Thomas J. Caperna

New models of hepatitis E virus replication in human and porcine hepatocyte cell lines.

Hepatitis E virus (HEV) causes acute, enterically transmitted hepatitis in human. It is associated with large epidemics in tropical and subtropical regions where it is endemic or with sporadic cases in non-endemic regions. Unlike other hepatitis viruses, HEV has several animal reservoirs. Phylogenetic studies on HEV human and animal sequences, and the identification of cases of direct transmission from animal to human strongly suggest that HEV is a zoonotic agent. The lack of efficient cell culture models limits studies on molecular and cellular aspects of HEV infection and species barrier crossing. The present study reports on the development of two new in vitro models of HEV replication using a human hepatoma-derived cell line, HepaRG, and a porcine embryonic stem cell-derived cell line, PICM-19. These two cell lines have morphological and functional properties similar to primary hepatocytes. These in vitro culture systems support HEV replication and release of encapsidated RNA. These new models represent a powerful tool for studying the viral replication cycle, species barrier crossing and virulence factors.

Identification of protein carbonyls in serum of the fetal and neonatal pig

Oxidation of serum proteins leads to non-reversible carbonyl formation which alters their function and is associated with stress-related disease processes. The primary objective of this study was to quantify and identify oxidized serum proteins in fetal and newborn piglets. Protein carbonyls were converted to hydrazones with dinitrophenyl hydrazine and quantified spectrophotometrically. For identification, serum protein carbonyls were derivatized with biotin hydrazide, separated by 2D PAGE and stained with FITC-avidin. Biotin-labeled proteins were excised from gels and identified by mass spectrometry. At birth, carbonyls were determined to be approximately 600 pmole/mg serum protein. Fetuses at 50 and 100 days of gestation had similar levels of protein carbonyls as newborns. Carbonyl levels were also similar for control and runt (<1 kg at birth) piglets between 1 and 21 days of age; however, distribution of many proteins varied by age and was also influenced by birth weight. Major oxidized proteins identified in fetal (f) and newborn (n) pigs included; albumin (f, n), transferrin (f, n), fetuin-A (f, n) alpha fetoprotein (f, n), plasminogen (f, n), fetuin-B (f), alpha-1-antitrypsin (f, n) alpha-1-acid glycoprotein (f) and immunoglobulins (n). While abundance and distribution of oxidized proteins changed over time, these changes appear to primarily reflect relative amounts of those proteins in serum.

Selective and organotypic culture of intrahepatic bile duct cells from adult pig liver

SummarySecondary culture of nontransformed bile duct epithelium has been difficult to achieve. STO feeder cell-dependent secondary cultures of adult pig bile duct cells were established from primary cultures of adult pig liver cells. Adult pig hepatocytes exhibited limited or no replication and were lost from the secondary culture at Passage 3 or 4. In contrast, adult pig bile duct cells replicated and were carried for 4–8 passages in secondary culture. A simple method to produce nearly pure pig intrahepatic bile duct cultures was first to freeze a relatively crude liver cell preparation. Upon subsequent thawing, all hepatocytes and most macrophages were lysed. Bile duct cells composed 95% of the surviving cells after the freeze/thaw, and they grew out rapidly. The bile duct cells grew on top of the STO feeder cells as closely knit epithelial, colonial outgrowths. Histocytochemical and biochemical analyses demonstrated high levels of gamma-glutamyltranspeptidase activity and low levels of P450 activity in the bile duct cultures. The bile duct cells spontaneously adopted a multicellular ductal morphology after 7–10 d in static culture which was similar to that found in in vivo pig liver. Transmission electron microscopic examination revealed complex junctions and desmosomes typical of epithelium, and lumenally projecting cilia typical of in vivo intrahepatic bile ductules. This simple method for the coculture of pig intrahepatic bile duct cells which adopt in vivo-like structure may facilitate biological studies of this important, but difficult to culture, cell type.

Adipokine gene transcription level in adipose tissue of runt piglets

Runt piglets were used as a model for neonatal stress to test the hypothesis that stress during the pre-weaning period can alter adipokine gene transcription levels. Runts were selected by birth mass <1kg and compared to littermates (controls) of mean litter weight. Subcutaneous (SQ) and perirenal (PR) adipose tissues were collected at d1 (n=5), d7 (n=7) or d21 (n=9) of age. Real time PCR was used to quantify mRNA abundance for: leptin, adiponectin, interleukin 1beta (IL1beta), IL6, IL8, IL10, IL15, tumor necrosis factor alpha, haptoglobin, macrophage migration inhibitory factor (MIF), monocyte chemotactic protein, vascular endothelial growth factor and cyclophilin. Leptin and adiponectin mRNA abundance were lower, while IL1beta, IL6, IL10 and MIF mRNA abundance were higher in SQ of runts than controls at d1 (P<0.05). Leptin, IL6, IL10, haptoglobin and MIF mRNA abundance were higher in PR from runts than controls at d7 (P<0.05) and MIF mRNA abundance was elevated by 30 fold in PR of runts at d21 (P<0.001). Thus, stressors affecting neonatal runts produce different responses in adipokine gene transcription by PR and SQ than in normal sized littermates.

Ontogeny of adipokine expression in neonatal pig adipose tissue

This study examined ontogeny of development for a range of adipokines in neonatal adipose tissue. Pigs (Sus scrofa) were selected across six litters for sampling subcutaneous (SQ) and perirenal (PR) adipose tissues at d1, d4, d7 or d21 of age and total RNA extraction. Reverse transcription and real-time PCR were used to quantify mRNA abundance for: leptin, adiponectin, interleukin 1beta (IL-1beta), IL-6, IL-8, IL-10, IL-15, tumor necrosis factor alpha (TNFalpha), haptoglobin, vascular endothelial growth factor (VEGF), macrophage migration inhibitory factor (MIF), monocyte chemoattractant protein 1 (MCP1) and cyclophilin. Leptin, adiponectin and IL-15 expression increased from d1 to d 21 of age in both SQ and PR. Haptoglobin, VEGF, MIF and IL-8 expression decreased between d1 and d4 of age in SQ. TNFalpha expression was unchanged from d1-7 and then increased at d21. IL-1beta, IL-6 and IL-10 expression were unchanged with age in SQ; whereas IL-1beta and IL-6 mRNA abundance in the PR increased with age. Analysis of the mRNA abundance for these adipokines within adipose tissue from d1 to d21 of age demonstrated that neonatal development of adipokine expression varies among the different adipokines and the internal and external sites of adipose tissue deposition (PR versus SQ).

Utility of proteomics techniques for assessing protein expression

Proteomic technologies are currently used as an effective analytical tool for examining modifications in protein profiles. Understanding the natural variation of soybean seed proteins is necessary to evaluate potential unintended (collateral) effects due to transgenic modifications in genetically modified (GMO) soybeans. We used two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry, and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) to separate, identify and quantify the different classes of soybean seed proteins. Sixteen soybean genotypes, including four wild and twelve cultivated genotypes, belonging to four different subgroups were used as models for protein profile evaluation. Significant variations of allergen and anti-nutritional protein profiles were observed between two different groups, cultivated and wild soybean genotypes. However, only minor variations in protein profiles were observed within the soybean samples from the same group (cultivated or wild). These results may be useful to scientists needing to compare GMO and non-GMO soybeans once additional data are generated on additional soybean varieties and the same varieties grown at different geographical locations.

A gel-based reference map of the porcine hepatocyte proteome

The overall goal of our research is to characterize and identify gene expression profiles of porcine hepatic cells. In this study, we have prepared two-dimensional electrophoresis maps of cytosol and membrane fractions from freshly prepared hepatocytes which were pooled from three crossbred pigs (35-69kg). Following isoelectric focusing with three pH range immobilized pH gradient strips (pH 3-6, 5-8 and 7-10) and staining the second dimension gels with colloidal Coomassie blue, 728 protein spots were picked and digested with trypsin. Extracted tryptic peptides were initially subjected to matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) analysis for identification of proteins by peptide mass fingerprinting (PMF). Proteins which were not identified by PMF were analyzed by liquid chromatography-tandem MS. Utilizing publicly available databases [NCBInr, Swiss Prot and expressed sequence tags (EST)], 648 proteins were identified. Of those, 282 were unique proteins and greater than 90% of proteins spots contained single proteins. These data represent the first comprehensive proteomic analysis of porcine hepatocytes and will provide a database for future investigations of endocrine regulation of gene expression and metabolic processes in vitro.

Identification and characterization of aquaporin-9 (AQP9) in porcine hepatic tissue and hepatocytes in monolayer culture☆

Aquaporins (AQPs) are members of a large family of integral membrane proteins involved in the rapid movement of water and neutral solutes across cell membranes. In this study, we have prepared an affinity-purified porcine-specific polyclonal antiserum to AQP9 and have investigated the distribution and expression of AQP9 in pig liver tissue and in hepatocytes in primary culture. Immunocytochemical analysis showed that AQP9 was primarily localized in the membrane structures of hepatocytes and was not associated with intrahepatic bile ducts or blood vessels. Western blot analysis indicated that AQP9 ranged in apparent molecular mass between 27 and 38 kD in whole liver and hepatocyte membrane fractions; minor components were also observed at approximately 34 kD in the cytosol compartment of hepatocytes, bile duct and gall bladder. A prominent immunoreactive band at 44 kD was shown to be an artifact of Western blot analysis. In primary cultures of porcine hepatocytes, glucagon enhanced absolute levels of AQP9 protein, while gene expression was enhanced by T3 and glucagon. Insulin alone had no discernable influence on AQP9 gene expression or its cellular protein levels. These data suggest that AQP9 is a major AQP in porcine hepatic tissue and appears to be primarily responsive to glucagon induction.

Growth and Development Symposium: Development, characterization, and use of a porcine epiblast-derived liver stem cell line: ARS-PICM-19.

Totipotent embryonic stem cell lines have not been established from ungulates; however, we have developed a somatic stem cell line from the in vitro culture of pig epiblast cells. The cell line, ARS-PICM-19, was isolated via colony cloning and was found to spontaneously differentiate into hepatic parenchymal epithelial cell types, namely hepatocytes and bile duct cells. Hepatocytes form as monolayers and bile duct cells as 3-dimensional bile ductules. Transmission electron microscopy revealed that the ductules were composed of radially arranged, monociliated cells with their cilia projecting into the lumen of the ductule whereas hepatocytes were arranged in monolayers with lateral canalicular structures containing numerous microvilli and connected by tight junctions and desmosomes. Extensive Golgi and rough endoplasmic reticulum networks were also present, indicative of active protein synthesis. Analysis of conditioned medium by 2-dimensional electrophoresis and mass spectrometry indicated a spectrum of serum-protein secretion by the hepatocytes. The PICM-19 cell line maintains a range of inducible cytochrome P450 activities and, most notably, is the only nontransformed cell line that synthesizes urea in response to ammonia challenge. The PICM-19 cell line has been used for several biomedical- and agricultural-related purposes, such as the in vitro replication of hepatitis E virus, a zoonotic virus of pigs, and a spaceflight experiment to evaluate somatic stem cell differentiation and liver cell function in microgravity. The cell line was also evaluated as a platform for toxicity testing and has been used in a commercial artificial liver rescue device bioreactor. A PICM-19 subclone, PICM-19H, which only differentiates into hepatocytes, was isolated and methods are currently under development to grow PICM-19 cells without feeder cells. Feeder-cell-independent growth will facilitate the study of mesenchymal-parenchymal interactions that influence the divergent differentiation of the PICM-19 cells, enhance our ability to genetically modify the cells, and provide a better model system to investigate porcine hepatic metabolism.

Identification and characterization of a nuclear factor-κ B-p65 proteolytic fragment in nuclei of porcine hepatocytes in monolayer culture.

Hepatic responses to proinflammatory signals are controlled by the activation of several transcription factors, including, nuclear factor-κ B (NF-κB). In this study, hepatocytes prepared from suckling pigs and maintained in serum-free monolayer culture were used to define a novel proinflammatory cytokine-specific NF-κB subunit modification. The immunoreactive p65 protein was detected by Western blot analysis at the appropriate molecular weight in the cytosol of control cultures and those incubated with tumor necrosis factor-α (TNF). However, in nuclei, the p65 antisera cross-reacted with a protein of approximately 38 kDa (termed p38) after TNF addition, which was not observed in the cytosol of control or cytokine-treated cells. Specifically, incubation with TNF also resulted in phosphorylation (P < 0.05) of the inhibitor complex protein (IκB), whereas incubation with other cytokines, IL-6, IL-17a, or oncostatin M was not associated with either phosphorylation of IκB or nuclear translocation of p65. Intracellular endothelial nitric oxide synthase was deceased (P < 0.05) and plasminogen activator inhibitor-1 secretion was increased (P < 0.05) after TNF incubation. The TNF-induced p38 protein was purified from hepatocyte nuclei by immunoprecipitation, concentrated by electrophoresis, and subsequently analyzed by mass spectrometry. Ten unique NF-κB p65 peptides were identified after digestion with trypsin and chymotrypsin; however, all were mapped to the N-terminus and within the first 310 amino acid residues of the intact p65 protein. Although low molecular weight immunoreactive p65 molecules were previously observed in various human and rodent systems, this is the first report to positively identify the p38 fragment within hepatocyte nuclei or after specific cytokine (TNF) induction.