to steatohepatitis and cirrhosis. Therapeutic strategies in NAFLD include lifestyle changes mainly related to dietary interventions and use of drugs or nutritional components that could improve plasma lipid profiles and insulin sensitivity and decrease the local inflammatory response. In this study, we tested the effects of monacolin K, an inhibitor of HMCoA reductase. In a prospective, uncontrolled, open study, we treated 24 patients with NAFLD and mild hypercholesterolemia with 10 mg/day of monacolin K. At baseline and after 26 weeks, we measured in plasma liver tests, lipids, malondialdehyde, and oxidized glutathione, and assessed biochemical steatosis scores, liver elastography, and body composition with bioimpedance analysis. Monacolin K significantly reduced plasma alanine aminotransferase, cholesterol, triglycerides and the homeostatic model assessment (HOMA) index that indicated improved insulin sensitivity. No significant changes were found in body fat mass and visceral fat, nor in liver elastography, while the fatty liver index (FLI) was significantly decreased. Plasma levels of both malondialdehyde and oxidized glutathione were markedly reduced by monacolin K treatment, suggesting a reduction in oxidative stress and lipid peroxidation. In summary, this pilot study suggests possible benefits of monacolin K use in NAFLD patients that could be linked to a reduction in oxidative stress. This hypothesis should be further investigated in future studies.

The growing demand for truffles has led to a global pursuit for their occurrence in the wild, and studies concerning their cultivation. Although European countries such as Italy, France and Spain have been known for truffle production, truffle hunting is new to Finland. The present study reports for the first time, the findings of Tuber maculatum in Finland on the basis of morphological and molecular analysis. The chemical characteristics of soil samples collected from the truffle sites have also been discussed. The species of the Tuber samples were identified primarily using morphological analysis. Molecular analysis was carried out for the confirmation of the identity of the species. Two phylogenetic trees were constructed based on internal transcribed spacer (ITS) sequences produced in this study and including representative sequences of whitish truffles available in GenBank. The truffles were identified as T. maculatum and T. anniae. This study could be considered as a basis for encouraging research on findings and identification of truffles in Finland., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background and Aims: Cardiac structural and functional changes have been demonstrated in patients with non-alcoholic fatty liver disease (NAFLD). Because of the frequent association of NAFLD with hypertension, we aimed to examine the relationship of liver steatosis with left ventricular (LV) changes in patients with hypertension., Materials and Methods: In a cross-sectional study, we included 360 untreated, essential hypertensive patients who were free of major cardiovascular and renal complications. Liver steatosis was assessed by three different biochemical scores (NAFLD Liver Fat Score, LFS; Fatty Liver Index, FLI; Hepatic Steatosis Index, HSI). Echocardiography was performed with standard B-mode and tissue-Doppler imaging., Results: LV hypertrophy was present in 19.4% and LV diastolic dysfunction in 49.2% of patients who had significantly higher body mass index (BMI), blood pressure (BP), and homeostatic model assessment (HOMA) index and higher frequency of the metabolic syndrome and liver steatosis that was defined by presence of 2 or more positive scores. LV mass index increased progressively across patients who had none, 1, or 2 or more liver steatosis scores, with associated progressive worsening of LV diastolic function. LV mass index was significantly and positively correlated with age, BMI, BP, HOMA-index, LFS, and HSI. Logistic regression analysis showed that age, BP, and liver steatosis scores independently predicted LV hypertrophy and diastolic dysfunction. Liver steatosis independently predicted LV dysfunction but not LV hypertrophy even after inclusion in analysis of the HOMA-index., Conclusion: NAFLD is associated with LV hypertrophy and diastolic dysfunction in untreated patients with hypertension. In hypertension, NAFLD could contribute to LV diastolic dysfunction with mechanisms unrelated to insulin resistance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Catena, Brosolo, Da Porto, Donnini, Bulfone, Vacca, Soardo and Sechi.)

Chronic HCV liver infection is considered one of the main causes of liver cirrhosis and hepatocellular carcinoma (HCC). For a selected group of patients, orthotopic liver transplantation (OLTx) is the most effective option to cure both liver diseases. After liver transplantation, patients may be at risk of viral infection reactivation and HCC recurrence. HCV recurrence on the transplanted organ can lead to graft cirrhosis and therefore the clearance of virus with antiviral therapies has a pivotal role on the prevention of graft damage. Nowadays, direct antiviral agents (DAAs) represent the choice treatment for HCV recurrence in liver transplanted patients, ensuring high eradication rates. We present the case of a liver transplant recipient who developed, 7 years after OLTx and immediately after a DAAs treatment, a subcutaneous abdominal mass with histological characteristics of HCC.

Sphaerosporella brunnea is a pioneer ectomycorrhizal fungus with facultative saprophytic capacities. Here, we sequenced the genome of S. brunnea strain Sb_GMNB300, which is estimated at 51.6 Mb in size with 872 assembled contigs accounting for 12,597 predicted coding genes. This genome will be useful for comparative studies of Pezizales ectomycorrhizal symbioses., (Copyright © 2019 Benucci et al.)

Background: Direct-acting antiviral agents (DAAs) are extremely effective in eradicating hepatitis C virus (HCV) in chronically infected patients. However, the protective role of the sustained virologic response (SVR) achieved by second- and third-generation DAAs against the onset of hepatocellular carcinoma (HCC) and mortality is less well established., Aim: To examine the occurrence of HCC or death from any cause in a retrospective-prospective study of patients treated with DAAs., Methods: Patients were enrolled from a tertiary academic hospital center for liver disease management that collects subject data mainly from northeastern Italy. The study was conducted in 380 patients (age: 60 ± 13 years, 224 males, 32% with cirrhosis) treated with DAAs with or without SVR (95/5%), with a median follow up of 58 wk (interquartile range: 38-117). The baseline anthropometric features, HCV viral load, severity of liver disease, presence of extra-hepatic complications, coinfection with HIV and/or HBV, alcohol consumption, previous interferon use, alpha-fetoprotein levels, and renal function were considered to be confounders., Results: The incidence rate of HCC in patients with and without SVR was 1.3 and 59 per 100 person-years, respectively (incidence rate ratio: 44, 95%CI: 15-136, P < 0.001). Considering the combined endpoint of HCC or death from any cause, the hazard ratio (HR) for the SVR patients was 0.070 (95%CI: 0.025-0.194, P < 0.001). Other independent predictors of HCC or death were low HCV viremia (HR: 0.808, P = 0.030), low platelet count (HR: 0.910, P = 0.041), and presence of mixed cryoglobulinemia (HR: 3.460, P = 0.044). Considering SVR in a multi-state model, the independent predictors of SVR achievement were absence of cirrhosis (HR: 0.521, P < 0.001) and high platelet count (HR: 1.019, P = 0.026). Mixed cryoglobulinemia predicted the combined endpoint in patients with and without SVR (HR: 5.982, P = 0.028 and HR: 5.633, P = 0.047, respectively)., Conclusion: DAA treatment is effective in inducing SVR and protecting against HCC or death. A residual risk of HCC persists in patients with advanced liver disease or with complications, such as mixed cryoglobulinemia or renal failure., Competing Interests: Conflict-of-interest statement: Authors do not have any conflict-of-interest to disclose., (©The Author(s) 2019. Published by Baishideng Publishing Group Inc. All rights reserved.)

There is an increased consumer interest in minimally processed vegetables that has led to the development of products, such as pre-cut sliced mushrooms. Few data are available on the hygienic condition and the presence of foodborne pathogens in such products. Therefore, the current study aimed to evaluate the safety and hygienic characteristics of both ready-to-eat and ready-to-cook, pre-cut sliced mushrooms obtained from a local Italian market. For the evaluation of the hygienic condition, the aerobic mesophilic bacteria, aerobic psychrotrophic bacteria and Escherichia coli enumerations were performed. Salmonella spp., Listeria monocytogenes and Campylobacter spp. were considered in the assessment of the foodborne pathogens. High microbial loads were detected, including counts higher than 5 log CFU/g for E. coli and 6 log CFU/g for the other bacteria counts considered, but no pathogens were found. Ready-to-eat and ready-to-cook products differed only for aerobic mesophilic counts (7.87 and 8.26 log CFU/g, respectively, P=0.003). Strategies to enhance the hygienic level of the mushrooms, particularly the ready-to-eat products, are needed., Competing Interests: Conflict of interests: the authors declare no potential conflict of interests.

Pecan, Carya illinoinensis, is an economically important nut producing tree that can establish ectomycorrhizal symbiosis with a high diversity of fungi. In the southern USA, truffles (Tuber spp.) sometimes fruit prolifically in cultivated pecan orchards and regularly associate with pecan roots as ectomycorrhizae (ECMs). It has been demonstrated that some valuable European truffle species (e.g., Tuber borchii and Tuber aestivum) can form ECMs with pecan seedlings in nursery conditions. Thus, pecan may represent an attractive alternative host to forest trees for truffle growers given the potential for co-cropping truffles and pecans. To further explore the capacity of pecan to host truffle symbionts, pecan seedlings were inoculated with species of black truffles that are economically important in Europe, T. melanosporum and T. brumale. Ectomycorrhizae were characterized molecularly and their morphology was described in detail. Mycorrhization rates on pecan roots were assessed over a 2-year period. Tuber melanosporum and T. brumale produced well-formed ECMs with a level of root colonization in the first year of 37.3 and 34.5%, respectively. After 24 months, the level of mycorrhization increased for T. brumale (49.4%) and decreased for T. melanosporum (10.5%) inversely to that of non-target ECM greenhouse contaminants (e.g., Sphaerosporella brunnea, Trichophaea woolhopeia, Pulvinula constellatio). To assess whether mating types segregated in T. melanosporum as been reported for other host species, we amplified the mating-type locus from single T. melanosporum ECM belonging to different seedlings over a 2-year period. The two mating idiomorphs were nearly equally represented along the 2-year time span: MAT 1-1-1 decreased from 59.4% in the first year to 48.5% in the second year after inoculation. Data reported in this study add to knowledge on the mycorrhization of pecan trees with commercial truffles and has application to truffle and nut co-cropping systems.

Tuber macrosporum Vittad. is a truffle with superb organoleptic properties, whose cultivation is still in its infancy. For the first time we have aimed to provide information on ectomycorrhizal communities in natural and cultivated T. macrosporum sites. Ectomycorrhizal morphotypes were identified using ITS nrDNA sequencing and sorted into molecular operational taxonomic unit (MOTU). We detected 16 MOTUs in the T. macrosporum cultivated plantation. Ascomycota were the most abundant (86.4%) with Helvellaceae, Pyronemataceae and Pezizaceae the most common. Twenty-two MOTUs were collected in the natural T. macrosporum site. Basidiomycota morphotypes were plentiful (70.6%) and Thelephoraceae dominated. Each site had different taxa belowground with only T. macrosporum in common, being more abundant in the natural (18.2%) than in the cultivated (14.4%) site. Species richness, Simpson and Shannon diversity indices, taxonomic diversity, distinctness and variation of taxonomic distinctness were lower in the cultivated than in the natural site.

Tuber macrosporum Vittad. is not a common truffle species, but with remarkable organoleptic qualities and much economic interest. After the addition of truffle spore slurry, 30 seedlings of Quercus robur L., Quercus cerris L. and Corylus avellana L. were grown inside a greenhouse for 11 months before evaluation of the mycorrhizal level. Two different potting mixes were used: a natural soil-based potting mix for Q. robur, Q. cerris and C. avellana and a peat-based potting mix for Q. robur. Quercus robur planted in soil potting mix was the most receptive towards the truffle spore inoculum, with a level of formation of T. macrosporum ectomycorrhizas (ECMs) of approximately 14 %, ranging from a minimum of ∼4 % to a maximum of ∼44 % in different seedlings. No T. macrosporum ECMs developed on Q. cerris (soil potting mix) or on Q. robur (peat potting mix), whereas a low percentage of ECMs was detected on only three C. avellana (soil potting mix) seedlings. The fungus Sphaerosporella brunnea (Alb. & Schwein.) Svrček & Kubička was also detected as a contaminant on almost half the truffle-inoculated seedlings. A new detailed description of the morphological and anatomical characteristics of T. macrosporum ECMs and their DNA-based verification with species-specific markers were also reported.

Background: Nonalcoholic fatty liver disease (NAFLD) is strongly associated to oxidative stress, metabolic syndrome, and cardiovascular risk. Hepatocytes overloaded with fatty acids (FA) could generate substances that interfere with endothelial function, providing a potential explanation for this association. We have investigated the response of cultured human hepatoblastoma cells (Hep-G2) that were exposed to FA by measuring markers of oxidative stress and thrombosis and expression of the insulin receptor., Methods: Hep-G2 cells were conditioned with a mixture of FA with or without N-acetyl-L-cysteine (NAC), glutathione (GSH), or adiponectin (ADN). After 7 days, we measured intracellular GSH (iGSH), nitric oxide (NO), malondialdehyde (MDA), and tissue plasminogen inhibitor-1 (PAI-1). Real-time polymerase chain reaction (PCR) was used to determine gene expression of inducible nitric oxide synthase (iNOS) and insulin receptor (INS-R)., Results: Exposure to FA decreased iGSH and NO levels in Hep-G2 cells and increased MDA and PAI-1 production. Gene expression of iNOS and INS-R in Hep-G2 cells was decreased by exposure to FA. Co-incubation with NAC and GSH prevented the change of iNOS mRNA levels, but not of INS-R; co-incubation with ADN restored the gene expression of INS-R, but not of i-NOS. ADN prevented also the FA-induced increase in MDA in cultured human endothelial cells., Conclusion: Exposure to FA activates oxidative stress and production of prothrombotic markers and decreases expression of insulin receptors in cultured human hepatocytes. These effects of FA are partially prevented by ADN and might contribute to the increased cardiovascular risk in patients with NAFLD and metabolic syndrome.

Background and Objectives: Combination therapy with interferon-α and ribavirin is considered the treatment of choice for chronic hepatitis C. However, interferon-α may induce severe depression. It has been suggested that interferon-α is able to modify cytochrome P450 (CYP) 1A2 and 2D6 activity. We therefore decided to study the effects of the interferon-α-2b pegylated derivative on fluoxetine disposition in patients receiving combination chemotherapy for chronic hepatitis C., Methods: After approval by the institutional ethics committee, 20 adult patients with chronic hepatitis C, but with no history of other liver diseases, were prospectively admitted to the study, which included phenotyping by means of a dextromethorphan test and evaluation of fluoxetine and norfluoxetine pharmacokinetic parameters (the area under the serum concentration-time curve, maximum serum concentration, time to reach the maximum serum concentration and terminal elimination half-life) before and after 2 months of continuous peginterferon-α-2b therapy., Results: The only statistically significant difference we observed was a significant reduction in the terminal elimination half-life of fluoxetine (from 47.30 to 33.23 hours; p = 0.014) after peginterferon-α-2b treatment., Conclusion: These data suggest that interferon-α may induce, rather than inhibit, the biotransformation of fluoxetine.

Alcohol-induced endothelial changes might contribute to an increase in blood pressure in regular alcohol consumers. Some antihypertensive drugs affect oxidative stress and endothelial function and might counteract the effects of alcohol at the cellular level. The aim of this study was to investigate in vitro the effects of three different types of antihypertensive agents on alcohol-induced endothelial responses and oxidative stress. Cultured human endothelial cells were exposed to increasing concentrations (1, 10, 60 micromol/L) of zofenoprilat, carvedilol, and lacidipine in the absence and in the presence of ethanol (140 mmol/L). Concentrations of endothelin (ET) and nitric oxide (NO) were measured in the culture media as markers of endothelial function, and malondialdehyde (MDA) and intracellular glutathione (GSHi) were measured as markers of oxidative stress. Exposure to alcohol increased the levels of ET, NO, and MDA, and decreased GSHi. Carvedilol and zofenoprilat were more effective than lacidipine in counteracting the effects of alcohol on ET production. Alcohol-induced NO production was enhanced by carvedilol, whereas zofenoprilat and lacidipine did not have a significant effect. The alcohol-induced increase in MDA concentrations was blunted by all three drugs, but only carvedilol restored a normal response. All three drugs increased GSHi levels, with the effect being greater for carvedilol and lacidipine than zofenoprilat. Carvedilol is more effective than zofenoprilat and lacidipine in counteracting alcohol-induced endothelial responses in vitro and in decreasing oxidative stress. These effects might be particularly beneficial in patients with alcohol-related hypertension.

Background: Epidemiological investigations have demonstrated a close association between heavy alcohol consumption and hypertension. The mechanisms of this association, however, remain elusive. We studied the effects of alcohol withdrawal on blood pressure, hormonal parameters, and circulating markers of endothelial activity., Methods: In 14 hypertensive heavy alcohol consumers (> 200 g/day) who agreed to participate in a hospital withdrawal programme we monitored, for 30 days, blood pressure, plasma levels of renin, aldosterone, cortisol, endothelin, and plasminogen activator inhibitor 1 (PAI-1), and urinary levels of catecholamines. Patients in the withdrawal group were compared with eight hypertensive heavy drinkers who refused to participate in the programme and maintained regular alcohol consumption and 11 normotensive teetotalers., Results: By the third day after withdrawal, blood pressure was significantly decreased and the normalization of levels was obtained in 13 of 14 patients by the end of the study. Alcohol withdrawal significantly decreased plasma aldosterone and cortisol levels, but did not affect levels of active renin and fractionated urinary catecholamines. At baseline, plasma endothelin and PAI-1 levels were significantly higher in alcoholic individuals than in teetotalers, and after the cessation of alcohol intake decreased progressively, reaching levels different from baseline within 1 week. A significant correlation was found between changes in endothelin and PAI-1, and blood pressure variations during alcohol abstinence that remained significant only for endothelin with the multivariate approach., Conclusion: Hypertension is rapidly reversible in the majority of heavy drinkers after the withdrawal of alcohol consumption. In these patients, hypertension is associated with an increased release of endothelial factors that might contribute to the increase in blood pressure.

Context: The recently discovered hormone resistin is linked to the development of insulin resistance, but direct evidence of resistin levels in humans with nonalcoholic fatty liver disease (NAFLD) is lacking., Methods: We conducted this study to assess the relationship between serum resistin and NAFLD. We measured serum resistin and biochemical, hormonal, and histological correlates in 28 NAFLD patients, 33 controls, and 30 obese patients [body mass index (BMI), >30 kg/m2] without NAFLD., Results: Resistin and adiponectin expression were measured in sc adipose tissue by quantitative RT-PCR. Resistin was higher in NAFLD patients compared with controls (5.87 +/- 0.49 vs. 4.30 +/- 0.20 ng/ml; P = 0.002) and obese patients (4.37 +/- 0.27 ng/ml; P = 0.002). Increased resistin mRNA was also found in the adipose tissue of NAFLD patients compared with controls and obese subjects., Conclusions: Both NAFLD and obese patients had lower adiponectin levels, whereas leptin was increased only in the obese group. No correlation was found between resistin and high-sensitivity C-reactive protein, BMI, homeostasis model assessment, insulin, glucose, transaminases, and lipid values. A positive correlation was found between resistin and histological inflammatory score. These data report increased resistin in NAFLD patients that is related to the histological severity of the disease, but do not support a link between resistin and insulin resistance or BMI in these patients.

Background: Although heavy alcohol drinkers are at an increased risk of developing cardiovascular events, moderate alcohol intake is associated with reduced incidence of cardiovascular death. This paradox might reflect a dose-related effect of different alcohol intakes on endothelial function and this, in turn, might depend on changes in oxidative stress., Methods: We tested the effects of alcohol withdrawal in heavy alcohol consumers and compared the plasma levels of endothelin-1, nitric oxide, plasminogen activator inhibitor-1, von Willebrand factor, malondialdehyde, and intracellular glutathione with those of alcoholics that did not modify their alcohol intake and teetotalers. In human endothelial cells that had been cultured for 2 weeks in the presence of different concentrations of ethanol, we assessed the same parameters after withdrawal of ethanol exposure., Results: Alcohol increased the levels of endothelin-1, nitric oxide, and plasminogen activator inhibitor-1 and decreased the levels of von Willebrand factor both in vivo and in vitro. These changes were dose dependent, rapidly reversed after withdrawal of exposure, and associated with the presence of increased oxidative stress as indicated by increased levels of both malondialdehyde and intracellular glutathione. Blockade of oxidative stress by incubation of endothelial cells in the presence of oxidants' scavengers prevented the alcohol-induced functional modifications of the endothelium., Conclusions: Alcohol affects endothelial function with an effect that is mediated by an activated oxidative stress and is rapidly reversed after withdrawal. Dose-related endothelial responses to different alcohol intakes might translate in either vascular protection or vascular damage.

Objectives: Nonalcoholic fatty liver disease (NAFLD) is a major cause of liver-related morbidity and is frequently associated with obesity and metabolic syndrome. The recently discovered hormone adiponectin is produced by adipose tissue, and low plasma adiponectin is considered a key factor in the development of the insulin resistance underlying metabolic syndrome. Animal studies suggest that adiponectin may protect against non-alcoholic steatohepatitis, but direct evidence in humans is lacking. We therefore conducted this study to assess the relationship between plasma adiponectin and nonalcoholic fatty liver disease to explore its role in the pathogenesis of this disease., Design and Methods: We measured plasma adiponectin and anthropometric, biochemical, hormonal and metabolic correlates in a group of 17 NAFLD patients with diagnosis confirmed by biopsy, and 20 controls with comparable age, body-mass index and sex. Furthermore we compared plasma adiponectin in patients with simple steatosis and steatohepatitis., Results: Plasma adiponectin was significantly lower in NAFLD patients than controls (5.93+/-0.45 vs 15.67+/-1.60ng/ml). Moreover, NAFLD patients were significantly more insulin resistant while having similar serum leptin. Adiponectin was similar in simple steatosis and in steatohepatitis (6.16+/-0.78 vs 5.69+/-0.49ng/ml). An inverse correlation was observed between adiponectin and homeostatic model assessment (HOMA) of insulin resistance (P = 0.008), while adiponectin did not correlate with serum transaminases and lipid values., Conclusions: These data support a role for low circulating adiponectin in the pathogenesis of NAFLD and confirm the strict association between reduced adiponectin production by adipose tissue, NAFLD and insulin resistance.

Apoptosis plays an important role within the endocrine system, particularly in the thyroid gland, although little is known about its regulation in normal thyroids. Because thyrotropin (TSH) regulates many thyroid-specific functions and cell proliferation, we investigated whether TSH can influence such mechanisms. To induce apoptosis we used UV-C radiation. The FRTL-5 rat thyroid cell strain, a cloned strain of differentiated and untransformed cells that reproduces many of the characteristics of the normal thyroid was chosen for this study. The FRTL-5 cells are a particularly suitable model because they actively proliferate when cultured in the presence of TSH (6H medium), while in TSH-free medium (5H medium) cells remain in a physiologic quiescent state for a long period of time. FRTL-5 cells in both culture conditions were irradiated with UV-C radiation (254 nm wavelength). At 48 hours after radiation, 6H cultured cells showed the characteristic signs of apoptosis. However, 5H cultured cells did not present macroscopic signs of damage, DNA fragmentation, or detectable apoptosis. Furthermore, the expression of 23 apoptosis-related genes was compared. Results indicate that Bcl2 and caspase-2 expression is enhanced, while bax, GADD45 and mdm-2 expression is reduced in irradiated cells. These data confirm that TSH plays a major role in regulating UV-induced apoptosis in FRTL-5 cells.

Several thrombogenic abnormalities are associated with diabetes. Since endothelial dysfunction occurs at early stages of disease, it may reflect pathophysiological changes that are responsible for alterations in vascular structure, growth and modifications of adhesivity to platelets and leukocytes, leading to atherosclerosis and thrombosis. Predisposing factors of vascular diseases, such as diabetes, are also associated with endothelial dysfunction. Restoration or replacement of endothelium-related factors like nitric oxide impede the progression of vascular thrombogenic diseases, and prevent the action of vasoconstrictor factors such as endothelin or other prothrombotic factors such as plasminogen-activator inhibitor-1. Since high glucose concentration in blood is the hallmark of diabetes and because the vascular lesions of atherosclerosis are localized in large artheries, we have cultured endothelial cells from the human aorta. Two endothelial cell strains from the same aortic tract that show different characteristics and behavior in high glucose were isolated. Such findings reflect the importance to have well characterized and standardized cell culture systems to carry out experiments to study the glucose-dependent atherosclerotic process in vitro. Our cell strains may represent a useful in vitro model to study the complex pathophysiology of diabetes-related atherosclerosis.

vasodilative Thyroid diseases have been associated with pathophysiological changes in the vasculature that may result from altered thyroid hormone production or to direct effect of elevated thyrotropin (TSH) levels on smooth muscle cells. A direct effect of TSH on vascular endothelium has not been considered. In the present study a strain of human aortic endothelial cells has been stimulated with TSH, and vascular parameters correlated with the atherosclerotic process have been analyzed. Addition of TSH induced an increase of cyclic AMP (cAMP) concentration in human aortic endothelial cells. Furthermore it induced a decrease of endothelin (from 30 +/- 2.5 to 13 +/- 1 fmol/mL) and of tissue plasminogen activator secretion (from 2,800 +/- 200 to 1,600 +/- 150 ng/mL). On the other hand, it increased nitric oxide (from 148 +/- 8 to 211 +/- 12 microM). TSH did not affect plasminogen activator inhibitor 1. Similar results were obtained when immunoglobulin Gs (IgGs) from Graves' disease patients were used. In conclusion, our findings suggest that TSH and IgGs from Graves' disease patients could stimulate endothelial cells, increasing the secretion of procoagulant and vasodilative factors, and that cAMP is involved in the transduction pathway. These findings are consistent with modifications of the fibrinolytic system reported in hypothyroidism and in Graves' disease. On the other hand, the increase of vascular resistance found in patients with hypothyroidism may be due to the altered thyroid hormone production and not to TSH directly, or to a different effect of TSH on peripheral vessels.

APE/Ref-1 is a multifunctional protein possessing both redox and DNA repair functions. Through its redox activity, APE/Ref-1 controls the DNA-binding function of several transcriptional regulators (AP1, NF-kappaB, p53, Pax proteins). We have previously shown that APE/Ref-1 upregulates the transcriptional activity of the thyroid-specific transcription factor Pax8. In thyroid cells, APE/Ref-1 can be detected both in the nuclear and cytoplasmatic compartments. In this study regulatory mechanisms acting on APE/Ref-1 were revealed using the FRTL-5 cell line. TSH induces both cytoplasm-to-nucleus translocation and neosynthesis of APE/Ref-1 protein. Interestingly, only neosynthesis is dependent on cAMP signalling. In contrast, the cytoplasm-to-nucleus translocation is dependent on redox-mediated mechanisms. Based upon the data shown in this study and in others, a bimodal control of APE/Ref-1 by TSH can be delineated.

Thyroid transcription factor-1 (TTF-1, also known as NKX2.1 and T/EBP), a transcription factor belonging to the NKX-2 family of homeodomain-containing genes, plays an essential role in the organogenesis of the thyroid gland, lung, and ventral forebrain. Nestin is an intermediate filament protein strongly expressed in multipotential neuroepithelial stem cells and rapidly down-regulated during postnatal life. Here we show that stable fibroblastic clones expressing TTF-1 acquire a phenotype reminiscent of neuroepithelial cells in culture and up-regulate the endogenous nestin gene. TTF-1 transactivates in HeLa and NIH3T3 cells a reporter gene driven by a central nervous system-specific enhancer element from the second intron of the rat nestin gene, where it recognizes a DNA-binding site (NestBS) whose sequence resembles a nuclear hormone/cAMP-responsive element very different from canonical TTF-1 binding sites. Nuclear extracts from the head of mouse embryos form a retarded complex with NestBS of the same mobility of the extracts obtained from TTF1-expressing clones, which is either abolished or supershifted in the presence of two different antibodies recognizing the TTF-1 protein. Thus, the neuroepithelial marker nestin is a direct central nervous system-specific target gene of TTF-1, leading to the hypothesis that it might be the effector through which TTF-1 plays its role in the organogenesis of the forebrain.

Recognition of virally infected cells by CD8+ T cells requires differentiation between self and nonself peptide-class I major histocompatibility complexes (pMHC). Recognition of foreign pMHC by host T cells is a major factor in the rejection of transplanted organs from the same species (allotransplant) or different species (xenotransplant). AHIII12.2 is a murine T cell clone that recognizes the xenogeneic (human) class I MHC HLA-A2.1 molecule (A2) and the syngeneic murine class I MHC H-2 D(b) molecule (D(b)). Recognition of both A2 and D(b) are peptide-dependent, and the sequences of the peptides recognized have been determined. Alterations in the antigenic peptides bound to A2 cause large changes in AHIII12.2 T cell responsiveness. Crystal structures of three representative peptides (agonist, null, and antagonist) bound to A2 partially explain the changes in AHIII12.2 responsiveness. Using class I pMHC octamers, a strong correlation is seen between T cell activity and the affinity of pMHC complexes for the T cell receptor. However, contrary to previous studies, we see similar half-lives for the pMHC multimers bound to the AHIII12.2 cell surface.

Vascular endothelial cells play an important role in coagulation regulation of vascular tone and in a variety of synthetic and metabolic functions. Endothelial cells also have a pivotal role in immunological diseases atherogenesis and tumor angiogenesis. Endothelial cells are often used as system to study the pathophysiology of late complications in diabetes mellitus atherosclerotic damages and leukocyte adhesion in inflammatory diseases. Most of the studies have been performed on primary arterial and venous endothelial cell cultures with problems such as availability of autoptic material and reproducibility of cell cultures. We have isolated and characterized a novel system of proliferating long-term cultures of human aortic endothelial cells that maintain their differentiated characteristics for many generations in vitro. They produce antithrombotic and thrombotic factors such as t-PA and PAI-1 and respond to TNFalpha, an important factor correlated with the inflammatory process by modifying growth characteristics by producing cytokines such as GM-CSF by expressing ICAM-1 on the surface and by producing large amounts of nitric oxide and endothelin. This new system may be very useful to understand and study the molecular mechanisms involved in many vascular alteration pathologies and in the aging process.

Alteration of the redox potential has been proposed as a mechanism influencing gene expression. Reduced glutathione (GSH) is one of the cellular scavengers involved in the regulation of the redox potential. To test the role that GSH may play in thyroid cells, we cultured a differentiated rat thyroid cell strain (FRTL-5) in the presence of L-buthionine-(S,R)-sulfoximine (BSO). BSO affects GSH synthesis by irreversibly inhibiting gamma-glutamylcysteine synthetase (EC 6.3.2.2), a specific enzyme involved in GSH synthesis. BSO-treated FRTL-5 cells show a great decrease in the GSH level, whereas malondialdehyde increases in the cell culture medium as a sign of lipid peroxidation. In these conditions the activity of two thyroid-specific promoters, thyroglobulin (Tg) and thyroperoxidase (TPO), is strongly reduced in transient transfection experiments. As both Tg and TPO promoters depend upon the thyroid-specific transcription factors, thyroid-specific transcription factor-1 (TTF-1) and Pax-8 for full transcriptional activity, we tested whether reduction of GSH concentration impairs the activity of these transcription factors. After BSO treatment of FRTL-5 cells, both transcription factors fail to trans-activate the respective chimerical targets, C5 and B-cell specific activating protein promoters, containing, respectively, multimerized TTF-1- or Pax-8-binding sites only as well as the Tg and TPO natural promoters. Northern analysis revealed that endogenous Tg messenger RNA (mRNA) expression is also reduced by BSO treatment, whereas endogenous TPO expression is not modified. Furthermore, the Pax-8 mRNA steady state concentration does not change in BSO-treated cells, whereas TTF-1 mRNA slightly decreases. Immunoblotting analysis of FRTL-5 nuclear extracts does not show significant modification of the Pax-8 concentration in BSO-treated cells, whereas a decrease of 25% in TTF-1 protein is revealed. Furthermore, BSO treatment decreases the DNA-binding activity to the respective consensus sequence of both transcription factors. Finally, different mechanisms seem to act on TTF-1 and Pax-8 functional impairment in BSO-treated cells. Indeed, with a lowered GSH concentration, the overexpressed Pax-8 still activates transcription efficiently, whereas, on the contrary, the overexpressed TTF-1 does not recover its transactivation capability when the respective chimerical target sequences are used (C5 and BSAP). When the natural Tg and TPO promoter sequences are used, overexpression of Pax-8 parallels the effect on both promoters observed using the chimeric target sequences, whereas overexpression of TTF-1 increases TPO promoter transcriptional activity only.

It has been reported that glucose may autooxidize generating free radicals which have been hypothesized to induce important cellular abnormalities. To investigate the cell damage induced by glucose-dependent oxidative stress, the FRTL5 cell strain was incubated in 10 or 20 mM glucose, either alone or in the presence of buthionine-sulfoximine, a transition state inhibitor that blocks glutathione synthesis. We found indeed that buthionine-sulfoximine greatly inhibited glutathione production and increased malondialdehyde (a marker of oxidative cell damage) levels, especially in 20mM glucose. We also found that, when glutathione production was inhibited, 10mM glucose induced apoptosis and 20 mM glucose induced necrosis. These data show that the glucose-dependent cell damage is a function of glutathione production. They also show that such glucose-dependent free radical production may be critical for determining cell damage, even for small variations as the ones we tested (from 10 to 20 mM glucose).