Anna Granato

Hepatocyte transplantation as a treatment for glycogen storage disease type 1a

Treatment of many inherited disorders of hepatic metabolism is still challenging. Hepatocyte transplantation was done in a 47-year-old woman who had glycogen storage disease type 1a and severe fasting hypoglycaemia. 2 billion viable hepatocytes were infused via an indwelling portal-vein catheter, followed by a triple immunosuppression regimen with mycophenolate mofetil, tacrolimus, and steroids. 9 months after transplantation, on only tacrolimus, she eats a normal diet and can fast for 7 h without experiencing hypoglycaemia. Our results show that hepatocyte transplantation might be an alternative to liver transplantation in glycogen storage disease type 1a.

Bilirubin inhibits bile acid induced apoptosis in rat hepatocytes.

Background and aims: Hydrophobic bile acids contribute to hepatocellular injury in cholestasis and rapidly induce apoptosis in vitro; however, unlike Fas agonists, cholestasis does not cause extensive hepatocyte apoptosis. As antioxidants provide protection against bile acid induced liver injury, our premise was that bilirubin, a free radical scavenger with increased plasma levels in the presence of liver disease, could protect hepatocytes against bile acid induced apoptosis. Methods: Freshly isolated rat hepatocytes were incubated for four hours with 100 μmol/l glycochenodeoxycholate (GCDC) alone or with increasing concentrations of unconjugated (UCB) or conjugated (CB) bilirubin. Results: Both UCB and CB inhibited GCDC induced apoptosis in a dose dependent fashion and suppressed the generation of reactive oxygen species by hepatocytes. Conclusions: The antiapoptotic effect of bilirubin associated with its antioxidant properties indicates that hyperbilirubinaemia may have a protective role in liver disease.

Liver repopulation with bone marrow-derived cells improves the metabolic disorder in the Gunn rat

Background: Reversible ischaemia/reperfusion (I/R) liver injury has been used to induce engraftment and hepatic parenchymal differentiation of exogenous β2-microglubulin−/Thy1+ bone marrow derived cells. Aim: To test the ability of this method of hepatic parenchymal repopulation, theoretically applicable to clinical practice, to correct the metabolic disorder in a rat model of congenital hyperbilirubinaemia. Methods and results: Analysis by confocal laser microscopy of fluorescence labelled cells and by immunohistochemistry for β2-microglubulin, 72 hours after intraportal delivery, showed engraftment of infused cells in liver parenchyma of rats with I/R, but not in control animals with non-injured liver. Transplantation of bone marrow derived cells obtained from GFP-transgenic rats into Lewis rats resulted in the presence of up to 20% of GFP positive hepatocytes in I/R liver lobes after one month. The repopulation rate was proportional to the number of transplanted cells. Infusion of GFP negative bone marrow derived cells into GFP positive transgenic rats resulted in the appearance of GFP negative hepatocytes, suggesting that the main mechanism underlying parenchymal repopulation was differentiation rather than cell fusion. Transplantation of wild type bone marrow derived cells into hyperbilirubinaemic Gunn rats with deficient bilirubin conjugation after I/R damage resulted in 30% decrease in serum bilirubin, the appearance of bilirubin conjugates in bile, and the expression of normal UDP-glucuronyltransferase enzyme evaluated by polymerase chain reaction. Conclusions: I/R injury induced hepatic parenchymal engraftment and differentiation into hepatocyte-like cells of bone marrow derived cells. Transplantation of bone marrow derived cells from non-affected animals resulted in the partial correction of hyperbilirubinaemia in the Gunn rat.

Intraportal hepatocyte transplantation in the pig: a hemodynamic and histopathological study1

Background. Hepatocyte transplantation is an attractive treatment for various liver diseases. The intraportal route of transplantation is favored, but little information is available on the possible adverse effects in this technique. We investigated the influence of intraportal loads of hepatocytes on portal, pulmonary, and systemic hemodynamics in 13 pigs. Methods. Under general anesthesia, pigs were provided with an arterial line, a Swan-Ganz catheter, and two intraportal catheters, one for cell infusion and one for heparin infusion and portal pressure measurement. Pig hepatocytes were infused at a rate of 25 million cells/min. Results. The first six animals were used to develop the infusion technique. In the last seven animals, portal pressure increased linearly with cell load upon infusion of 400–2400×106 hepatocytes (r2=0.704;P <0.05). Portal flow measured by Doppler sonography decreased by 23–66% below basal values. An inverse linear relationship was found between portal pressure and portal flow (r2=0.679;P <0.05), portal flow approaching zero for portal pressure >40 mmHg. Pulmonary arterial pressure increased by 11–62%. AST increased up to 10-fold, and platelets decreased by 22–58%. Hepatocytes-containing thrombi were present in segmental and in smaller portal branches. Hepatocytes were always identified in lung sinusoids 48 hr after infusion, and a small basal pulmonary infarction was found in one animal. Conclusions. These data suggest that up to 2.4% of total hepatocyte mass can be infused in this large animal model. However, the risk of significant thrombotic complications should be considered for clinical applications.

Functional polarity of Na+/H+ and Cl-/HCO3/- exchangers in a rat cholangiocyte cell line

Intrahepatic bile duct cells (cholangiocytes) play an important role in the secretion and alkalinization of bile. Both Na+/H+exchange (NHE) and Cl-/[Formula: see text]exchange (AE) contribute to these functions, but their functional distribution between the apical and basolateral membrane domains remains speculative. We have addressed this issue in a normal rat cholangiocyte cell line (NRC-1), which maintains a polarized distribution of membrane markers. Gene expression of AE and NHE isoforms was studied by RT-PCR. For functional studies, cells were placed in a chamber that allowed separate perfusion of the apical and basolateral aspect of the epithelial sheet; intracellular pH (pHi) was measured by 2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein microfluorometry. In[Formula: see text]-CO2free medium and in the presence of apical amiloride, pHi recovery from an acid load was Na+ dependent and was inhibited by basolateral amiloride and by HOE-642 (10 μM), consistent with basolateral localization of the NHE1 isoform, which had clearly expressed mRNA. Apical Na+readmission induced a slow pHirecovery that was inhibited by apical administration of 1 mM HOE-642 or amiloride. Among the apical NHE isoforms, NHE2 but not NHE3 gene expression was detected. The AE1 gene was not expressed, but two different variants of AE2 mRNAs (AE2a and AE2b) were detected; pHi experiments disclosed AE activities at both sides of the membrane, but only apical AE was activated by cAMP. In conclusion, these studies provide the first functional description of acid-base transporters in a polarized cholangiocyte cell line. NHE1, NHE2, AE2a, and AE2b isoforms are expressed and show different membrane polarity, functional properties, and sensitivity to inhibitors. These observations add a considerable level of complexity to current models of electrolyte transport in cholangiocytes.

Comparison of pig, human and rat hepatocytes as a source of liver specific metabolic functions in culture systems--implications for use in bioartificial liver devices.

The limited availability of human hepatocytes results in the use of animal cells in most bioartificial liver support devices. In the present work, clinically relevant liver specific metabolic activities were compared in rat, pig and human hepatocytes cultured on liver-derived biomatrix to optimize the expression of differentiated functions. Pig hepatocytes showed higher rates of diazepam metabolism (2.549±0.821 μg/h/million cells vs. 0.474±0.079 μg/h/million cells rats, p<0.005, and vs. 0.704±0.171 μg/h/million cells in man, p<0.005) and of bilirubin conjugation (21.60116±8.433237 μmoles/l/24 h vs. 6.786809±2.983758 in man, p<0.001 and vs. 9.956538±1.781016 μmoles/l/24 h in rats, p<0.005). Urea synthesis was similar in pig and in human hepatocytes (150±46.3 vs. 144.8±21.46 nmoles/h/million cells) and it was lower in rats (84.38±35.2; p<0.001 vs. man, p<0.02 vs. pig). High liver specific metabolic activities in cultured pig hepatocytes further support their use as a substitue for human cells in bioartificial liver devices

RNA-sequence analysis of gene expression from honeybees (Apis mellifera) infected with Nosema ceranae

Honeybees (Apis mellifera) are constantly subjected to many biotic stressors including parasites. This study examined honeybees infected with Nosema ceranae (N. ceranae). N. ceranae infection increases the bees energy requirements and may contribute to their decreased survival. RNA-seq was used to investigate gene expression at days 5, 10 and 15 Post Infection (P.I) with N. ceranae. The expression levels of genes, isoforms, alternative transcription start sites (TSS) and differential promoter usage revealed a complex pattern of transcriptional and post-transcriptional gene regulation suggesting that bees use a range of tactics to cope with the stress of N. ceranae infection. N. ceranae infection may cause reduced immune function in the bees by: (i)disturbing the host amino acids metabolism (ii) down-regulating expression of antimicrobial peptides (iii) down-regulation of cuticle coatings and (iv) down-regulation of odorant binding proteins.

DNA and RNA isolation from canine oncologic formalin-fixed, paraffin-embedded tissues for downstream “-omic” analyses possible or not?

Formalin-fixed, paraffin-embedded (FFPE) tissues represent a unique source of archived biological material, but obtaining suitable DNA and RNA for retrospective “-omic” investigations is still challenging. In the current study, canine tumor FFPE blocks were used to 1) compare common commercial DNA and RNA extraction kits; 2) compare target gene expression measured in FFPE blocks and biopsies stored in a commercial storage reagent; 3) assess the impact of fixation time; and 4) perform biomolecular investigations on archival samples chosen according to formalin fixation times. Nucleic acids yield and quality were determined by spectrophotometer and capillary electrophoresis, respectively. Quantitative real-time polymerase chain reaction assays for the following genes: BCL-2–associated X protein, B-cell lymphoma extra large, antigen identified by monoclonal antibody Ki-67, proto-oncogene c-KIT (c-kit). Two internal control genes (Golgin A1 and canine transmembrane BAX inhibitor motif containing 4), together with direct sequencing of c-kit exons 8, 9, 11, and 17, were used as end points. Differences in DNA/RNA yield and purity were noticed among the commercial kits. Nucleic acids (particularly RNA) extracted from paraffin blocks were degraded, even at lower fixation times. Compared to samples held in the commercial storage reagent, archived tissues showed a poorer amplification. Therefore, a gold standard protocol for DNA/RNA isolation from canine tumor FFPE blocks for molecular investigations is still troublesome. More standardized storage conditions, including time between sample acquisition and fixation, fixation time, and sample thickness, are needed to guarantee the preservation of nucleic acids and, then, their possible use in retrospective transcriptomic analysis.

Spring mortality in honey bees in northeastern Italy: detection of pesticides and viruses in dead honey bees and other matrices

In spring there is often a rise in honey bee mortality incidents. The aim of this study was to investigate the potential correlation, in the reported incidents, between exposure to pesticide treatments and virus infections. Here we summarize the situation in northeastern Italy during spring 2014, evaluated by monitoring 150 active ingredients and three honey bee viruses in dead honey bees and other matrices. At least one active ingredient was found in 72.2% of the 79 dead honey bee samples, with the most abundant (59.4%) being insecticides, mainly belonging to the class of neonicotinoids (41.8%), followed by fungicides (40.6%). Imidacloprid, chlorpyrifos, tau-fluvalinate, and cyprodinil were the most frequently detected active ingredients. Multiple virus infections were monitored, revealing a high prevalence of chronic bee paralysis virus (CBPV) and deformed wing virus (DWV), detected in all samples except one. 71 and 37% of the CBPV- and DWV positive samples, respectively, showed a high number of viral copies per bee (>107). This work emphasizes the possible relationship between spring mortality in honey bees and pesticide treatments. Honey bee viruses could synergistically exacerbate the negative impact of pesticides on honey bee health, endangering the survival of colonies.

Introduction of Aethina tumida (Coleoptera: Nitidulidae) in the regions of Calabria and Sicily (southern Italy)

Aethina tumida (small hive beetle, SHB) was first detected in September 2014 in Calabria region, southern Italy, and in a single apiary in Sicily in November 2014. In September 2015, SHB was again recorded in Calabria, and in 2016, only sentinel honey bee nucleus colonies were found to be infested. Its phylogenetic relationship and possible origin were investigated comparing the cox1 sequences with the corresponding region available in the GenBank database. The neighbour-joining method revealed that the first Italian specimen belonged to a group also containing an African specimen from Cameroon. The Italian specimens differ from the SHBs spread worldwide and are split into two different groups: group B1 includes the AfricCam3 sequence and the first SHB identified in Calabria; group B2 includes specimens from Calabria and the only one from Sicily which share identical cox1 sequences. SHB in Italy appears to have been introduced from Africa and includes independent or contemporary incursions in the two concerned regions. The most likely scenario is that SHB was introduced into Calabria followed by man-mediated migration to Sicily.