Necrosome-positive granulovacuolar degeneration is associated with TDP-43 pathological lesions in the hippocampus of ALS/FTLD cases.

AIM: Granulovacuolar degeneration (GVD) in Alzheimer's disease (AD) involves the necrosome, which is a protein complex consisting of phosphorylated receptor-interacting protein kinase 1 (pRIPK1), pRIPK3 and phosphorylated mixed lineage kinase domain-like protein (pMLKL). Necrosome-positive GVD was associated with neuron loss in AD. GVD was recently linked to the C9ORF72 mutation in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with transactive response DNA-binding protein (TDP-43) pathology (FTLD-TDP). Therefore, we investigated whether GVD in cases of the ALS-FTLD-TDP spectrum (ALS/FTLD) shows a similar involvement of the necrosome as in AD, and whether it correlates with diagnosis, presence of protein aggregates and cell death in ALS/FTLD. METHODS: We analysed the presence and distribution of the necrosome in post-mortem brain and spinal cord of ALS and FTLD-TDP patients (n = 30) with and without the C9ORF72 mutation, and controls (n = 22). We investigated the association of the necrosome with diagnosis, the presence of pathological protein aggregates and neuronal loss. RESULTS: Necrosome-positive GVD was primarily observed in hippocampal regions of ALS/FTLD cases and was associated with hippocampal TDP-43 inclusions as the main predictor of the pMLKL-GVD stage, as well as with the Braak stage of neurofibrillary tangle pathology. The central cortex and spinal cord, showing motor neuron loss in ALS, were devoid of any accumulation of pRIPK1, pRIPK3 or pMLKL. CONCLUSIONS: Our findings suggest a role for hippocampal TDP-43 pathology as a contributor to necrosome-positive GVD in ALS/FTLD. The absence of necroptosis-related proteins in motor neurons in ALS argues against a role for necroptosis in ALS-related motor neuron death.

Dietary tryptophan supplementation induces a transient immune enhancement of gilthead seabream (Sparus aurata) juveniles fed fishmeal-free diets.

European aquaculture is an industry with a high sustainability profile contributing to the supply of safe seafood. However, several diseases can affect farmed fish and it is imperative to find alternatives for chemotherapeutic treatments when disease outbreaks occur. Maintenance of health through nutrition is well-establish in modern animal farming, and amino acids (AA) are promising candidates as functional additives to improve fish health. Therefore, the goal of this research is to provide a better understanding of the influence of tryptophan supplementation on nutritional condition and immune mechanisms in fish. Triplicate groups of fish (13.3 ±â€¯0.3g) previously fed with a fishmeal-based diet were either fed a control diet with an extreme formulation (0% fishmeal) but meeting the AA requirements (CTRL), or the SUP diet, formulated as the CTRL with an increase in tryptophan (TRP) content. After 2 and 13 weeks of feeding, head-kidney (HK), liver (L) and white skeletal muscle (WSM) were collected for gene expression, whereas plasma was suited for humoral immune parameters. A holistic approach using transcriptomic, humoral and zootechnical parameters was undertaken. The expression of 29-31 genes for WSM, L or HK confirms an effect due to the treatment across time. A two-way ANOVA analysis revealed that 15-24 genes varied significantly depending on the tissue, and the multivariate analysis by means of PLS-DA explained (R2) and predicted (Q2) with four components up to 93% and 78% of total variance, respectively. Component 1 (R2 = 50.06%) represented the time effects, whereas components 2 (24.36%) and 3 (13.89%) grouped fish on the basis of dietary treatment, at early sampling. The HK results in particular suggest that fish fed SUP diet displayed an immunostimulated state at 2 weeks. No major differences were observed in plasma humoral parameters, despite an increase in antiprotease and peroxidase activities after 13 weeks regardless of dietary treatment. These results suggest that tryptophan supplementation may improve the seabream immune status after 2 weeks. Hence, the use of functional feeds is especially relevant during a short-term feeding period before a predictable stressful event or disease outbreak, considering that these putative advantageous effects seem to disappear after a 13 weeks feeding period.

Enhanced dietary formulation to mitigate winter thermal stress in gilthead sea bream (Sparus aurata): a 2D-DIGE plasma proteome study.

Low water temperatures during winter are common in farming of gilthead sea bream in the Mediterranean. This causes metabolic disorders that in extreme cases can lead to a syndrome called "winter disease." An improved immunostimulatory nutritional status might mitigate the effects of this thermal metabolic stress. A trial was set up to assess the effects of two different diets on gilthead sea bream physiology and nutritional state through plasma proteome and metabolites. Four groups of 25 adult gilthead sea bream were reared during winter months, being fed either with a control diet (CTRL) or with a diet called "winter feed" (WF). Proteome results show a slightly higher number of proteins upregulated in plasma of fish fed the WF. These proteins are mostly involved in the immune system and cell protection mechanisms. Lipid metabolism was also affected, as shown both by plasma proteome and by the cholesterol plasma levels. Overall, the winter feed diet tested seems to have positive effects in terms of fish condition and nutritional status, reducing the metabolic effects of thermal stress.

Identification of proteins with potential osteogenic activity present in the water-soluble matrix proteins from Crassostrea gigas nacre using a proteomic approach.

Nacre, when implanted in vivo in bones of dogs, sheep, mice, and humans, induces a biological response that includes integration and osteogenic activity on the host tissue that seems to be activated by a set of proteins present in the nacre water-soluble matrix (WSM). We describe here an experimental approach that can accurately identify the proteins present in the WSM of shell mollusk nacre. Four proteins (three gigasin-2 isoforms and a cystatin A2) were for the first time identified in WSM of Crassostrea gigas nacre using 2DE and LC-MS/MS for protein identification. These proteins are thought to be involved in bone remodeling processes and could be responsible for the biocompatibility shown between bone and nacre grafts. These results represent a contribution to the study of shell biomineralization process and opens new perspectives for the development of new nacre biomaterials for orthopedic applications.

Dietary Histidine, Threonine, or Taurine Supplementation Affects Gilthead Seabream (Sparus aurata) Immune Status.

AAs have become interesting feed ingredients to be used in functional fish feeds as not only are they protein building blocks, but they also participate in several other key metabolic processes. In the present study, a comprehensive analysis of transcriptomics, hematology, and humoral immune parameters (plasma and skin mucus) were measured twice over the course of the feeding trial (four weeks). Plasma antiprotease activity increased in fish fed Thr compared to those fed the CTRL and Tau treatments, regardless of sampling time. The bactericidal activity in skin mucus decreased in fish fed Tau and His treatments compared to those fed the CTRL diet after two weeks. The membrane IgT (mIgT) was upregulated in fish fed Tau after four weeks, while C-type lectin domain family domain 10 member (clec10a) was downregulated in fish fed Thr after two weeks of feeding. By comparing the molecular signatures of head-kidney by means of a PLS-DA, it is possible to visualize that the main difference is between the two sampling points, regardless of diet. Altogether, these results suggest that dietary supplementation with these AAs at the tested levels causes mild immune-modulation effects in gilthead seabream, which should be further studied under disease challenge conditions.

Short-Term Supplementation of Dietary Arginine and Citrulline Modulates Gilthead Seabream (Sparus aurata) Immune Status.

Several amino acids (AA) are known to regulate key metabolic pathways that are crucial for immune responses. In particular, arginine (ARG) appears to have important roles regarding immune modulation since it is required for macrophage responses and lymphocyte development. Moreover, citrulline (CIT) is a precursor of arginine, and it was reported as an alternative to ARG for improving macrophage function in mammals. The present study aimed to explore the effects of dietary ARG and CIT supplementation on the gilthead seabream (Sparus aurata) immune status. Triplicate groups of fish (23.1 ± 0.4 g) were either fed a control diet (CTRL) with a balanced AA profile, or the CTRL diet supplemented with graded levels of ARG or CIT (i.e., 0.5 and 1% of feed; ARG1, CIT1, ARG2, and CIT2, respectively). After 2 and 4 weeks of feeding, fish were euthanized and blood was collected for blood smears, plasma for humoral immune parameters and shotgun proteomics, and head-kidney tissue for the measurement of health-related transcripts. A total of 94 proteins were identified in the plasma of all treatments. Among them, components of the complement system, apolipoproteins, as well as some glycoproteins were found to be highly abundant. After performing a PLS of the expressed proteins, differences between the two sampling points were observed. In this regard, component 1 (61%) was correlated with the effect of sampling time, whereas component 2 (18%) seemed associated to individual variability within diet. Gilthead seabream fed ARG2 and CIT2 at 4 weeks were more distant than fish fed all dietary treatments at 2 weeks and fish fed the CTRL diet at 4 weeks. Therefore, data suggest that the modulatory effects of AA supplementation at the proteome level were more effective after 4 weeks of feeding and at the higher inclusion level (i.e., 1% of feed). The bactericidal activity increased in fish fed the highest supplementation level of both AAs after 4 weeks. Peripheral monocyte numbers correlated positively with nitric oxide, which showed an increasing trend in a dose-dependent manner. The colony-stimulating factor 1 receptor tended to be up-regulated at the final sampling point regardless of dietary treatments. Data from this study point to an immunostimulatory effect of dietary ARG or CIT supplementation after 4 weeks of feeding in the gilthead seabream, particularly when supplemented at a 1% inclusion level.

How tryptophan levels in plant-based aquafeeds affect fish physiology, metabolism and proteome.

Fish meal replacement by plant-protein sources is a priority in aquaculture feeds. Within this framework, dietary supplementation with essential amino acids (EAA), as tryptophan (TRP), is strategic to ensure that the individual nutritional needs are met, besides promoting enhanced immunological status. The purpose of this study was to examine the beneficial effects of TRP incorporation in plant-protein source diets on fish growth performance and nutritional status. We tested diets with 20% lower (LTRP) and 27% higher (HTRP) of the putative requirements of TRP for seabream (Sparus aurata) and assessed its impact on fish physiology and liver metabolism and proteome. After 12 weeks, growth performance, body proximate, hepatic composition and liver metabolic profiling were similar between diets. Nevertheless, liver proteome analysis indicated a higher accumulation of proteins involved in acute-phase responses, typically triggered by infection, inflammation or trauma, in fish fed with HTRP diet as compared with those fed with LTRP. The overall results obtained suggest a potential beneficial effect of TRP supplementation in terms of immune stimulation, without compromising growth or feed intake. Moreover, proteomics and metabolic profiling demonstrate to be valuable tools in this endeavour. SIGNIFICANCE: Nutritional needs are hard to assess in aquaculture fisheries, and many times controversial depending on the methodology employed. The estimated amino acid requirements depend on both fish species and stage development, making it extremely hard to standardise. On the other hand, the substitution of fish-based to plant-based protein sources diets towards a sustainable aquaculture, may imbalance these requirements, being necessary further studies to assess the impact on fish growth and development. Finally, the incorporation of crystalized amino acids such as TRP into diets aims global better performance both at fish health/immune condition and growth development. This work focused on the potential beneficial effects of TRP supplementation into diets with a plant-based protein source, addressing the effects on the liver metabolism and proteome, and on growth performance of Gilthead seabream juveniles, a species with special relevance and economical importance in the Mediterranean region. The present study by employing proteomics together with metabolic profiling shows that TRP supplementation at the tested doses, does not compromise growth performance, and seems to stimulate the immune system. Our findings can contribute to the development of new feed formulations for Gilthead seabream species, therefore, reinforcing the resilience and competitiveness of the on-growing aquaculture industry and impact directly the sustainability of living resources with the decrease of the fisheries needs to fulfil the human search for quality proteins consume.

Safety of an immunosuppressant protocol based on sirolimus in liver transplant recipients with malignancies or high risk of tumor recurrence.

BACKGROUND: The mammalian target of rapamycin inhibitors are immunosuppressive agents with antiproliferative effects and consequent potential application as anticancer agents. The safety and tolerance of calcineurin inhibitor (CNI)-free sirolimus-based immunosuppressant protocols in liver transplant recipients with malignancies or high risk of tumor recurrence has been scarcely evaluated. PATIENTS AND METHODS: Fourteen liver transplant recipients, including 12 men, of overall mean age of 57.4 +/- 12.4 years were distributed into two groups: group I, corresponding to 11 patients with malignant neoplasia, eight de novo neoplasia, and three recurrent hepatocarcinoma and; group II, three patients with high risk of tumor recurrence due to cholangiocarcinoma. Sirolimus was initiated at 2 mg od, with target levels of 3 to 9 ng/mL. Withdrawal of CNI was performed after reaching target levels of sirolimus. Periodic examinations of weight, arterial pressure, liver function tests, serum creatinine, triglycerides, cholesterol, sirolimus blood levels, and creatinine clearance were performed at 30, 60, 90, 180, and 360 days. RESULTS: After a median follow-up of 221.5 days, eight group I patients (72.7%) were alive, including six with stable disease. All group II patients were alive without evidence of tumor recurrence after a median follow-up of 560 days. CNI was withdrawn in 11 patients (78.6%). Sirolimus was withdrawn in only one case due to severe symptomatic oral ulcers. No vascular complications or rejection episodes were observed. CONCLUSIONS: A sirolimus-based immunosuppressant protocol was well tolerated and safe in liver transplant recipients with malignancies or a high risk of recurrence of neoplastic disease.

STC1 and PTHrP Modify Carbohydrate and Lipid Metabolism in Liver of a Teleost Fish.

Stanniocalcin 1 (STC1) and parathyroid hormone-related protein (PTHrP) are calciotropic hormones in vertebrates. Here, a recently hypothesized metabolic role for these hormones is tested on European sea bass treated with: (i) teleost PTHrP(1-34), (ii) PTHrP(1-34) and anti-STC1 serum (pro-PTHrP groups), (iii) a PTHrP antagonist PTHrP(7-34) or (iv) PTHrP(7-34) and STC1 (pro-STC1 groups). Livers were analysed using untargeted metabolic profiling based on proton nuclear magnetic resonance (1H-NMR) spectroscopy. Concentrations of branched-chain amino acid (BCAA), alanine, glutamine and glutamate increased in pro-STC1 groups suggesting their mobilization from the muscle to the liver for degradation and gluconeogenesis from alanine and glutamine. In addition, only STC1 treatment decreased the concentrations of succinate, fumarate and acetate, indicating slowing of the citric acid cycle. In the pro-PTHrP groups the concentrations of glucose, erythritol and lactate decreased, indicative of gluconeogenesis from lactate. Taurine, trimethylamine, trimethylamine N-oxide and carnitine changed in opposite directions in the pro-STC1 versus the pro-PTHrP groups, suggesting opposite effects, with STC1 stimulating lipogenesis and PTHrP activating lipolysis/ß-oxidation of fatty acids. These findings suggest a role for STC1 and PTHrP related to strategic energy mechanisms that involve the production of glucose and safeguard of liver glycogen reserves for stressful situations.

Orthotopic liver transplantation for hepatocellular carcinoma: a thirteen-year single-center experience.

BACKGROUND: When restrictive selection criteria are applied orthotopic liver transplantation (OLT) is the most efficient option for the treatment of hepatocellular carcinoma (HCC) in terms of survival and recurrence rate. Nevertheless, tumor recurrence may occur in 3.5%-21% of recipients, with a consequent negative impact on prognosis. The aim of this study was to analyze the long-term survival and tumor recurrence rate among a cohort of liver transplant recipients with HCC. METHODS: During the period 1994-2007, 130 HCC patients, including 111 males with a mean overall age of 57.8 +/- 7.1 years (range, 38-70), underwent cadaveric donor-OLT. The etiology of liver disease was alcoholic cirrhosis in 66 patients (50.8%) and viral infection in 52 patients (40%). Baseline alpha fetoprotein values were 53.4 +/- 280.9 ng/mL (range, 1-2593). Median interval between inclusion date and transplantation was 179.5 days. RESULTS: After a median follow-up of 40.8 months, 93 recipients (71.5%) were alive. Tumor recurrence was detected in 11 patients (8.5%). Neoplasm recurrence sites were as follows: liver graft (45.4%), bone (36.4%), lymphoadenopathies (27.3%), adrenal glands (27.3%), and lung (27.3%). Overall survival rates at 1, 3, 5, and 10 years were 85.1%, 78.3%, 70.1%, and 57%, respectively. After examination of the explanted liver, Milan criteria were surpassed in 32 recipients (24.6%). Nevertheless, no differences in survival were observed according to fulfilment or not of Milan criteria (log-rank test, P > .05). Hepatitis C virus (HCV) infection, female gender, and tumor recurrence were associated with a worse survival rate (log-rank test, < .05). CONCLUSIONS: OLT is an effective option for the treatment of HCC with good long-term survival and low recurrence rates. In this series, survival was not affected by findings of poor prognostic factors in the explanted liver.

Protein degradation and peptide release from milk proteins in human jejunum. Comparison with in vitro gastrointestinal simulation.

Human jejunal digests after oral ingestion of casein and whey protein were collected by a nasogastric tube and protein degradation and peptide release was compared with that found in the digests of the same substrates using a standardised protocol. No intact casein was detected in the jejunal nor in the in vitro samples taken during the intestinal phase, while ß-lactoglobulin was found in one hour-jejunal samples in agreement with the in vitro digestion. In vivo and in vitro digests showed comparable peptide profiles and high number of common sequences. A selective precipitation step was used to strengthen the identification of phosphorylated peptides. Most of the sequences found in jejunum, some of them not previously described, were also identified in the simulated digests. Common resistant regions to digestion were identified, revealing that the in vitro protocol constitutes a good approximation to the physiological gastrointestinal digestion of milk proteins.

Human intestinal pro-inflammatory CD11chighCCR2+CX3CR1+ macrophages, but not their tolerogenic CD11c-CCR2-CX3CR1- counterparts, are expanded in inflammatory bowel disease.

Although macrophages (Mϕ) maintain intestinal immune homoeostasis, there is not much available information about their subset composition, phenotype and function in the human setting. Human intestinal Mϕ (CD45+HLA-DR+CD14+CD64+) can be divided into subsets based on the expression of CD11c, CCR2 and CX3CR1. Monocyte-like cells can be identified as CD11chighCCR2+CX3CR1+ cells, a phenotype also shared by circulating CD14+ monocytes. On the contrary, their Mϕ-like tissue-resident counterparts display a CD11c-CCR2-CX3CR1- phenotype. CD11chigh monocyte-like cells produced IL-1ß, both in resting conditions and after LPS stimulation, while CD11c- Mϕ-like cells produced IL-10. CD11chigh pro-inflammatory monocyte-like cells, but not the others, were increased in the inflamed colon from patients with inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis. Tolerogenic IL-10-producing CD11c- Mϕ-like cells were generated from monocytes following mucosal conditioning. Finally, the colonic mucosa recruited circulating CD14+ monocytes in a CCR2-dependent manner, being such capacity expanded in IBD. Mϕ subsets represent, therefore, transition stages from newly arrived pro-inflammatory monocyte-like cells (CD11chighCCR2+CX3CR1+) into tolerogenic tissue-resident (CD11c-CCR2-CX3CR1-) Mϕ-like cells as reflected by the mucosal capacity to recruit circulating monocytes and induce CD11c- Mϕ. The process is nevertheless dysregulated in IBD, where there is an increased migration and accumulation of pro-inflammatory CD11chigh monocyte-like cells.

Serum cytokeratins in alcoholic liver disease: contrasting levels of cytokeratin-18 and cytokeratin-19.

Serum cytokeratin (CK) levels are widely used as tumor markers. Serum levels of CK-18, a tumor marker also known as tissue polypeptide specific antigen (TPS), are increased in patients with alcoholic liver disease. Cytokeratin-18 is the main component of Mallory bodies, a hallmark of alcoholic hepatitis, which may also contain CK-19. Serum levels of CK-18 and CK-19, a tumor marker also known as CYtokeratin FRAgment 21-1 (CYFRA 21-1) were investigated in (a) heavy drinkers with alcoholic liver disease (n=15), (b) patients with malignancy (n=22), and (c) healthy controls (n=10). Serum levels of CYFRA 21-1 (CK-19) were markedly increased in patients with malignancy, but were similar in heavy drinkers and healthy controls. In contrast, serum levels of TPS (CK-18) in heavy drinkers were higher than those of healthy controls, and even tended to be higher than those of patients with malignancy. Both CK-19 and CK-18 levels were higher in cases of alcoholic hepatitis than in cases of fatty liver. Correlation with hepatocyte CK inclusions was stronger for serum TPS (CK-18) than for CYFRA 21-1 (CK-19). In conclusion, serum CYFRA 21-1 (CK-19) and TPS (CK-18) show a different pattern of increase that could reflect the composition of the altered hepatocyte CK network in alcoholic liver disease. Their usefulness as tumor markers, particularly that of serum TPS (CK-18), may be limited in patients with alcoholic liver disease.

Visualization and Differential Analysis of Protein Expression Data Using R.

Data analysis is essential to derive meaningful conclusions from proteomic data. This chapter describes ways of performing common data visualization and differential analysis tasks on gel-based proteomic datasets using a freely available statistical software package (R). A workflow followed is illustrated using a synthetic dataset as example.

Nutritional mitigation of winter thermal stress in gilthead seabream: Associated metabolic pathways and potential indicators of nutritional state.

A trial was carried out with gilthead seabream juveniles, aiming to investigate the ability of an enhanced dietary formulation (diet Winter Feed, WF, containing a higher proportion of marine-derived protein sources and supplemented in phospholipids, vitamin C, vitamin E and taurine) to assist fish in coping with winter thermal stress, compared to a low-cost commercial diet (diet CTRL). In order to identify the metabolic pathways affected by WF diet, a comparative two dimensional differential in-gel electrophoresis (2D-DIGE) analysis of fish liver proteome (pH 4­7) was undertaken at the end of winter. A total of 404 protein spots, out of 1637 detected, were differentially expressed between the two groups of fish. Mass spectrometry analysis of selected spots suggested that WF diet improved oxidative stress defense, reduced endoplasmic reticulum stress, enhanced metabolic flux through methionine cycle and phenylalanine/tyrosine catabolism, and induced higher aerobic metabolism and gluconeogenesis. Results support the notion that WF diet had a positive effect on fish nutritional state by partially counteracting the effect of thermal stress and underlined the sensitivity of proteome data for nutritional and metabolic profiling purposes. Intragroup variability and co-measured information were also used to pinpoint which proteins displayed a stronger relation with fish nutritional state. SIGNIFICANCE: Winter low water temperature is a critical factor for gilthead seabream farming in the Mediterranean region, leading to a reduction of feed intake, which often results in metabolic and immunological disorders and stagnation of growth performances. In a recent trial, we investigated the ability of an enhanced dietary formulation (diet WF) to assist gilthead seabream in coping with winter thermal stress, compared to a standard commercial diet (diet CTRL). Within this context, in the present work, we identified metabolic processes that are involved in the stress-mitigating effect observed with diet WF, by undertaking a comparative analysis of fish liver proteome at the end of winter. This study brings information relative to biological processes that are involved in gilthead seabream winter thermal stress and shows that these can be mitigated through a nutritional strategy, assisting gilthead seabream to deal better with winter thermal conditions. Furthermore, the results show that proteomic information not only clearly distinguishes the two dietary groups from each other, but also captures heterogeneities that reflect intra-group differences in nutritional state. This was exploited in this work to refine the variable selection strategy so that protein spots displaying a stronger correlation with "nutritional state" could be identified as possible indicators of gilthead seabream metabolic and nutritional state. Finally, this study shows that gel-based proteomics seems to provide more reliable information than transmissive FT-IR spectroscopy, for the purposes of nutritional and metabolic profiling.

Prospective histopathological analysis of hepatocellular carcinoma treated with percutaneous ethanol injection in patients on the waiting list for liver transplantation.

INTRODUCTION: Liver transplantation (LT) improves survival in selected patients suffering from hepatocellular carcinoma (HCC). Unfortunately, the long time lapse between indication and LT may cause tumor progression. Thus, percutaneous ethanol injection (PEI) has been proposed as adjuvant therapy of HCC in patients awaiting LT. The efficacy of PEI assessed using histopathological analysis of hepatectomy specimens has not been adequately evaluated. PATIENTS AND METHODS: Twenty-nine nodules of HCC in 27 patients (21 men; mean age, 58.1 +/- 7.3 years) listed for LT were treated with PEI. Pretreatment mean serum alpha-fetoprotein (AFP) was 11 +/- 13.4 ng/mL. Mean tumor diameter was 30.8 +/- 12.9 mm. Data from the explanted livers after transplantation included percentage tumor necrosis, presence of satellite and distant nodules, vascular invasion, tumor capsule, and grade of differentiation. RESULTS: Nineteen patients with 20 treated lesions underwent transplantation. The median interval PEI-LT was 3 months. Complete necrosis was observed in 13 nodules (65%). Satellite nodules were present in 10% of lesions. Previously unrecognized distant lesions were seen in 15.8% of patients. Only 1 nodule presented microscopic vascular invasion. Most HCC were well differentiated (90%), and completely encapsulated (80%). No tumor-related deaths occurred. Seventeen patients are alive and recurrence-free after a median follow-up of 15 months. CONCLUSIONS: PEI may achieve significant necrosis in cases of HCC awaiting LT. Nevertheless, previously unrecognized satellite and distant lesions may be observed. Further studies are needed to evaluate the influence of tumor necrosis on overall survival of these patients.

Circadian variability of blood pressure in liver transplant recipients without antihypertensive therapy.

Hypertension is a frequent cardiovascular risk factor in liver transplant recipients. The usefulness of ambulatory blood pressure monitoring (ABPM) in these patients is unknown. This study was aimed at evaluating the circadian rhythms of blood pressure in liver allograft recipients. In 53 liver transplant patients blood pressure was measured with the Spacelabs device program. No patient received antihypertensive therapy for at least 15 days beforehand. Clinical blood pressure measurement showed 26 patients to be hypertensive. Of these, ABPM verified the diagnosis in 23. Overall, 72% of the patients were hypertensive, and 39.5% showed a nondipper pattern. Diastolic hypertension was more frequent than systolic hypertension. No differences were found in renal function, immunosuppressive therapy, or corticosteroids.

Prospective interventional study to evaluate the efficacy and safety of liposomal amphotericin B as prophylaxis of fungal infections in high-risk liver transplant recipients.

INTRODUCTION: Invasive fungal infections are a life-threatening complication in transplant recipients. The prevalence of fungal infection after orthotopic liver transplantation (OLT) is 5% to 42%. The most common isolated pathogens are Candida and Aspergillus species. High-risk liver transplant recipients are more susceptible to the development of invasive fungal infections, with prevalence >40% and mortality rates of 78% to 100%. The strategy for fungal prophylaxis in this population has not been defined. PATIENTS AND METHODS: Among 100 consecutive OLT followed for 28 months, 21 recipients (15 men, overall mean age of 48.5 years, range 23-65 years) were considered to be high risk for the development of fungal infections when they presented at least one of the following criteria: acute liver failure, assisted ventilation >7 days, retransplantation, relaparotomy, antibiotic therapy >14 days, transfusion requirements >20 red blood cells units, and/or biliary leakage. This group received intravenous liposomal amphotericin B (1 mg/kg/d for 7-10 days). RESULTS: One-year survival in the high-risk group was 80%. Prevalence of invasive fungal infection was 9.5%. No Candida infection was observed. Two patients developed Aspergillus infection: an abdominal aspergillosis treated with percutaneous drainage and liposomal amphotericin B (5 mg/kg/d) showed a favorable clinical outcome. The other patient who developed brain aspergillosis died 25 days after OLT. Adverse events related to the drug were hypokalemia (n = 2), back pain (n = 3), and renal dysfunction (n = 2). None of these events required withdrawal of the prophylaxis regimen. CONCLUSION: In our series, prophylaxis with liposomal amphotericin B in high-risk liver graft recipients showed a low rate of severe fungal infections. More studies are needed in order to determine the highest risk population and the best drug dosage.

Safety of percutaneous ethanol injection as neoadjuvant therapy for hepatocellular carcinoma in waiting list liver transplant candidates.

UNLABELLED: Orthotopic liver transplantation (OLT) as therapy of hepatocellular carcinoma (HCC) improves the survival of a selected group of patients. Unfortunately, the progressive increase in waiting time for OLT may allow tumor progression. Percutaneous ethanol injection (PEI) has been proposed as neoadjuvant therapy for HCC in patients awaiting OLT, but its safety has not been defined. PATIENTS AND METHODS: During a 60-month period, 34 patients (27 men, overall mean age of 58.5 years, range 41-67) with HCC, were listed for OLT. Ultrasonography-guided PEI was delivered into 39 nodules at 117 sessions on an inpatient basis. Written informed consent was obtained from all patients before PEI. Doppler-ultrasonography was done before PEI, immediately after, and 4 weeks later. Noninvasive monitoring of arterial pressure, cardiac rate, and temperature was performed during the procedure and during a 24-hour period after each session. Pain was considered significant if analgesia was required or discontinuation of PEI necessary. Fever was defined as a temperature > or =37.5 degrees C after PEI. RESULTS: Minor complications included pain in 45 sessions (38.5%), fever in 17 (14.5%), arterial hypertension in 14 (12%), hypotension in 7 (7%), and vomiting in 2 (1.7%). The major complications were segmental liver infarction (n = 3), portal branch venous thrombosis (n = 2), ascites (n = 2), and one case each of subcapsular hematoma, duodenal ulcer, pneumonia, hepatic encephalopathy, and hepatic artery thrombosis. In all cases, clinical outcomes were favorable with conservative treatment. No evidence of tumor seeding in the needle track was reported and no PEI-related mortality observed. CONCLUSIONS: PEI is a safe neoadjuvant therapy for HCC on waiting list liver transplant candidates. In our series, pain and self-limited fever were the most frequent complications. Clinically significant severe complications were uncommon, and nonconservative treatments were not required.

Review article: current management of alcoholic liver disease.

Alcoholic liver disease, including acute alcoholic hepatitis and alcoholic cirrhosis, is a major cause of morbidity and mortality in the Western world. Abstinence remains the cornerstone of management of all forms of alcoholic liver disease. Recent research, which has elucidated the mechanisms of alcohol-induced liver injury, offers the prospect of advances in the management of alcoholic liver disease. We review the most recent data on the efficacy of treatment of acute alcoholic injury, including nutritional support, corticosteroids, anti-inflammatory agents and antioxidants, and agents that are directed against the progression to fibrosis, such as colchicines, propylthiouracil and antioxidants. Although these therapies offer a tantalizing glimpse into a future that may include therapies that directly alter the process of injury and repair in the liver, none has been shown consistently to improve the course of alcoholic liver damage. Consequently, liver transplantation remains an ultimate option for selected patients with liver failure due to chronic alcoholic liver damage.