HEV infection in swine from Eastern Brazilian Amazon: Evidence of co-infection by different subtypes

Hepatitis E virus (HEV) is a fecal-orally transmitted member of the genus Hepevirus that causes acute hepatitis in humans and is widely distributed throughout the world. Pigs have been reported as the main source of genotypes 3 and 4 infection to humans in non-endemic areas. To investigate HEV infection in pigs from different regions of Para state (Eastern Brazilian Amazon), we performed serological and molecular analyses of serum, fecal and liver samples from 151 adult pigs slaughtered between April and October 2010 in slaughterhouses in the metropolitan region of Belem, Para. Among the animals tested, 8.6% (13/151) were positive for anti-HEV IgG but not for anti-HEV IgM. HEV RNA was detected in 4.8% (22/453) of the samples analyzed and 9.9% (15/151) of the animals had at least one positive sample. Phylogenetic analysis showed that all sequences belonged to genotype 3 that were related to human isolates from other non-endemic regions, suggesting that the isolates had zoonotic potential. Subtypes 3c and 3f were simultaneously detected in some pigs, suggesting co-infection by more than one strain and/or the presence of a recombinant virus. These results constitute the first molecular and serologic evidence of swine HEV circulation in the Eastern Brazilian Amazon. (C) 2012 Elsevier Ltd. All rights reserved.

LIVER DAMAGES AND NUCLEAR ABNORMALITIES IN ERYTHROCYTES OF Atherinella brasiliensis (ACTYNOPTERIGII, ATHERINOPSIDADE) FROM TWO BEACHES IN SOUTHEAST OF BRAZIL

There are few environmental studies using biomarkers for the species Atherinella brasiliensis in Brazil. In the present work, the presence of hepatic histopathological lesions and nuclear abnormalities in erythrocytes were investigated in A. brasiliensis from Lamberto, a beach under influence of domestic wastes and marine activities. For comparison, fish were also sampled in Puruba, a non-polluted beach, located in the northeastern of Sao Paulo State. The frequency of lesions found in liver was in higher numbers in individuals from Lamberto than Puruba beach. The most critical injuries observed in A. brasiliensis were the presence of necrotic areas, leucocytes infiltration and piknotic nucleus. A high occurrence of cells with vacuolization was also observed. The hepatic lesion index of the fish from Lamberto beach showed significant high values (I(org)=13) when compared with fish from Puruba beach (I(org)=7) suggesting the influence of the several human activities in the studied site. Notched and blebed nucleous were observed in this study, and significant differences were found between the studied sites. However, these differences did not reflect the total nuclear alterations.

Patients on the Waiting List for Liver Transplantation: Caregiver Burden and Stress

Over the last few decades, informal caregivers of patients with chronic diseases have received more attention, and there is a growing volume of studies demonstrating high rates of burden, stress, and mental disorders in this group of individuals. The objective of this study was to evaluate the burden, stress, and psychosocial characteristics of informal caregivers of liver transplantation candidates. Participants were assessed by individual evaluations with the following instruments: a semi-structured interview, the Caregiver Burden Scale, the Inventario de Sintomas de Stress para Adultos de Lipp, and the Beck Depression Inventory. The Mann-Whitney test was used for statistical analysis with a significance level of 0.05. The characteristics of the study group (n = 61) were similar to those of groups in other studies with respect to gender (82% were women), kinship (64% were spouses), and age (the mean age was 47.6 years). The main stressors identified by the participants were as follows: doubts about ways to react in a crisis or in emergency situations (42.6%), mood swings of the patient (29.5%), and care involving food and medications (27.9%). Approximately 25% of the caregivers reported that they felt unprepared to adequately perform their roles. Data analysis indicated a greater burden overall on caregivers when the patient`s Model for End-Stage Liver Disease score was greater than or equal to 15 points (P = 0.041). Furthermore, caregivers of patients with alcoholic liver disease showed higher depression (P = 0.034) and overall burden scores (P = 0.031) versus caregivers of patients with liver disease due to other etiologies. In conclusion, the participants showed significantly high levels of burden, stress, and depression. Support measures and caregiver preparation should be implemented by health care providers. Liver Transpl 16: 1164-1168, 2010. (C) 2010 AASLD.

Tauroursodeoxycholic Acid Affects PPAR gamma and TLR4 in Steatotic Liver Transplantation

Numerous steatotic livers are discarded for transplantation because of their poor tolerance to ischemia-reperfusion (I/R). We examined whether tauroursodeoxycholic acid (TUDCA), a known inhibitor of endoplasmic reticulum (ER) stress, protects steatotic and nonsteatotic liver grafts preserved during 6 h in University of Wisconsin (UW) solution and transplanted. The protective mechanisms of TUDCA were also examined. Neither unfolded protein response (UPR) induction nor ER stress was evidenced in steatotic and nonsteatotic liver grafts after 6 h in UW preservation solution. TUDCA only protected steatotic livers grafts and did so through a mechanism independent of ER stress. It reduced proliferator-activated receptor-gamma(PPAR gamma) and damage. When PPAR gamma was activated, TUDCA did not reduce damage. TUDCA, which inhibited PPAR gamma, and the PPAR gamma antagonist treatment up-regulated toll-like receptor 4 (TLR4), specifically the TIR domain-containing adaptor inducing IFN beta (TRIF) pathway. TLR4 agonist treatment reduced damage in steatotic liver grafts. When TLR4 action was inhibited, PPAR gamma antagonists did not protect steatotic liver grafts. In conclusion, TUDCA reduced PPAR gamma and this in turn up-regulated the TLR4 pathway, thus protecting steatotic liver grafts. TLR4 activating-based strategies could reduce the inherent risk of steatotic liver failure after transplantation.

Effects of selective bile duct ligation on liver parenchyma in young animals: histologic and molecular evaluations

Background/Purpose: The mechanisms of increased collagen production and liver parenchyma fibrosis are poorly understood. These phenomena are observed mainly in children with biliary obstruction (BO), and in a great number of patients, the evolution to biliary cirrhosis and hepatic failure leads to the need for liver transplantation before adolescence. However, pediatric liver transplantation presents with biliary complications in 20% to 30% of cases in the postoperative period. Intra-or extrahepatic stenosis of bile ducts is frequent and may lead to secondary biliary cirrhosis and the need for retransplantation. It is unknown whether biliary stenosis involving isolated segments or lobes may affect the adjacent nonobstructed lobes by paracrine or endocrine means, leading to fibrosis in this parenchyma. Therefore, the present study aimed to create an experimental model of selective biliary duct ligation in young animals with a subsequent evaluation of the histologic and molecular alterations in liver parenchyma of the obstructed and nonobstructed lobes. Methods: After a pilot study to standardize the surgical procedures, weaning rats underwent ligation of the bile ducts of the median, left lateral, and caudate liver lobes. The bile duct of the right lateral lobe was kept intact. To avoid intrahepatic biliary duct collaterals neoformation, the parenchymal connection between the right lateral and median lobes was clamped. The animals were divided into groups according to the time of death: 1, 2, 3, 4, and 8 weeks after surgical procedure. After death, the median and left lateral lobes (with BO) and the right lateral lobe (without BO [NBO]) were harvested separately. A group of 8 healthy nonoperated on animals served as controls. Liver tissues were subjected to histologic evaluation and quantification of the ductular proliferation and of the portal fibrosis. The expressions of smooth muscle alpha-actin (alpha-SMA), desmin, and transforming growth factor beta 1 genes were studied by molecular analyses (semiquantitative reverse transcriptase-polymerase chain reaction and real-time polymerase chain reaction, a quantitative method). Results: Histologic analyses revealed the occurrence of ductular proliferation and collagen formation in the portal spaces of both BO and NBO lobes. These phenomena were observed later in NBO than BO. Bile duct density significantly increased 1 week after duct ligation; it decreased after 2 and 3 weeks and then increased again after 4 and 8 weeks in both BO and NBO lobes. The portal space collagen area increased after 2 weeks in both BO and NBO lobes. After 3 weeks, collagen deposition in BO was even higher, and in NBO, the collagen area started decreasing after 2 weeks. Molecular analyses revealed increased expression of the alpha-SMA gene in both BO and NBO lobes. The semiquantitative and quantitative methods showed concordant results. Conclusions: The ligation of a duct responsible for biliary drainage of the liver lobe promoted alterations in the parenchyma and in the adjacent nonobstructed parenchyma by paracrine and/or endocrine means. This was supported by histologic findings and increased expression of alpha-SMA, a protein related to hepatic fibrogenesis. (C) 2012 Elsevier Inc. All rights reserved.

The effects of high-intensity resistance exercise on the blood lipid profile and liver function in hypercholesterolemic hamsters

It is well established that atherogenic dyslipidemia, characterized by high levels of triglycerides (TG), total cholesterol (TC), and low-density lipoprotein (LDL) cholesterol and low levels of high-density lipoprotein (HDL) cholesterol, constitutes important risk factors for cardiovascular disease. Regular exercise has been associated with a reduced risk for metabolic diseases. However, studies supporting the concept that resistance exercise is a modifier of blood lipid parameters are often contradictory. The aim of this study was to investigate the effects of high-intensity resistance exercise on the serum levels of TG, TC, HDL and non-HDL cholesterol, glucose, and the liver function enzymes alanine aminotransferase (ALT, EC 2.6.1.2) and aspartate aminotransferase (AST, EC 2.6.1.1) in golden Syrian hamsters (Mesocricetus auratus (Waterhouse, 1839)) fed a hypercholesterolemic diet. Sedentary groups (S) and exercise groups (E) were fed a standard diet (SS and ES) or a cholesterol-enriched diet (standard plus 1% cholesterol, SC and EC). Resistance exercise was performed by jumps in the water, carrying a load strapped to the chest, representing 10 maximum repetitions (10 RM, 30 s rest, five days per week for five weeks). Mean blood sample comparisons were made by ANOVA + Tukey or ANOVA + Kruskal-Wallis tests (p < 0.05) to compare parametric and nonparametric samples, respectively. There were no differences in blood lipids between the standard diet groups (SS and ES) (p > 0.05). However, the EC group increased the glucose, non-HDL, and TC levels in comparison with the ES group. Moreover, the EC group increased the TG levels versus the SC group (p < 0.05). In addition, the ALT levels were increased only by diet treatment. These findings indicated that high-intensity resistance exercise contributed to dyslipidemia in hamsters fed a hypercholesterolemic diet, whereas liver function enzymes did not differ in regards to the exercise protocol.

Rice oil as a soybean oil replacement in weanling pig diets

The motivation for including oils in the diets of weanling pigs is to increase dietary energy density to meet the pigs' energy requirements. The purpose of this study was to determine the digestible and metabolizable energy values of semi-refined rice oil (RO) and refined soybean oil (SO) for weanling pigs (Experiment 1), as well as to evaluate how replacing SO with RO in weanling pig diets affects performance, intestinal histology, organ morphometry and the occurrence of diarrhea (Experiment 2). In Experiment 1,21 barrows, averaging 19.63 +/- 0.37 kg body weight (BW), were used in a randomized complete block design experiment, with seven replications per treatment and one pig per experimental unit (metabolism cage). The treatments consisted of a reference diet and two test diets. The test diets were obtained by using 90% of the reference diet and 10% of RO or SO. The experimental period consisted of a 7 days adaptation period, followed by a 5 days collection period of feces and urine. The values of the apparent digestible energy, apparent metabolizable energy and N-corrected apparent metabolizable energy of the oils were, respectively, 26.24, 25.78 and 26.01 MJ kg(-1) for RO and 31.74, 31.35 and 31.51 MJ kg(-1) for SO. In Experiment 2, 120 weaned pigs, castrated males and females, averaging 6.74 +/- 0.42 kg BW, were used in a randomized complete block design experiment, with eight replications per treatment and three pigs per experimental unit (pen) to evaluate the effects of five replacement levels of SO with RO in isonutritive weanling pig diets. The treatments were as follows: 4:0 basal diet with 4% of SO; 3:1 basal diet with 3% of SO and 1% of RO; 2:2 basal diet with 2% of SO and 2% of RO; 1:3 basal diet with 1% of SO and 3% of RO; and 0:4 basal diet with 4% of RO. No effects of the replacement levels were observed in the weanling pigs' performance, organ morphometry or in their occurrence of diarrhea. However, a quadratic effect on the villus width and a cubic effect on the villus height:crypt depth ratio were observed in the duodenum. Therefore, if the determined values of energy are used in the feed formulation, refined soybean oil can be replaced with semi-refined rice oil in weanling pig diets without affecting their growth performance. (C) 2011 Elsevier B.V. All rights reserved.

Centrilobular necrosis as a manifestation of venous outflow block in pediatric malnourished liver transplant recipients - case reports

CLN is a frequent histological finding in biopsies after pediatric: LT, and its pathogenesis has not yet been fully clarified and has different causes. Among the vascular causes, VOB is sometimes difficult to diagnose, especially when technical variants such as split-liver, reduced-liver, or living-related LT are utilized. Three liver-transplanted malnourished children (ages 12, 20, and 28 months) developed altered LFTs and post-operative ascites with right pleural effusion (two cases) and jaundice (one case). Doppler ultrasound examinations were normal and liver biopsies showed CLN interpreted as severe ACR. There were no responses to the medical treatment. Additional investigation with CT angiography suggested obstructed hepatic vein drainage, which was confirmed by interventional radiology and angioplasty of the anastomosis between the hepatic vein and the inferior vena cava, with clinical and histological resolution. It is concluded that in malnourished children undergoing LT with technical variations, in which the occurrence of severe ACR is usually less common because of the severity of the patient condition, the finding of CLN should raise the possibility of VOB, so that excessive immunosuppression and its consequences can be avoided.

Experimental hyperprolinemia induces mild oxidative stress, metabolic changes, and tissue adaptation in rat liver

The present study investigated the effects of chronic hyperprolinemia on oxidative and metabolic status in liver and serum of rats. Wistar rats received daily subcutaneous injections of proline from their 6th to 28th day of life. Twelve hours after the last injection the rats were sacrificed and liver and serum were collected. Results showed that hyperprolinemia induced a significant reduction in total antioxidant potential and thiobarbituric acid-reactive substances. The activities of the antioxidant enzymes catalase and superoxide dismutase were significantly increased after chronic proline administration, while glutathione (GSH) peroxidase activity, dichlorofluorescin oxidation, GSH, sulfhydryl, and carbonyl content remained unaltered. Histological analyses of the liver revealed that proline treatment induced changes of the hepatic microarchitecture and increased the number of inflammatory cells and the glycogen content. Biochemical determination also demonstrated an increase in glycogen concentration, as well as a higher synthesis of glycogen in liver of hyperprolinemic rats. Regarding to hepatic metabolism, it was observed an increase on glucose oxidation and a decrease on lipid synthesis from glucose. However, hepatic lipid content and serum glucose levels were not changed. Proline administration did not alter the aminotransferases activities and serum markers of hepatic injury. Our findings suggest that hyperprolinemia alters the liver homeostasis possibly by induction of a mild degree of oxidative stress and metabolic changes. The hepatic alterations caused by proline probably do not implicate in substantial hepatic tissue damage, but rather demonstrate a process of adaptation of this tissue to oxidative stress. However, the biological significance of these findings requires additional investigation. J. Cell. Biochem. 113: 174183, 2012. (C) 2011 Wiley Periodicals, Inc.

Detection of simulated incipient furcation involvement by CBCT: an in vitro study using pig mandibles

The aim of the present study was to test the reproducibility, sensitivity, and specificity of cone-beam computed tomography (CBCT) in detecting incipient furcation involvement. Fifteen macerated pig mandibles, with intact second molar teeth and preserved adjacent cortical areas, were used. Simulated lesions were created in the furcation region of these teeth by applying 70% perchloric acid in up to four possible buccal/lingual sites in the right/left sides of each mandible. The mandibles were then submitted to a CBCT scan. Two blinded and calibrated experienced oral and maxillofacial radiologists interpreted the exams. Furcation involvement was also assessed in the regions without simulated lesions. CBCT showed high levels of accuracy, ranging from 78% to 88%. The variations in Kappa values for intra- and inter-observer agreement (0.41-0.59) were considered moderate. CBCT can be considered a reliable and accurate method for detecting incipient furcation involvement.

Transmission of clear cell tumor in a graft liver from cadaveric donor: Case report

Neoplasms in children after organ transplantation are related to the type and intensity of immunosuppression and the donorrecipient serostatus, especially in relation to the EpsteinBarr virus. The patient was a two-yr-old female child with biliary atresia who underwent a liver transplantation from a female cadaver donor. Two adults received kidney transplants from the same donor. Nine months after transplantation, one of the adult recipients developed an urothelial tumor in the kidney graft. Imaging tests were repeated monthly in the liver-transplanted child and revealed no abnormalities. However, one yr and two months after the transplantation, the patient developed episodes of fever. At that time, imaging and liver biopsy showed a clear cell tumor of urothelial origin in the graft and the disease was limited to the liver. The patient underwent liver retransplantation, and she is currently free of tumor recurrence. Although rare, the occurrence of tumors in the post-transplant period from cadaver donors, without previously diagnosed tumors, is one of the many problems encountered in the complex world of organ transplantation.

Increased Glyceride-Glycerol Synthesis in Liver and Brown Adipose Tissue of Rat: In-Vivo Contribution of Glycolysis and Glyceroneogenesis

We have previously shown that a high-protein, carbohydrate-free diet can decrease the production of glycerol-3-phosphate (G3P) from glucose and increase glyceroneogenesis in both brown (BAT) and epididymal (EAT) adipose tissue. Here, we utilized an in-vivo approach to examine the hypothesis that there is reciprocal regulation in the G3P synthesis from glucose (via glycolysis) and glyceroneogenesis in BAT, EAT and liver of fasted rats and cafeteria diet-fed rats. Glyceroneogenesis played a prominent role in the generation of G3P in the liver (similar to 70 %) as well as in BAT and EAT (similar to 80 %) in controls rats. The cafeteria diet induced an increase in the total glyceride-glycerol synthesis and G3P synthesis from glucose and a decrease in glyceroneogenesis in BAT; this diet did not affect either the total glyceride-glycerol synthesis or G3P generation from glyceroneogenesis or glycolysis in the liver or EAT. Fasting induced an increase in total glyceride-glycerol synthesis and glyceroneogenesis and a decrease in G3P synthesis from glucose in the liver but did not affect either the total glyceride-glycerol synthesis or G3P synthesis from glyceroneogenesis in BAT and EAT, despite a reduction in glycolysis in these tissues. These data demonstrate that reciprocal changes in the G3P generation from glucose and from glyceroneogenesis in the rat liver and BAT occur only when the synthesis of glycerides-glycerol is increased. Further, our data suggest that this increase may be essential for the systemic recycling of fatty acids by the liver from fasted rats and for the maintenance of the thermogenic capacity of BAT from cafeteria diet-fed rats.

Repression of Ppargc1a Gene in Liver of Hyperglycemic Rats Induced with High Fat Diet Combined with Streptozotocin

Type 2 diabetes mellitus implies deregulation of multiple metabolic processes, being the maintenance of glycemia one of the most important. Many genes are involved in the deregulation of this particular process. Therefore, the aim of this study was to evaluate gene expression of genes related to type 2 diabetes mellitus, in the liver and pancreas of rats with hyperglycemia induced by high fat diet along with a low single dose of streptozotocin. Ahsg and Ppargc1a genes were studied in liver, whereas Kcnj11 and Slc2a2 genes were analyzed in pancreas. For this purpose, 210-240 g female rats were fed a high fat diet or a control diet for three weeks. At day 14, animals fed with high fat diet were injected with a single low dose of streptozotocin (35 mg/kg) and the control group rats were injected only with the vehicle. Plasmatic glucose, triglycerides and total cholesterol levels were measured at the beginning, day 14 and end of treatment. Body weight was also measured. Once the treatment was complete, rats were appropriately euthanized and then, pancreas and liver were surgically removed and frozen in liquid nitrogen. Total RNA was isolated using TRIzol reagent, treated with DNase land reversely transcribed to cDNA. Gene expression analysis was performed using SYBR Green - Real time PCR and comparative Cq method, using three reference genes. Rats fed with high fat diet and treated with streptozotocin showed higher values of plasmatic glucose (17.09 +/- 0.43 vs. 5.91 +/- 0.29 mmol/L, p < 0.01) and a minor expression of Ppargc1a versus the control group (2-fold less expressed, p < 0.05) in liver. We conclude that repression of Ppargc1a gene may be an important process in the establishment of chronic hyperglycemia, probably through deregulation of hepatic gluconeogenesis. However, further studies need to be performed in order to clarify the role of Ppargc1a deregulation in liver glucose homeostasis.

Transforming growth factor alpha immunoreactivity. A study in hepatocellular carcinoma and in non-neoplastic liver tissue

Background. Transforming growth factor alpha (TGF alpha) is an important mitogen that binds to epidermal growth factor receptor and is associated with the development of several tumors. Aims. Assessment of the immunoexpression of TGF alpha in hepatocellular carcinoma (HCC) and in non-neoplastic liver tissue and its relationship to morphological patterns of HCC. Material and methods. The immunohistochemical expression of TGF alpha was studied in 47 cases of HCC (27 multinodular, 20 nodular lesions). Five lesions measured up to 5 cm and 15 lesions above 5 cm. Thirty-two cases were graded as I or II and 15 as III or IV. The non-neoplastic tissue was examined in 40 cases, of which 22 had cirrhosis. HBsAg and anti-HCV were positive in 5/38 and 15/37 patients, respectively. The statistical analysis for possible association of immunostaining of TGF alpha and pathological features was performed through chi-square test. Results. TGF alpha was detected in 31.9% of the HCC and in 42.5% of the non-neoplastic. There was a statistically significant association between the expression of TGF alpha and cirrhosis (OR = 8.75, 95% CI = [1.93, 39.75]). The TGF alpha was detected more frequently in patients anti-HCV(+) than in those HBsAg(+). The immunoexpression of TGF alpha was not found related to tumor size or differentiation. In conclusion the TGF alpha is present in hepatocarcinogenesis in HBV negative patients. Further analysis is needed to examine the involvement of TGF alpha in the carcinogenesis associated with HCV and other possible agents. In addition, TGF alpha has an higher expression in hepatocyte regeneration and proliferation in cirrhotic livers than in HCC.

Time sequence of the intensification of the liver glucose production induced by high-fat diet in mice

It is well established that the development of insulin resistance shows a temporal sequence in different organs and tissues. Moreover, considering that the main aspect of insulin resistance in liver is a process of glucose overproduction from gluconeogenesis, we investigated if this metabolic change also shows temporal sequence. For this purpose, a well-established experimental model of insulin resistance induced by high-fat diet (HFD) was used. The mice received HFD (HFD group) or standard diet (COG group) for 1, 7, 14 or 56?days. The HFD group showed increased (P?<?0.05 versus COG) epididymal, retroperitoneal and inguinal fat weight from days 1 to 56. In agreement with these results, the HFD group also showed higher body weight (P?<?0.05 versus COG) from days 7 to 56. Moreover, the changes induced by HFD on liver gluconeogenesis were progressive because the increment (P?<?0.05 versus COG) in glucose production from l-lactate, glycerol, l-alanine and l-glutamine occurred 7, 14, 56 and 56 days after the introduction of the HFD schedule, respectively. Furthermore, glycaemia and cholesterolemia increased (P?<?0.05 versus COG) 14?days after starting the HFD schedule. Taken together, the results suggest that the intensification of liver gluconeogenesis induced by an HFD is not a synchronous all-or-nothing process but is specific for each gluconeogenic substrate and is integrated in a temporal manner with the progressive augmentation of fasting glycaemia. Copyright (c) 2012 John Wiley & Sons, Ltd.