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Financiado para publicación en acceso aberto: Universidade da Coruña/CISUG [Abstract] Current treatment for corneal endothelial dysfunction consists in the replacement of corneal endothelium by keratoplasty. Owing to the scarcity of donor corneas and the increasing number of transplants, alternative treatments such as cell-based therapies are necessary. In this article, we highlight the biological aspects of the cornea and the corneal endothelium, as well as the context that surrounds the need for new alternatives to conventional keratoplasty. We then review some of those experimental treatments in more detail, focusing on the development of the in vitro and preclinical phases of two cell-based therapies: tissue-engineered endothelial keratoplasty (TE-EK) and cell injection. In the case of TE-EK graft construction, we analyse the current progress, considering all the requirements it must meet in order to be functional. Moreover, we discuss the inherent drawbacks of endothelial keratoplasties, which TE-EK grafts should overcome in order to make surgical intervention easier and to improve the outcomes of current endothelial keratoplasties. Finally, we analyse the development of preclinical trials and their limitations in terms of performing an optimal functional evaluation of cell-based therapy, and we conclude by discussing early clinical trials in humans. Xunta de Galicia; R2016/036 Xunta de Galicia; ED431B 2020/55 Xunta de Galicia; ED481B 2017/029 Xunta de Galicia; ED481A-2019/206 Xunta de Galicia; ED481A-2017/280 This work was carried out thanks to funding from the Rede Galega de Terapia Celular 2016 (R2016/036) and Grupos con Potencial de Crecemento 2020 (ED431B 2020/55) both from Xunta de Galicia. This work was supported by one postdoctoral and two predoctoral fellowships from the Xunta de Galicia and the European Union (European Regional Development Fund) [grant numbers ED481B 2017/029, ED481A-2019/206, and ED481A-2017/280, respectively], as well as by two predoctoral fellowships for research stays from INDITEX-University of A Coruña-2019.

The haemorrhagic disease virus (RHDV) is a non‐cultivable virus that promotes in rabbits an acute disease which accomplishes many characteristics of an animal model of fulminant hepatic failure (FHF). Beneficial effects of melatonin have been reported in RHDV‐infected rabbits. This study investigated whether protection against viral‐derived liver injury by melatonin is associated with modulation of mitophagy, innate immunity and clock signalling. Rabbits were experimentally infected with 2 × 104 haemagglutination units of a RHDV isolate and killed at 18, 24 and 30 hours after infection (hpi). Melatonin (20 mg/kg body weight ip) was administered at 0, 12 and 24 hpi. RHDV infection induced mitophagy, with the presence of a high number of mitophagosomes in hepatocytes and increased expression of mitophagy genes. Greater expression of main innate immune intermediaries and inflammasome components was also found in livers with RHDV‐induced FHF. Both mitophagy and innate immunity activation was significantly hindered by melatonin. FHF induction also elicited an early dysregulation in clock signalling, and melatonin was able to prevent such circadian disruption. Our study discloses novel molecular routes contributing to RHDV‐induced damage progression and supports the potential of melatonin as a promising therapeutic option in human FHF.

To evaluate the causes of IOL explantation, techniques for secondary IOL implantation, visual outcomes and complications. Setting: Department of Ophthalmology Complexo Hospitalario Universitario A Coruna, Spain. Design: Retrospective study. All explanted IOLs from January 2010 to June 2018 were included. Medical records were reviewed to determine the surgical indication for IOL explantation, type of IOL implanted, time between surgeries, visual outcomes and surgical complications. One hundred forty-one IOLs were explanted (134 patients). Mean time from original surgery to IOL explantation was 7.89 ± 5.81 years. Causes of IOL explantation were IOL dislocation (81.56%)—in-the-bag IOL dislocation (71.63%), out-of-the-bag IOL dislocation (9.9%)—corneal decompensation (12.05%), refractive surprise (3.5%), uveitis-glaucoma-hyphema syndrome (1.4%), IOL opacification (1.4%). Procedures for secondary IOL implantation were retropupillar iris-claw IOL (63.8%), flanged scleral fixated IOL (9.2%), three-piece IOL in ciliary sulcus (8.5%), angle-supported anterior chamber IOL (7.1%), in-the-bag IOL (3.5%), scleral fixated IOL with sutures (0.7%). Ten cases (7.1%) were left aphakic. Mean preoperative and postoperative logMAR CDVA were 1.34 ± 0.87 and 0.63 ± 0.69, respectively (p = 0.000). Mean preoperative IOP and postoperative IOP were 16.78 ± 4.49 and 15.53 ± 3.476 mmHg, respectively (p = 0.005). Complications include cystoid macular edema (7.8%), glaucoma (7.1%), IOL luxation (2.1%), retinal detachment (1.4%), trophic ulcer and leucoma (1.4%), corneal decompensation (1.4%). In-the-bag IOL dislocation was the most frequent indication for IOL explantation, followed by pseudophakic bullous keratopathy. Simultaneous IOL exchange for a retropupillar iris-claw IOL was the most frequent procedure for secondary IOL implantation. Mean CDVA improved significantly and IOP decreased significantly after IOL explantation. The most frequent postoperative complication was cystoid macular edema.

The sphingosine kinase 1/sphingosine 1-phosphate (SphK1/S1P) system is involved in different pathological processes, including fibrogenesis. Melatonin abrogates activation of hepatic stellate cells (HSCs) and attenuates different profibrogenic pathways in animal models of fibrosis, but it is unknown if protection associates with its inhibitory effect on the SphK1/S1P axis. Mice in treatment groups received carbon tetrachloride (CCl4 ) 5 μL g-1 body wt i.p. twice a week for 4 or 6 weeks. Melatonin was given at 5 or 10 mg kg-1 day-1 i.p, beginning 2 weeks after the start of CCl4 administration. At both 4 and 6 weeks following CCl4 treatment, liver mRNA levels, protein concentration and immunohistochemical labelling for SphK1 increased significantly. S1P production, and expression of S1P receptor (S1PR)1, S1PR3 and acid sphingomyelinase (ASMase) were significantly elevated. However, there was a decreased expression of S1PR2 and S1P lyase (S1PL). Melatonin attenuated liver fibrosis, as shown by a significant inhibition of the expression of α-smooth muscle actin (α-SMA), transforming growth factor (TGF)-β and collagen (Col) Ι. Furthermore, melatonin inhibited S1P production, lowered expression of SphK1, S1PR1, SP1R3, and ASMase, and increased expression of S1PL. Melatonin induced a reversal of activated human HSCs cell line LX2, as evidenced by a reduction in α-SMA, TGF-β, and Col I expression. Melatonin-treated cells also exhibited an inhibition of the SphK1/S1P axis. Antifibrogenic effect of SphK1 inhibition was confirmed by treatment of LX2 cells with PF543. Abrogation of the lipid signaling pathway by the indole reveals novel molecular pathways that may account for the protective effect of melatonin in liver fibrogenesis. © 2016 BioFactors, 43(2):272-282, 2017.

25 p. The sphingosine kinase (SphK)1/sphingosine-1-phosphate (S1P) pathway is involved in multiple biological processes, including liver diseases. This study investigate whether modulation of the SphK1/S1P system associates to the beneficial effects of melatonin in an animal model of acute liver failure (ALF) induced by the rabbit hemorrhagic disease virus (RHDV). Rabbits were experimentally infected with 2x104 hemagglutination units of a RHDV isolate and received 20 mg/kg of melatonin at 0 hr, 12 hr and 24 hr postinfection. Liver mRNA levels, protein concentration and immunohistochemical labelling for SphK1 increased in RHDV-infected rabbits. S1P production and protein expression of the S1PR1 receptor were significantly elevated following RHDV infection. These effects were significantly reduced by melatonin. Rabbits also exhibited increased expression of toll-like receptor (TLR)4, tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, nuclear factor-kappa B (NF-κB) p50 and p65 subunits and phosphorylated inhibitor of kappa B (IκB)α. Melatonin administration significantly inhibited those changes and induced a decreased immunoreactivity for RHDV viral VP60 antigen in the liver. Results obtained indicate that the SphK1/S1P system activates in parallel to viral replication and the inflammatory process induced by the virus. Inhibition of the lipid signaling pathway by the indole reveals novel molecular pathways that may account for the protective effect of melatonin in this animal model of ALF, and supports the potential of melatonin as an antiviral agent

Autophagy is an important survival pathway and participates in the host response to infection. Beneficial effects of melatonin have been previously reported in an animal model of acute liver failure (ALF) induced by the rabbit hemorrhagic disease virus (RHDV). This study was aimed to investigate whether melatonin protection against liver injury induced by the RHDV associates to modulation of autophagy. Rabbits were infected with 2 × 104 hemagglutination units of a RHDV isolate and received 20 mg/kg melatonin at 0, 12, and 24 hr postinfection. RHDV induced autophagy, with increased expression of beclin‐1, ubiquitin‐like autophagy‐related (Atg)5, Atg12, Atg16L1 and sequestrosome 1 (p62/SQSTM1), protein 1 light chain 3 (LC3) staining, and conversion of LC3‐I to autophagosome‐associated LC3‐II. These effects reached a maximum at 24 hr postinfection, in parallel to extensive colocalization of LC3 and lysosome‐associated membrane protein (LAMP)‐1. The autophagic response induced by RHDV infection was significantly inhibited by melatonin administration. Melatonin treatment also resulted in decreased immunoreactivity for RHDV viral VP60 antigen and a significantly reduction in RHDV VP60 mRNA levels, oxidized to reduced glutathione ratio (GSSG/GSH), caspase‐3 activity, and immunoglobulin‐heavy‐chain‐binding protein (BiP) and CCAAT/enhancer‐binding protein homologous protein (CHOP) expression. Results indicate that, in addition to its antioxidant and antiapoptotic effects, and the suppression of ER stress, melatonin induces a decrease in autophagy associated with RHDV infection and inhibits RHDV RNA replication. Results obtained reveal novel molecular pathways accounting for the protective effect of melatonin in this animal model of ALF.

8 páginas, 2 tablas, 3 figuras., Outbreaks of abortions and lambs born with nervous clinical signs and/or congenital malformations affected different sheep farms in Spain. Initial diagnosis of ‘border disease-like’ was established, based on clinical signs, serology and/or RNA detection by a pan-pestivirus RT-PCR. However, further investigation using immunohistochemical and molecular techniques identified BVDV-2b as the aetiological agent.

The objective of the present study was to investigate the effect of melatonin on the liver inflammatory and regenerative response in an animal model of fulminant hepatic failure (FHF) of viral origin. Rabbits were experimentally infected with 2×10(4) hemagglutination units of a rabbit hemorrhagic disease virus (RHDV) isolate and received melatonin at two concentrations of 10 or 20mg/kg at 0, 12 and 24hr postinfection. RHDV infection induced an inflammatory response, with increased expression of toll-like receptor 4, high-mobility group box (HMGB)1, interleukin (IL)-1β, IL-6, tumor necrosis factor-α, and C-reactive protein, and decreased expression of decay accelerating factor (DAF/CD55). These effects were significantly reduced by melatonin. Matrix metalloproteinase-9 expression was also lowered in melatonin-treated rabbits. RHDV infection inhibited the hepatic regenerative/proliferative response, with a reduced expression of hepatocyte growth factor (HGF), epidermal growth factor, platelet-derived growth factor (PDGF)-B and vascular endothelial growth factor and their receptors; these responses were prevented by melatonin administration. Melatonin treatment also resulted in reduced expression of phosphorylated Janus kinase and enhanced expression of extracellular mitogen-activated protein kinase (ERK) and signal transducer and activator of transcription (STAT) 3. Our findings show that anti-inflammatory effects and stimulation of regenerative mechanisms contribute to the beneficial effects of melatonin in rabbits with experimental infection by RHDV and support a potential hepatoprotective role of melatonin in FHF.

Antibodies against non-structural protein 3 (NS3, p80) of bovine viral diarrhoea virus (BVDV) were determined in milk from cows vaccinated with an inactivated BVDV vaccine and compared to serum antibody levels. Animals in one herd were vaccinated with an inactivated BVDV vaccine according to the standard protocol and animals from a second herd with an intensive schedule. Serum and milk samples were tested for BVDV NS3 antibodies using five commercial ELISAs. With a few exceptions, vaccination according to the standard schedule did not induce BVDV NS3-specific antibodies in serum or milk. However, after vaccination according to the intensive schedule, anti-NS3 antibodies were detected for a short time in serum and, to a lesser extent, in milk. Bulk milk was a suitable substrate for BVDV monitoring of herds vaccinated with the inactivated BVD vaccine.

Hepatocyte apoptosis plays an important role in the development of fulminant hepatic failure (FHF). The objective of this study was to investigate the antiapoptotic effect of melatonin in an animal model of FHF of viral origin induced by the rabbit hemorrhagic disease virus (RHDV). Rabbits were experimentally infected with 2 × 104 hemagglutination units of a RHDV isolate and received melatonin at two concentrations of 10 and 20 mg/kg at 0, 12, and 24 hr postinfection. RHDV infection induced liver apoptosis, with increased caspase-3 immunoexpression and activity and poly(ADP-ribose)polymerase-1 (PARP-1) proteolysis. These effects were attenuated by melatonin in a concentration-dependent manner. Antia-poptotic effects of melatonin were related to a reduced expression of Bax and cytosolic cytochrome c release, increased expression of Bcl-2 and Bcl-xL, and inhibition of caspase-9 activity. Increased thiobarbituric reactive acid substances concentration and oxidized-to-reduced glutathione ratio were significantly prevented by melatonin administration. Melatonin treatment also resulted in a reduction in caspase-8 activity, tumor necrosis factor receptor-1 (TNF-R1) expression, and phosphorylated Janus kinase (JNK) expression, and increased expression of cellular FLICE-inhibitory protein (c-FLIP). Our findings show that inhibition of apoptotic mechanisms contributes to the beneficial effects of melatonin in rabbits with experimental infection by RHDV and supports a potential hepatoprotective role of melatonin in FHF.

Disruption of circadian rhythms, which are regulated by the circadian clock machinery, plays an important role in different long-term diseases including hepatocellular carcinoma (HCC). Melatonin has been reported to alleviate promotion and progression of HCC, but the potential contribution of circadian clock modulation is unknown. We investigated the effects of melatonin in mice which received diethylnitrosamine (DEN) (35 mg/kg body weight ip) once a week for 8 weeks. Melatonin was given at 5 or 10 mg kg-1 d-1 ip beginning 4 weeks after the onset of DEN administration and ending at the sacrifice time (10, 20, 30, or 40 weeks). Liver expression of Bmal1, Clock, Npas2, Rorα, and Sirt1 increased, whereas Cry1, Per1, Per2, Per3, CK1e, Rev-erbα, and Rev-erbβ decreased following DEN administration. Melatonin treatment prevented changes in the expression of clock genes, and this effect was accompanied by an upregulation of the MT1 receptor and reduced levels of the hypoxia-inducible factors Hif-1α and Hif-2α. An increased expression of p21, p53, and PARP1/2, a higher Bax/Bcl-2 ratio, and a lower expression of Cyclin D1, CDK6, HSP70, HSP90, and GRP78 proteins were also observed in melatonin-treated mice. Melatonin significantly potentiated the suppression of proliferation and cell cycle arrest induced by the synthetic REV-ERB agonist SR9009 in human Hep3B cells, and BMAL1 knocking down attenuated the pro-apoptotic and antiproliferative effect of melatonin. Results support a contribution of changes in the circadian clock components to the beneficial effects of melatonin in HCC and highlight the usefulness of strategies modulating the circadian machinery in hepatocarcinogenesis.

In order to evaluate whether cattle vaccinated with an inactivated vaccine against bovine viral diarrhoea virus (BVDV) can be differentiated serologically from BVDV infected animals, two different aspects were investigated. Firstly the antibody response against non-structural proteins (NS) was measured after multiple vaccinations of cattle with a single or double dose of a commercially available inactivated BVDV vaccine. In a second study, the animals were first vaccinated with the product, and then infected with BVDV. The antibody response was determined in four different commercial ELISA systems. It can be concluded, that the inactivated BVD vaccine exhibits properties of a marker vaccine when an appropriate antibody NS3 ELISA is applied: after vaccination NS3-specific antibody levels are low or undetectable, but the vaccination does in the present study not show any interference with the development of antibodies against NS3 after subsequent field virus infection.

The study was undertaken to evaluate the effects of dietary protein sources on lipogenesis and fat deposition in a marine teleost, the European seabass (Dicentrarchus labrax). Four isonitrogenous (crude protein (CP, Nx6.25), 44% DM) and isoenergetic (22-23 kJ/g DM) diets were formulated to contain one of the following as the major protein source: fish meal (FM), one of two soy protein concentrates (SPC) and corn gluten meal (CGM). Apparent digestibility coefficients of the diets and raw ingredients, as well as soluble nitrogen (ammonia and urea) and phosphorus excretion were measured. Growth rates of seabass fed plant protein-based diets were significantly lower than those fed fish meal based diet. The protein utilisation was strongly correlated to the dietary essential amino acids index. Measurements of N excretion (ammonia and urea nitrogen) confirmed these data. Daily fat gain at the whole body level ranged between 1.1 to 1.7 g/kg BW, with the highest values being recorded in fish fed the fish meal based diet. Levels of plasma triglycerides and cholesterol were lower in fish fed soy protein diets than in those fed the diet solely based on fish meal. Soy protein rich diets decreased the activities of selected hepatic lipogenic enzymes (glucose 6-phosphate dehydrogenase, malic enzyme, ATP-citrate lysase, acetylcoenzyme A carboxylase and fatty acid synthetase). Highest lipogenic enzyme activities where found in fish fed the fish meal diet, except for fatty acid synthetase which was increased in seabass fed the corn-gluten meal based diets. Overall data suggest that dietary protein sources affects fat deposition and the lipogenic potential in European seabass.

Forty-four bovine viral diarrhoea virus (BVDV) isolates collected from the north of Spain between 1989 and 2000 were characterised at the molecular level. For 24 of these isolates the entire N pro gene sequence was determined. Phylogenetic analysis revealed that 21 isolates belong to subgroup BVDV-1b, while two BVDV-1c isolates and one BVDV-1h isolate were found. Furthermore, 20 additional virus isolates were analysed by differential reverse transcription-polymerase chain reaction (RT-PCR) and classified as BVDV-1. The results from our study demonstrate that BVDV-1b is the most prevalent subgroup present in bovine dairy herds of Spain, while there is no evidence for the presence of BVDV-2. © 2003 Elsevier B.V. All rights reserved.

Radiological features and biochemical changes were investigated during the parenchymal and ductal phases of chronic Fasciola hepatica infection in sheep. The activities of plasma aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and gamma-glutamyl transpeptidase (GGT), plasma levels of IgG anti- F. hepatica and serial ultrasound, computed tomography (CT) and magnetic resonance imaging (MRI) findings were studied in eight sheep infected with 150 F. hepatica metacercariae. Experimental fluke infection provoked an increase in plasma level of IgG directed against F. hepatica and in plasma LDH and AST activities from 4 weeks after infection. Enzyme activities did not significantly differ from the baseline after 15 and 12 weeks for LDH and AST, respectively. GGT activity increased from 9 weeks postinfection and still remained significantly elevated at 18 weeks. In the parenchymal phase, both CT and MRI showed nodular lesions in five animals and MRI could also detect early tracks in the subcapsular area in three sheep. Ultrasound findings were nonspecific in this phase. Ductal dilatation was shown by ultrasound, CT and MRI in almost all animals, although MRI was inferior to CT in depicting a mild ductal dilatation. Moving echogenic forms in the dilated bile ducts were observed by ultrasound from 9 weeks postinfection in seven of the eight sheep. Moving worms were only demonstrated in four sheep at CT and in a single sheep at MRI. This study shows that radiological findings may be useful in studies of fluke-induced liver damage.

In this study we sought to characterize a novel model of fulminant liver failure (FLF) by means of experimental infection of rabbits with the rabbit hemorrhagic disease virus (RHDV). Thirty-seven 9-week-old rabbits were injected intramuscularly with 2 × 104 hemagglutination units of an RHDV isolate. Eighty-five percent of rabbits died 36 to 54 hours after infection. From 36 hours after infection we noted marked increases in transaminases, lactate dehydrogenase, and total bilirubin. The rabbits exhibited hypoglycemia and coagulation abnormalities, with a significant decrease in factor V, factor VII, and prothrombin. Plasma aromatic amino acids and taurine showed progressive increases, and the Fischer index was significantly reduced. Expression of hepatocyte growth factor messenger RNA was inhibited from 36 hours after infection. Prostration and side recumbency were present at later stages, and neurologic symptoms rapidly progressed to coma. Onset of brain death was associated with a significant increase in intracranial pressure and blood ammonia. RHDV infection reproduces clinical, biochemical, and histologic features of the FLF syndrome and satisfies criteria for a suitable animal model. Rabbit hemorrhagic viral disease could provide a useful tool for the study of FLF and the evaluation of new liver-support technologies in human subjects. (J Lab Clin Med 2003;141:272-8)

A growth trial was conducted with groups of European seabass having an initial weight of 6 g to study the lipogenic action of dietary protein and non-protein energy supplies. Six experimental diets were formulated to contain one of two crude protein levels (43 and 52%) with digestible protein (DP) to digestible energy (DE) ratios ranging from 19 to 26 mg/kJ. At the end of the growth trial (12 weeks), the activities of liver glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49), malic enzyme (ME, EC 1.1.1.40), ATP citrate lyase (ACL, EC 4.1.3.8), acetyl-CoA carboxylase (ACoAC, EC 6.4.1.2) and fatty acid synthetase (FAS, EC 2.3.1.38) were measured. Digestibility of main dietary components was also determined over a three-week period. At each protein level, an increase in dietary DE led to improved growth performance, protein efficiency, daily N deposition and to a reduction of N loss. Best results were achieved at 40% DP and a DP/DE ratio of 19–20 mg/kJ. G6PD, ME and ACoAC were found to be the key regulatory enzymes in the lipogenic pathway, with G6PD being the main NADPH-generating enzyme. Activities of G6PD, ME, ACL and FAS were reduced with increasing fat intake. Activities of G6PD, ME and ACL were increased with increasing starch intake. ACoAC activity was negatively correlated with starch intake and positively with fat intake.

International audience; The Rabbit Hemorrhagic Disease Virus (RHDV) induces a severe disease that fulfils many requirements of an animal model of fulminant hepatic failure. However, a better knowledge of molecular mechanisms contributing to liver damage is required, and it is unknown whether the RHDV induces liver autophagy and how it relates to apoptosis. In this study, we attempted to explore which signalling pathways were involved in the autophagic response induced by the RHDV and to characterize their role in the context of RHDV pathogenesis. Rabbits were infected with 2 × 104 hemmaglutination units of a RHDV isolate. The autophagic response was measured as presence of autophagic vesicles, LC3 staining, conversion of LC3-I to autophagosome-associated LC3-II and changes in expression of beclin-1, UVRAG, Atg5, Atg12, Atg16L1 and p62/SQSTM1. RHDV-triggered autophagy reached a maximum at 24 hours post-infection (hpi) and declined at 30 and 36 hpi. Phosphorylation of mTOR also augmented in early periods of infection and there was an increase in the expression of the endoplasmic reticulum chaperones BiP/GRP78, CHOP and GRP94. Apoptosis, measured as caspase-3 activity and expression of PARP-1, increased significantly at 30 and 36 hpi in parallel to the maximal expression of the RHDV capsid protein VP60. These data indicate that RHDV infection initiates a rapid autophagic response, perhaps in an attempt to protect liver, which associates to ER stress development and is independent from downregulation of the major autophagy suppressor mTOR. As the infection continues and the autophagic response declines, cells begin to exhibit apoptosis.

Hepatocyte apoptosis plays an important role in the development of fulminant hepatic failure (FHF). The objective of this study was to investigate whether endoplasmic reticulum (ER) stress and unfolded protein response (UPR) inhibition is an underlying mechanism of melatonin anti-apoptotic effects in an animal model of FHF of viral origin induced by the rabbit hemorrhagic disease virus (RHDV). Rabbits were experimentally infected with 2 9 10 4 hemagglutination units of a RHDV isolate and received melatonin at two concentrations of 10 mg/kg and 20 mg/kg at 0 hr, 12 hr and 24 hr postinfection. RHDV infection induced increased expression of CCAAT/ enhancer-binding protein homologous protein (CHOP), immunoglobulin heavy chain binding protein (BiP/GRP78), glucose-regulated protein 94 (GRP94), phospho-c-Jun N-terminal kinase (JNK) and caspase-12. These effects were attenuated by melatonin. Double immunofluorescence staining showed colocalization of CHOP and cleaved caspase-3 in liver sections of RHDV-infected rabbits, while immunostaining decreased markedly with melatonin treatment. RHDV infection resulted in significant increases in the mRNA levels of activating transcription factor 6 (ATF6), ATF4, inositol- requiring enzyme 1 (IRE1), spliced X-box binding protein-1 (XBP1s) and tumor necrosis factor receptor-associated factor 2 (TRAF2). Melatonin attenuated the extent of the changes. Data obtained provide evidence that in rabbits with experimental infection by RHDV, reduction in apoptotic liver damage by melatonin is associated with attenuation of ER stress through a modulation of the three arms of UPR signaling and further support a potential hepatoprotective role of melatonin in FHF.

Antitumor and antiviral properties of the antimalaria drug artemisinin from Artemisia annua have been reported. Novel artemisinin derivatives (AD1-AD8) have been synthesized and evaluated using in vitro models of liver/colon cancer and viral hepatitis B and C. Cell viability assays after treating human cell lines from hepatoblastoma (HepG2), hepatocarcinoma (SK-HEP-1), and colon adenocarcinoma (LS174T) with AD1-AD8 for a short (6h) and long (72h) period revealed that AD5 combined low acute toxicity together with high antiproliferative effect (IC50=1-5μM). Since iron-mediated activation of peroxide bond is involved in artemisinin antimalarial activity, the effect of iron(II)-glycine sulfate (ferrosanol) and iron(III)-containing protoporphyrin IX (hemin) was investigated. Ferrosanol, but not hemin, enhanced antiproliferative activity of AD5 if the cells were preloaded with AD5, but not if both compouds were added together. Five derivatives (AD1>AD2>AD7>AD3>AD8) were able to inhibit the cytopathic effect of bovine viral diarrhoea virus (BVDV), a surrogate in vitro model of hepatitis C virus (HCV), used here to evaluate the anti-Flaviviridae activity. Moreover, AD1 and AD2 inhibited the release of BVDV-RNA to the culture medium. Co-treatment with hemin or ferrosanol resulted in enhanced anti-Flaviviridae activity of AD1. In HepG2 cells permanently infected with hepatitis B virus (HBV), AD1 and AD4, at non-toxic concentrations for the host cells were able to reduce the release of HBV-DNA to the medium. In conclusion, high pharmacological interest deserving further evaluation in animal models has been identified for novel artemisinin-related drugs potentially useful for the treatment of liver cancer and viral hepatitis B and C.

Seven bacterial strains capable of utilizing monochloroacetate (MCA) at a concentration of 50 mM as the sole carbon source were isolated from soil and displayed MCA dehalogenase activity. Three of them were identified as Pseudomonas spp., and the remaining four strains as Alcaligenes sp., Agrobacterium sp., Arthrobacter sp., and Azotobacter sp. This latter is the first reported example of a bacterium fixing atmospheric nitrogen under aerobic conditions that also uses a chloro-organic compound as sole source of carbon and energy. MCA dehalogenase activity in these strains was found to be inducible under different growth conditions. Crude extracts from all seven isolated strains also displayed dehalogenating activity with a relatively wide range of halogenated organic compounds (aliphatic acids, ketones, alcohols, alkanes, and aromatics), which, depending on the strain, were dehalogenated to different extents. The estimated Kmvalues for MCA were used to classify the dehalogenase activities into three groups: high affinity (30–40 μM) in Alcaligenes and Agrobacterium species, medium affinity (100–180 μM) in Pseudomonas and Azotobacter species, and low affinity (100 mM) in Arthrobacter sp. Both the optimal pH range for MCA dehalogenase activity (between pH 8 and 10) and the pH profile of stability (in the neutral–basic range) were found to be similar in all strains, whereas the thermal stability profiles were variable.Key words: dehalogenase, halohydrolase, monochloroacetate, soil.

Neutralizing antibodies to 9 rotavirus strains representing serotypes G1, G3, G5, G6, G8, G9, and G10 were investigated in 212 ovine serum samples from 3 age groups, 1-week-old lambs, 2- to 3-months-old lambs and adult sheep. All sera from 1-week-old lambs had neutralizing antibodies to all 9 rotavirus strains. Both neutralizing antibody titers and prevalences to all 9 strains markedly decreased in the 2- to 3-months-old lamb group and increased again in the adult sheep group. Also, adult sheep sera neutralized a larger number of rotavirus strains than 2- to 3-months-old lamb sera. The highest neutralizing antibody titers and prevalences were found to strains B223 and K923, representing serotype G10, to strain RRV, representing serotype G3, and to strain NCDV, representing serotype G6, indicating that these could be the predominant 3 rotavirus serotypes in Spanish sheep. The rotavirus serotypes infecting sheep observed by us differ from those described for cattle, where G6 is the most prevalent serotype followed by G10, and G3 has been seldom found. Very low prevalences were observed for strains WA and OSU representing serotypes G1 and G5 respectively, suggesting that they probably do not infect sheep and neutralizing antibodies found are derived from heterotypic responses to other serotypes. Intermediate prevalences and titers were found to strains UK (serotype G6), 69M (serotype G8) and WI61 (serotype G9). Neutralizing antibodies distinguished between different strains sharing their VP7 specificity: B223 and K923, a bovine and an ovine serotype G10 strains, and NCDV and UK, two serotype G6 bovine rotavirus strains with different VP4 antigen.

A cross-sectional study was carried out in a dairy goat herd, investigating the presence of rotavirus by means of ELISA, polyacrylamide gel electrophoresis (PAGE) and two latex agglutination tests in feces of 63 goat kids younger than 1 month, with and without diarrhea, and in feces of 19 adult goats during the first few days after parturition. All animals belonged to a herd located in the mountains of the León province (NW Spain). Rotaviruses were found in 18 out of 63 goat kid fecal samples but no significant association between shedding of rotavirus and presence of diarrhea could be established. Rotaviruses were found in kids aged 6 to 21 days, and more frequently between 6 and 10 days. No shedding of virus was detected in any of the adults. Considering ELISA as the reference test, PAGE and both latex agglutination tests were less sensitive. One of the latex tests was also highly non-specific. All PAGE-positive samples showed the typical electropherotype of group A rotavirus. Feces were also screened for other pathogens includingSe realizó un estudio de corte en una explotación de ganado caprino lechero investigando la presencia de rotavirus mediante ELISA, electroforesis en gel de poliacrilamida (PAGE) y 2 pruebas de aglutinación en látex en las heces de 63 cabritos menores de un mes, con y sin diarrea, y en las heces de 19 cabras durante los primeros días tras el parto. Todos los animales pertenecían a una explotación situada en la montaña de la provincia de León (NO de España). Se encontraron rotavirus en 18 de las 63 muestras fecales de cabritos analizadas, aunque no pudo demostrarse la existencia de asociación estadísticamente significativa entre la eliminación del virus y la presencia de diarrea. Las edades de los cabritos en que se detectó eliminación de rotavirus estaban comprendidas entre los 6 y los 21 días y más frecuentemente entre los 6 y los 10. Considerando ELISA como la prueba de referencia tanto PAGE como ambas pruebas de aglutinación en látex fueron menos sensibles. Una de las pruebas de aglutinación en látex fue también muy inespecífica. En todas las muestras positivas mediante electroforesis se observó el electroferotipo característico de los rotavirus del grupo A. También se estudió la presencia en las heces de otros patógenos como

Rabbit hemorrhagic disease virus (RHDV) causes lethal fulminant hepatitis closely resembling acute liver failure (ALF) in humans. In this study, we investigated whether cardiotrophin-1 (CT-1), a cytokine with hepatoprotective properties, could attenuate liver damage and prolong survival in virus-induced ALF. Twenty-four rabbits were infected with 2 × 10 4 hemagglutination units of RHDV. Twelve received five doses of CT-1 (100 μg/kg) starting at 12 h postinfection (hpi) (the first three doses every 6 h and then two additional doses at 48 and 72 hpi), while the rest received saline. The animals were analyzed for survival, serum biochemistry, and viral load. Another cohort ( n = 22) was infected and treated similarly, but animals were sacrificed at 30 and 36 hpi to analyze liver histology, viral load, and the expression of factors implicated in liver damage and repair. All infected rabbits that received saline died by 60 hpi, while 67% of the CT-1-treated animals survived until the end of the study. Treated animals showed improved liver function and histology, while the viral loads were similar. In the livers of CT-1-treated rabbits we observed reduction of oxidative stress, diminished PARP1/2 and JNK activation, and decreased inflammatory reaction, as reflected by reduced expression of tumor necrosis factor alpha, interleukin-1β , Toll-like receptor 4, VCAM-1, and MMP-9. In addition, CT-1-treated rabbits exhibited marked upregulation of TIMP-1 and increased expression of cytoprotective and proregenerative growth factors, including platelet-derived growth factor B, epidermal growth factor, platelet-derived growth factor receptor β, and c-Met. In conclusion, in a lethal form of acute viral hepatitis, CT-1 increases animal survival by attenuating inflammation and activating cytoprotective mechanisms, thus representing a promising therapy for ALF of viral origin.

This work was undertaken to investigate whether treatment with melatonin prevents oxidative stress and changes in the expression and activity of factor erythroid 2-related factor 2 (Nrf2)-mediated antioxidant enzymes in an animal model of fulminant hepatic failure of viral origin. Rabbits were experimentally infected with 2 x 10(4) hemagglutination units of a rabbit hemorrhagic disease virus (RHDV) isolate and received melatonin at two concentrations of 10 mg/kg and 20 mg/kg at 0, 12 and 24 hr postinfection. Blood transaminases, blood lactate dehydrogenase, liver concentration of thiobarbituric reactive acid substances and the liver oxidized to reduced glutathione ratio significantly increased at 36 hr postinfection in infected animals. Significant decreases were found in the mRNA levels and in the liver activities of Mn-superoxide dismutase, glutathione peroxidase and glutathione-S-transferase in infected rabbits. These effects were prevented by melatonin administration in a concentration-dependent manner. Melatonin treatment was not accompanied by changes in protein levels of Kelch-like ECH-associating protein 1 (Keap1) but resulted in an increased protein expression of Nrf2 in the cytoplasm and the nucleus, which was confirmed by the results of Nrf2 immunostaining. Nuclear extracts from livers of melatonin-treated rats displayed an enhanced antioxidant responsive element (ARE)-binding activity of Nrf2. Our results suggest a potential hepatoprotective role of melatonin in fulminant hepatic failure, partially mediated through the abrogation of oxidative stress and the prevention of the decreased activity of antioxidant enzymes via the Nrf2 pathways.

International audience; Management of fulminant hepatic failure (FHF) continues to be one challenging problem, and experimental animal models resembling its clinical conditions are still needed. Rabbit hemorrhagic disease (RHD) fullfils many requirements of an animal model of FHF. This work investigated changes in MAPK, NF-κB, AP-1 and STAT pathways during RHD-induced liver injury. Rabbits were infected with 2 × 104 hemagglutination units of an RHD virus isolate. Apoptosis was documented by the presence of caspase-3 activity and substantial PARP proteolysis at 36 and 48 h postinfection (pi). Infection induced a marked and maintained expression of TNF-α from 12 h pi, while there was only a transitory increase in IL-6 expression. Expression of phosphorylated (p)-JNK, p-p38 and p-ERK1/2 was significantly elevated at 12 h pi. At 48 h pi p-JNK expression was maintained at a maximum level, while that of p-p38 returned to normality and there was no p-ERK1/2 expression. Activation of NF-κB and AP-1 and increased expression of VCAM-1 and COX-2 were observed. No significant changes were detected in activation of STAT1 and STAT3, while SOCS3 expression increased significantly. The current findings suggest that activation of JNK is an essential component in liver injury mediated by the RHD virus and that lack of activation of STAT3, probably mediated by SOCS3 over-expression, would contribute to the inhibition of the regenerative response. Data show the presence of molecular mechanisms contributing to liver damage and the lack of regeneration and they support the usefulness of this model to investigate novel therapeutical modalities in FHF.

The prevalence of border disease virus (BDV) viraemia in Spanish lambs was determined from 2089 sera randomly collected at two slaughterhouses in 2001 and 2003, as well as in 126 sera obtained in 2004 from a fattening unit with an acute disease problem. BDV was detected with an indirect peroxidase monolayer assay (IPMA), and for the fattening unit sera also by an antigen ELISA. A subset of sera was additionally tested for BDV antibodies. The BDV prevalence in the slaughterhouse sera was 0.24%, whereas 7.1% of randomly selected and 38.6% of sera from clinically affected lambs in the fattening unit were virus positive. Pestivirus antibodies were found in 17.6% of the slaughterhouse sera and 28.6% of those from randomly selected lambs in the fattening unit. In total, 33 virus isolates and 3 antigen positive samples were identified. Genetic typing of 5'-UTR sequences classified all 36 pestiviruses as of BDV type 4. This shows that from a low BDV prevalence in apparently healthy lambs in the entire sheep population, clinical problems associated with BDV can develop when viraemic sheep are brought into intense rearing units.

The effect as antiviral agents versus viral hepatitis B and C of three compounds purified from natural products commonly used as remedies in traditional Chinese medicine, cantharidin, cephalotaxine and homoharingtonine, was investigated. To assess the activity of these compounds against flavivirus, we used bovine viral diarrhoea virus (BVDV) as a surrogate for hepatitis C virus (HCV). Anti-BVDV activity was determined by reduction in BVDV-RNA production and protection of infected embryonic bovine trachea (EBTr) cells against the cytopathic effect of BVDV. The effect versus hepatitis B virus (HBV) was investigated by measuring HBsAg and HBV-DNA release from hepatoblastoma HepG2 2.2.15 cells infected with HBV. As positive control we used the standard anti-HBV and anti-HCV drugs, lamivudine and ribavirin, respectively. Up to 100 microM lamivudine and ribavirin did not induce cell toxicity, whereas they induced dose-dependent anti-HBV and anti-BVDV effects, respectively. In the same range, cantharidin, cephalotaxine and homoharringtonine induced toxicity in EBTr cells and had no protective effect against BVDV. In contrast, they were able to inhibit HBV production at concentrations 10- to 100-fold lower than those inducing cell toxicity, which suggests that they are useless for the treatment of infection by flaviviruses, but potentially useful in combined therapy against hepatitis B.

Background: This work was undertaken to investigate whether treatment with N-acetyl-cysteine (NAC) prevents oxidative stress and inhibits the apoptotic pathways in an animal model of fulminant hepatic failure. Methods: Rabbits were experimentally infected with 2×104 hemagglutination units of a rabbit hemorrhagic disease virus isolate. Results: The spontaneous mortality rate of infected animals was 67% at 36 h post infection (pi) and 90% at 48 h pi. This percentage decreased significantly in animals receiving an i.p. injection of NAC (150 mg/kg body way/daily), for 7 days prior to infection. From 36 h pi marked increases were detected in blood levels of transaminases, lactate dehydrogenase, bilirubin and the oxidised/reduced glutathione ratio. All these effects were significantly prevented by NAC treatment. The Bax to Bcl-2 relative expression, the expression of FasL, cytochrome c and PARP-1, and the activity of caspase 3 were significantly increased at 36 and 48 h pi in infected animals. These changes were markedly reduced in animals treated with NAC, with the exception of FasL. Conclusion: Our results suggest a potential hepatoprotective role of NAC in fulminant hepatic failure, mediated partially through the modulation of the intrinsic pathway of apoptosis.

The antiviral activity versus flaviviruses of artemisinin, a safe drug obtained from Artemisia annua and commonly used to treat malaria, has been investigated using as an IN VITRO model bovine epithelial cells from embryonic trachea (EBTr) infected with the cytopathic strain Oregon C24V, of bovine viral diarrhoea virus (BVDV), which is a member of the Flaviviridae family. Antiviral activity was estimated by the degree of protection against the cytopathic effect of BVDV on host cells and by the reduction in BVDV-RNA release to the culture medium. To induce an intermediate cytopathic effect in non-treated cells, EBTr cells were first exposed to BVDV for 48 h and then incubated with virus-free medium for 72 h. Ribavirin and artemisinin (up to 100 microM) induced no toxicity in host cells, whereas a slight degree of toxicity was observed for IFN-alpha at concentrations above 10 U/mL up to 100 U/mL. Treatment of infected cells with IFN-alpha, ribavirin and artemisinin markedly reduced BVDV-induced cell death. A combination of these drugs resulted in an additive protective effect. These drugs induced a significant reduction in the production/release of BVDV virions by infected EBTr cells; there was also an additive effect when combinations of them were assayed. These results suggest a potential usefulness of artemisinin in combination with current pharmacological therapy for the treatment of human and veterinary infections by flaviviruses.

The pathophysiology of the diabetic kidney (e.g., hypertrophy, increased urinary albumin excretion (UAE)) is still ill-defined. Parathyroid hormone-related protein (PTHrP) is overexpressed in several nephropathies, but its role remains unclear. We evaluated the effect of high glucose on PTHrP and the PTH1 receptor (PTH1R) protein (by Western blot and immunohistochemistry) in the kidney of mice with streptozotocin-induced diabetes, and in several mouse renal cells in vitro. Diabetic mice showed a significantly increased renal expression of PTHrP and PTH1R proteins within 2–8 weeks from the onset of diabetes. These animals exhibited an intense immunostaining for both proteins in the renal tubules and glomeruli. Using transgenic mice overexpressing PTHrP targeted to the renal proximal tubule, we found a significant increase in the renal hypertrophy index and in UAE in these diabetic mice relative to their control littermates. Moreover, logistic regression analysis showed a significant association between both PTHrP and PTH1R protein levels and UAE in all diabetic mice throughout the study. High-glucose (25 mM) medium was found to increase PTHrP and PTH1R in tubuloepithelial cells, mesangial cells, and podocytes in vitro. Moreover, this increase in PTHrP (but not that of PTH1R) was inhibited by the AT1 receptor antagonist losartan. Collectively, these results indicate that the renal PTHrP/PTH1R system is upregulated in streptozotozin-induced diabetes in mice, and appears to adversely affect the outcome of diabetic renal disease. Our findings also suggest that angiotensin II might have a role in the PTHrP upregulation in this condition.

In this study we sought to determine whether molecular mechanisms involved in the pathogenesis of fulminant hepatic failure are present in rabbits experimentally infected with rabbit hemorrhagic disease virus (RHDV). The activities of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase, as well as bilirubin concentration, were found to be significantly increased 36 hours after infection. Infected animals also demonstrated significant decreases in factor VII activity, in the Fischer index, and in the deterioration of prothrombin time. The concentration of reduced glutathione was significantly decreased 36 hours after infection, and we noted a marked increase in the ratio of oxidized to reduced glutathione. Infected animals showed progressive decreases in liver activity of the antioxidant enzyme superoxide dismutase. Expression of hepatocyte growth factor and c-met was found to be progressively reduced from 24 hours after infection, during which time we detected no modification in messenger RNA (mRNA) levels of transforming growth factor (TGF)-alpha. TFG-beta 1 was overexpressed 24 and 36 hours after infection, and 36 hours after infection we detected a significant increase in TNF-alpha mRNA levels. Experimental RHDV infection also induced marked activation of nuclear factor-kappaB and a significant increase in inducible nitric oxide synthase mRNA levels from 24 hours after infection. Data obtained from this animal model support its usefulness in the investigation of potential novel therapeutical modalities aimed at neutralizing reactive oxygen species and hepatocyte growth inhibitors or enhancing hepatocyte responsiveness to mitogens.

The presence of rotaviruses was investigated in the faeces of 31 goat kids in a dairy herd that experienced an outbreak of severe diarrhoea which caused dehydration, anorexia and prostration in seven (22·6 per cent) of them. All the affected animals were two to three days old. A group A-specific elisa failed to detect rotaviruses in any of the samples but the characteristic electropherotype of group B rotaviruses was observed by electrophoresis in polyacrylamide gels in six of the animals. A highly significant statistical association between the shedding of rotavirus and the occurrence of diarrhoea was demonstrated. All the rotaviruses were detected in animals three to four days old. Cryptosporidium parvum, Clostridium perfringens and enterotoxigenic Escherichia coli were not detected in the seven diarrhoeic animals.

This study was designed to explore the effect of protein source on muscle susceptibility to lipid peroxidation in two representative species of fish farmed for human consumption: the freshwater rainbow trout and the seawater European sea bass. Four isoproteic diets (digestible protein in the range 366 to 392 for rainbow trout and 391 to 415 g/kg for European sea bass) were formulated to contain one of the following as the main protein source: fish meal, warm water alcohol-extracted or toasted soya protein concentrates or maize gluten meal. Highest daily growth indices were always achieved using the diets based on fish meal as the main source of protein (P< 0·05). Fish of both species given diets containing maize gluten and the toasted soya protein concentrate showed slowest growth. The depressant growth effect of the vegetable protein concentrates was greater in sea bass than in rainbow trout. Dietary treatment was not correlated with any significant effect on whole-body composition or intramuscular fat content except for ash concentration in European sea bass. Under conditions of forced peroxidation in vitro for 240 min, muscle specimens of trout and sea bass given diets containing fish protein as the main source of protein showed the highest peroxidation levels (P< 0·05); while the lowest peroxidation values were found in fish given maize gluten-containing diets (P< 0·05). In the present case, the partial substitution of fish meal with vegetable proteins in diets led to a lower susceptibility of fish flesh to peroxidation. This finding may have applications in the production of fish of improved quality and longer shelf life.

We evaluated the influence of dietary gelatinized starch and protein on the fatty acid composition of muscle in rainbow trout and European sea bass and on the susceptibility of flesh to lipid peroxidation. The possibility that flesh peroxidation could be accounted for by lipogenesis and the deposition of fat was also explored. The inclusion of gelatinized starch in the diet of rainbow trout improved growth with respect to that observed in fish fed crude starch (P

The effects of dietary fish oil and digestible protein (DP) levels on muscle fatty acid composition and susceptibility to lipid peroxidation were studied in two representative fish species for human nutrition, from fresh and seawater, rainbow trout (Oncorhynchus mykiss) and European sea bass (Dicentrarchus labrax). In rainbow trout, higher concentrations of dietary fat and DP led to higher weight gain (g/d) (P = 0.001 and P = 0.043 respectively). Additionally, an interaction effect was observed in this species, since the effect of DP was only evident when the dietary fat concentration was low (P = 0.043). A similar tendency was also observed in European sea bass, although with less marked differences among nutritional treatments. Trout fed on diets with a higher concentration of dietary fat had higher concentrations of intramuscular total and neutral lipids in the dorsal muscle (P = 0.005). Increased levels of dietary DP led to significantly lower concentrations of polar lipids in the dorsal muscle of both rainbow trout (P = 0.005) and European sea bass (P = 0.006). In the neutral fraction of intramuscular lipids of dorsal muscle the concentration of n-3 fatty acids was positively affected by the dietary fat concentration in both rainbow trout (P = 0.04) and sea bass (P = 0.001). Muscle homogenates from trout and sea bass fed on diets rich in fish oil showed a significantly higher susceptibility to oxidation than muscle homogenates from fish fed on low-fat diets (P = 0.001). The higher DP concentration also increased susceptibility to oxidation. Moreover, in rainbow trout an interaction effect was observed where the pro-oxidant effect was of higher magnitude when the dietary concentration of both nutrients, fat and protein, was high (P = 0.004).

SummaryFaeces samples from diarrhoeic and non-diarrhoeic lambs and goat kids aged 1–45 days were examined for enteric pathogens.Cryptosporidium parvumwas detected in both diarrhoeic lambs (45%) and goat kids (42%) but not in non-diarrhoeic animals. F5+(K99+) and/or F41+Escherichia colistrains were isolated from 26% and 22% of the diarrhoeic lambs and goat kids, respectively, although these strains, which did not produce enterotoxins ST I or LT I, were found with similar frequencies in non-diarrhoeic animals. A F5−F41−ST I+E. colistrain was isolated from a diarrhoeic lamb (0·6%). VerotoxigenicE. coliwas isolated from both diarrhoeic and non-diarrhoeic lambs (4·1% and 8·2%, respectively) and there was no association between infection and diarrhoea. The prevalence of group A rotavirus infection in diarrhoeic lambs was very low (2·1%). Groups A and B rotaviruses were detected in three (8·1%) and five (13·5%) diarrhoeic goat kids from two single outbreaks. Group C rotaviruses were detected in four non-diarrhoeic goat kids. An association of diarrhoea and infection was demonstrated only for group B rotavirus.Clostridium perfringenswas isolated from 10·8% of the diarrhoeic goat kids but not from non-diarrhoeic goat kids or lambs.Salmonella arizonaewas isolated from a diarrhoeic goat kid (2·7%) and the clinical characteristics of the outbreaks where these two latter enteropathogens were found different from the rest. Picobirnaviruses were detected in a diarrhoeic lamb. No coronaviruses were detected using a bovine coronavirus ELISA. No evidence was found of synergistic effect between the agents studied. Enteric pathogens were not found in four (8·7%) and three (20%) outbreaks of diarrhoea in lambs and goat kids, respectively.

The stability and catalytic efficacy of the L-2-haloacid dehalogenase isolated from Azotobacter sp. RC26 were studied after immobilisation on a DEAE Sephacel solid matrix. While the optimum temperature for the soluble dehalogenase falls in the range of 30-40 degrees C, the activity of the immobilised enzyme shows a four-fold increase at 60 degree C. Immobilisation on a plug-flow bioreactor extends the range of usable substrate concentration. The improved catalytic characteristics after immobilisation of the haloacid dehalogenase may be relevant for its possible utilization in biotechnological applications ranging from waste treatment to synthesis of stereoisomers.

Acute hepatic failure (AHF) is a severe liver injury accompanied by hepatic encephalopathy which causes multiorgan failure with an extremely high mortality rate, even if intensive care is provided. Management of severe AHF continues to be one of the most challenging problems in clinical medicine. Liver transplantation has been shown to be the most effective therapy, but the procedure is limited by shortage of donor organs. Although a number of clinical trials testing different liver assist devices are under way, these systems alone have no significant effect on patient survival and are only regarded as a useful approach to bridge patients with AHF to liver transplantation. As a result, reproducible experimental animal models resembling the clinical conditions are still needed. The three main approaches used to create an animal model for AHF are: surgical procedures, toxic liver injury and infective procedures. Most common models are based on surgical techniques (total/partial hepatectomy, complete/transient devascularization) or the use of hepatotoxic drugs (acetaminophen, galactosamine, thioacetamide, and others), and very few satisfactory viral models are available. We have recently developed a viral model of AHF by means of the inoculation of rabbits with the virus of rabbit hemorrhagic disease. This model displays biochemical and histological characteristics, and clinical features that resemble those in human AHF. In the present article an overview is given of the most widely used animal models of AHF, and their main advantages and disadvantages are reviewed.

Rainbow trout (Oncorhynchus mykiss) hepatocytes were cultured under simulated conditions of varying nutritional status to explore the short-term modulation by dietary substrates of the main lipogenic enzymes: glucose-6-phosphate dehydrogenase (G6PD), malic enzyme (ME), ATP-citrate lyase (ACL), acetyl-CoA carboxylase (ACoAC) and fatty acid synthetase (FAS). Primary cultures were individually exposed to varying amounts of glucose, hydrolysed casein and long-chain polyunsaturated fatty acids (PUFA) for 12 h. A second set of experiments was designed to evaluate the effects of mixing different relative amounts of these macronutrients in the culture medium. Glucose concentrations of up to 20–25 mM SHOWED A STIMULATORY EFFECT ON G6PD, ME, ACL AND ACOAC ACTIVITY (Pin vivodietary conditions. It is felt that such an approach may serve to investigate the macronutrient regulation of other metabolic pathways.

A monomeric 29 kDa protein showing dehalogenase activity on several halogenated carboxylic acids has been purified from Azotobacter sp. strain RC26. The purified enzyme is specific for the L isomer of optically active 2-haloacids leading to the inversion of the product configuration. The dehalogenase is active at temperatures ranging from 30 to 60°C and shows a relatively high affinity for the substrate. The combined thermal stability, high substrate affinity and resistance to enzyme inhibitors found for the RC26 dehalogenase may be relevant for its use as catalyst in biotransformation processes.