Detection of apoptosis in vivo using antibodies against caspase-induced neo-epitopes

Cell death induction by apoptosis is an important process in the maintenance of tissue homeostasis as well as tissue destruction during various pathological processes. Consequently, detection of apoptotic cells in situ represents an important technique to assess the extent and impact of cell death in the respective tissue. While scoring of apoptosis by histological assessment of apoptotic cells is still a widely used method, it is likely biased by sensitivity problems and observed-based variations. The availability of caspase-mediated neo-epitope-specific antibodies offers new tools for the detection of apoptosis in situ. Here, we discuss the use of immunohistochemical detection of cleaved caspase 3 and lamin A for the assessment of apoptotic cells in paraffin-embedded liver tissue. Furthermore, we evaluate the effect of tissue pretreatment and antigen retrieval on the sensitivity of apoptosis detection, background staining and maintenance of tissue morphology.

Analysis of mRNA transcripts improves the success rate of molecular genetic testing in OTC deficiency

BACKGROUND: Ornithine transcarbamylase (OTC) deficiency is the most common inborn error of urea metabolism that can lead to hyperammonemic crises and orotic aciduria. To date, a total of 341 causative mutations within the OTC gene have been described. However, in about 20% of the patients with enzymatically confirmed OTC deficiency no mutation can be detected when sequencing of genomic DNA analyzing exons and adjacent intronic segments of the OTC gene is performed. METHODS: Standard genomic DNA analysis of the OTC gene in five consecutive patients from five families revealed no mutation. Hence, liver tissue was obtained by needle sampling or open biopsy and RNA extracted from liver was analyzed. RESULTS: Complex rearrangements of the OTC transcript (three insertions and two deletions) were found in all five patients. CONCLUSION: In patients with a strong suspicion of OTC deficiency despite normal results of sequencing exonic regions of the OTC gene, characterization of liver OTC mRNA is highly effective in resolving the genotype. Liver tissue sampling by needle aspiration allows for both enzymatic analysis and RNA based diagnostics of OTC deficiency.

Tumor necrosis factor-alpha upregulates 11beta-hydroxysteroid dehydrogenase type 1 expression by CCAAT/enhancer binding protein-beta in HepG2 cells

The enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyzes the conversion of inactive to active glucocorticoids. 11beta-HSD1 plays a crucial role in the pathogenesis of obesity and controls glucocorticoid actions in inflammation. Several studies have demonstrated that TNF-alpha increases 11beta-HSD1 mRNA and activity in various cell models. Here, we demonstrate that mRNA and activity of 11beta-HSD1 is increased in liver tissue from transgenic mice overexpressing TNF-alpha, indicating that this effect also occurs in vivo. To dissect the molecular mechanism of this increase, we investigated basal and TNF-alpha-induced transcription of the 11beta-HSD1 gene (HSD11B1) in HepG2 cells. We found that TNF-alpha acts via p38 MAPK pathway. Transient transfections with variable lengths of human HSD11B1 promoter revealed highest activity with or without TNF-alpha in the proximal promoter region (-180 to +74). Cotransfection with human CCAAT/enhancer binding protein-alpha (C/EBPalpha) and C/EBPbeta-LAP expression vectors activated the HSD11B1 promoter with the strongest effect within the same region. Gel shift and RNA interference assays revealed the involvement of mainly C/EBPalpha, but also C/EBPbeta, in basal and only of C/EBPbeta in the TNF-alpha-induced HSD11B1 expression. Chromatin immunoprecipitation assay confirmed in vivo the increased abundance of C/EBPbeta on the proximal HSD11B1 promoter upon TNF-alpha treatment. In conclusion, C/EBPalpha and C/EBPbeta control basal transcription, and TNF-alpha upregulates 11beta-HSD1, most likely by p38 MAPK-mediated increased binding of C/EBPbeta to the human HSD11B1 promoter. To our knowledge, this is the first study showing involvement of p38 MAPK in the TNF-alpha-mediated 11beta-HSD1 regulation, and that TNF-alpha stimulates enzyme activity in vivo.

Molecular detection of hepatitis E virus in German domestic pigs

In industrial countries, Hepatitis E virus (HEV) transmission to humans is predominantly assumed to be a zoonotic infection. Recently, it has been demonstrated that about 50% of domestic pigs in Germany carry HEV-specific antibodies. However, further investigations concerning the distribution of HEV in different age groups of German domestic pigs, phylogenetic analyses and the viral load in the porcine liver are still pending. Liver samples of all age groups from herds in a pig-dense region in north-western Germany were investigated for the presence and quantity of HEV RNA and subsequently genotyped. Out of 251 liver samples, 34 contained ORF2-specific RNA, whereas 19 samples were positive using ORF1-specific primers, resulting in an overall detection rate of 13.5% and 7.6%, respectively. Especially nursery pigs and growers were tested positive for viral RNA. Furthermore, determination of the HEV copy numbers revealed high replication levels. Up to 10(9) genome copies per g of liver tissue could be detected suggesting a likely high degree of viral spread to the environment. In the HEV-positive liver samples we found no hints for pathohistological changes reflecting the HEV status.The HEV sequences showed marked diversity but could be assigned to HEV genotype 3 without exception. However, by comparing two different genomic fragments, we found indications for infections with two different HEV variants in domestic pigs. Apart from this, the current study confirms the outcome of our recent serological HEV survey and for the first time gives direct proof of HEV infections in the German domestic pig population.

Effects of colostrum versus formula feeding on hepatic glucocorticoid and α1- and β2-adrenergic receptors in neonatal calves and their effect on glucose and lipid metabolism

Neonatal energy metabolism in calves has to adapt to extrauterine life and depends on colostrum feeding. The adrenergic and glucocorticoid systems are involved in postnatal maturation of pathways related to energy metabolism and calves show elevated plasma concentrations of cortisol and catecholamines during perinatal life. We tested the hypothesis that hepatic glucocorticoid receptors (GR) and α₁- and β₂-adrenergic receptors (AR) in neonatal calves are involved in adaptation of postnatal energy metabolism and that respective binding capacities depend on colostrum feeding. Calves were fed colostrum (CF; n=7) or a milk-based formula (FF; n=7) with similar nutrient content up to d 4 of life. Blood samples were taken daily before feeding and 2h after feeding on d 4 of life to measure metabolites and hormones related to energy metabolism in blood plasma. Liver tissue was obtained 2 h after feeding on d 4 to measure hepatic fat content and binding capacity of AR and GR. Maximal binding capacity and binding affinity were calculated by saturation binding assays using [(3)H]-prazosin and [(3)H]-CGP-12177 for determination of α₁- and β₂-AR and [(3)H]-dexamethasone for determination of GR in liver. Additional liver samples were taken to measure mRNA abundance of AR and GR, and of key enzymes related to hepatic glucose and lipid metabolism. Plasma concentrations of albumin, triacylglycerides, insulin-like growth factor I, leptin, and thyroid hormones changed until d 4 and all these variables except leptin and thyroid hormones responded to feed intake on d 4. Diet effects were determined for albumin, insulin-like growth factor I, leptin, and thyroid hormones. Binding capacity for GR was greater and for α₁-AR tended to be greater in CF than in FF calves. Binding affinities were in the same range for each receptor type. Gene expression of α₁-AR (ADRA1) tended to be lower in CF than FF calves. Binding capacity of GR was related to parameters of glucose and lipid metabolism, whereas β₂-AR binding capacity was negatively associated with glucose metabolism. In conclusion, our results indicate a dependence of GR and α₁-AR on milk feeding immediately after birth and point to an involvement of hepatic GR and AR in postnatal adaptation of glucose and lipid metabolism in calves.

Fatal hyperammonemia and carbamoyl phosphate synthetase 1 (CPS1) deficiency following high-dose chemotherapy and autologous hematopoietic stem cell transplantation

Fatal hyperammonemia secondary to chemotherapy for hematological malignancies or following bone marrow transplantation has been described in few patients so far. In these, the pathogenesis of hyperammonemia remained unclear and was suggested to be multifactorial. We observed severe hyperammonemia (maximum 475 μmol/L) in a 2-year-old male patient, who underwent high-dose chemotherapy with carboplatin, etoposide and melphalan, and autologous hematopoietic stem cell transplantation for a neuroblastoma stage IV. Despite intensive care treatment, hyperammonemia persisted and the patient died due to cerebral edema. The biochemical profile with elevations of ammonia and glutamine (maximum 1757 μmol/L) suggested urea cycle dysfunction. In liver homogenates, enzymatic activity and protein expression of the urea cycle enzyme carbamoyl phosphate synthetase 1 (CPS1) were virtually absent. However, no mutation was found in CPS1 cDNA from liver and CPS1 mRNA expression was only slightly decreased. We therefore hypothesized that the acute onset of hyperammonemia was due to an acquired, chemotherapy-induced (posttranscriptional) CPS1 deficiency. This was further supported by in vitro experiments in HepG2 cells treated with carboplatin and etoposide showing a dose-dependent decrease in CPS1 protein expression. Due to severe hyperlactatemia, we analysed oxidative phosphorylation complexes in liver tissue and found reduced activities of complexes I and V, which suggested a more general mitochondrial dysfunction. This study adds to the understanding of chemotherapy-induced hyperammonemia as drug-induced CPS1 deficiency is suggested. Moreover, we highlight the need for urgent diagnostic and therapeutic strategies addressing a possible secondary urea cycle failure in future patients with hyperammonemia during chemotherapy and stem cell transplantation.

Chimpanzee: A predictive model for the human cytochrome P450 3A subfamily

The human cytochrome P450 3A (CYP3A) subfamily is responsible for most of the metabolism of therapeutic drugs; however, an adequate in vivo model has yet to be discovered. This study begins with an investigation of a controversial topic surrounding the human CYP3As--estrogen regulation. A novel approach to this topic was used by defining expression in the estrogen-responsive endometrium. This study shows that estrogen down-regulates CYP3A4 expression in the endometrium. On the other hand, analogous studies showed an increase in CYP3A expression as age increases in liver tissue. Following the discussion of estrogen regulation, is an investigation of the cross-species relationships among all of the CYP3As was completed. The study compares isoforms from piscines, avians, rodents, canines, ovines, bovines, and primates. Using the traditional phylogenetic analyses and employing a novel approach using exon and intron lengths, the results show that only another primate could be the best animal model for analysis of the regulation of the expression of the human CYP3As. This analysis also demonstrated that the chimpanzee seems to be the best available human model. Moreover, the study showed the presence and similarities of one additional isoform in the chimpanzee genome that is absent in humans. Based on these results, initial characterization of the chimpanzee CYP3A subfamily was begun. While the human genome contains four isoforms--CYP3A4, CYP3A5, CYP3A7, and CYP3A43--the chimpanzee genome has five, the four previously mentioned and CYP3A67. Both species express CYP3A4, CYP3A5, and CYP3A43, but humans express CYP3A7 while chimpanzees express CYP3A67. In humans, CYP3A4 is expressed at higher levels than the other isoforms, but some chimpanzee individuals express CYP3A67 at higher levels than CYP3A4. Such a difference is expected to alter significantly the total CYP3A metabolism. On the other hand, any study considering individual isoforms would still constitute a valid method of study for the human CYP3A4, CYP3A5, and CYP3A43 isoforms. ^

An integrated molecular biology and computational approach to CYP1A1 expression in human brain

The cytochromes P450 comprise a superfamily of heme-containing mono-oxygenases. These enzymes metabolize numerous xenobiotics, but also play a role in metabolism of endogenous compounds. The P450 1A1 enzyme generally metabolizes polycyclic aromatic hydrocarbons, and its expression can be induced by aryl hydrocarbon receptor (AhR) activation. CYP1A1 is an exception to the generality that the majority of CYPs demonstrate highest expression in liver; CYP1Al is present in numerous extrahepatic tissues, including brain. This P450 has been observed in two forms, wildtype (WT) and brain variant (BV), arising from alternatively spliced mRNA transcripts. The CYP1A1 BV mRNA presented an exon deletion and was detected in human brain but not liver tissue of the same individuals. ^ Quantitative PCR analyses were performed to determine CYP1A1 WT and BV transcript expression levels in normal, bipolar disorder or schizophrenic groups. In our samples, we show that CYP1A1 BV mRNA, when present, is found alongside the full-length form. Furthermore, we demonstrate a significant decrease in expression of CYP1A1 in patients with bipolar disorder or schizophrenia. The expression level was not influenced by post-mortem interval, tissue pH, age, tobacco use, or lifetime antipsychotic medication load. ^ There is no indication of increased brain CYP1A1 expression in normal smokers versus non-smokers in these samples. We observed slightly increased CYP1A1 expression only in bipolar and schizophrenic smokers versus non-smokers. This may be indicative of complex interactions between neuronal chemical environments and AhR-mediated CYP1A1 induction in brain. ^ Structural homology modeling demonstrated that P450 1A1 BV has several alterations to positions/orientations of substrate recognition site residues compared to the WT isoform. Automated substrate docking was employed to investigate the potential binding of neurological signaling molecules and neurotropic drugs, as well as to differentiate specificities of the two P450 1A1 isoforms. We consistently observed that the BV isoform produced energetically favorable substrate dockings in orientations not observed for the same substrate in the WT isoform. These results demonstrated that structural differences, namely an expanded substrate access channel and active site, confer greater capacity for unique compound docking positions suggesting a metabolic profile distinct from the wildtype form for these test compounds. ^

Intercellular adhesion and membrane polarity in rat hepatocytes: Localization of cell -CAM 105 and other domain-specific membrane proteins {\it in situ\/} and {\it in vitro\/} by electron microscopy

Cell-CAM 105 has been identified as a cell adhesion molecule (CAM) based on the ability of monospecific and monovalent anti-cell-CAM 105 antibodies to inhibit the reaggregation of rat hepatocytes. Although one would expect to find CAMs concentrated in the lateral membrane domain where adhesive interactions predominate, immunofluorescence analysis of rat liver frozen sections revealed that cell-CAM 105 was present exclusively in the bile canalicular (BC) domain of the hepatocyte. To more precisely define the in situ localization of cell-CAM 105, immunoperoxidase and electron microscopy were used to analyze intact and mechanically dissociated fixed liver tissue. Results indicate that although cell-CAM 105 is apparently restricted to the BC domain in situ, it can be detected in the pericanalicular region of the lateral membranes when accessibility to lateral membranes is provided by mechanical dissociation. In contrast, when hepatocytes were labeled following incubation in vitro under conditions used during adhesion assays, cell-CAM 105 had redistributed to all areas of the plasma membrane. Immunofluorescence analysis of primary hepatocyte cultures revealed that cell-CAM 105 and two other BC proteins were localized in discrete domains reminscent of BC while cell-CAM 105 was also present in regions of intercellular contact. These results indicate that the distribution of cell-CAM 105 under the experimental conditions used for cell adhesion assays differs from that in situ and raises the possibility that its adhesive function may be modulated by its cell surface distribution. The implications of these and other findings are discussed with regard to a model for BC formation.^ Analysis of molecular events involved in BC formation would be accelerated if an in vitro model system were available. Although BC formation in culture has previously been observed, repolarization of cell-CAM 105 and two other domain-specific membrane proteins was incomplete. Since DMSO had been used by Isom et al. to maintain liver-specific gene expression in vitro, the effect of this differentiation system on the polarity of these membrane proteins was examined. Based on findings presented here, DMSO apparently prolongs the expression and facilitates polarization of hepatocyte membrane proteins in vitro. ^

Governing step of metastasis visualized in vitro

Metastasis is the ultimate life-threatening stage of cancer. The lack of accurate model systems thwarted studies of the metastatic cell’s basic biology. To follow continuously the succeeding stages of metastatic colony growth, we heritably labeled cells from the human lung adenocarcinoma cell line ANIP 973 with green fluorescent protein (GFP) by transfection with GFP cDNA. Labeled cells were then injected intravenously into nude mice, where, by 7 days, they formed brilliantly fluorescing metastatic colonies on mouse lung [Chishima, T., Miyagi, Y., Wang, X., Yang, M., Tan, Y., Shimada, H., Moossa, A. R. & Hoffman, R. M. (1997) Clin. Exp. Metastasis 15, 547–552]. The seeded lung tissue was then excised and incubated in the three-dimensional sponge-gel-matrix-supported histoculture that maintained the critical features of progressive in vivo tumor colonization while allowing continuous access for measurement and manipulation. Tumor progression was continuously visualized by GFP fluorescence in the same individual cultures over a 52-day period, during which the tumors spread throughout the lung. Histoculture tumor colonization was selective for lung cancer cells to grow on lung tissue, because no growth occurred on histocultured mouse liver tissue, which was also observed in vivo. The ability to support selective organ colonization in histoculture and visualize tumor progression by GFP fluorescence allows the in vitro study of the governing processes of metastasis [Kuo, T.-H., Kubota, T., Watanbe, M., Furukawa, T., Teramoto, T., Ishibiki, K., Kitajima, M., Moossa, A. R., Penman, S. & Hoffman, R. M. (1995) Proc. Natl. Acad. Sci. USA 92, 12085–12089]. The results presented here provide significant, new opportunities to understand and to develop treatments that prevent and possibly reverse metastasis.

Gain of imprinting at chromosome 11p15: A pathogenetic mechanism identified in human hepatocarcinomas

Genomic imprinting is a reversible condition that causes parental-specific silencing of maternally or paternally inherited genes. Analysis of DNA and RNA from 52 human hepatocarcinoma samples revealed abnormal imprinting of genes located at chromosome 11p15 in 51% of 37 informative samples. The most frequently detected abnormality was gain of imprinting, which led to loss of expression of genes present on the maternal chromosome. As compared with matched normal liver tissue, hepatocellular carcinomas showed extinction or significant reduction of expression of one of the alleles of the CDKN1C, SLC22A1L, and IGF2 genes. Loss of maternal-specific methylation at the KvDMR1 locus in hepatocarcinoma correlated with abnormal expression of CDKN1C and IGF2, suggesting a function for KvDMR1 as a long-range imprinting center active in adult tissues. These results point to the role of epigenetic mechanisms leading to loss of expression of imprinted genes at chromosome region 11p15 in human tumors.

Identification of two flavivirus-like genomes in the GB hepatitis agent.

A subtractive PCR methodology known as representational difference analysis was used to clone specific nucleotide sequences present in the infectious plasma from a tamarin infected with the GB hepatitis agent. Eleven unique clones were identified, seven of which were examined extensively. All seven clones appeared to be derived from sequences exogenous to the genomes of humans, tamarins, Saccharomyces cerevisiae, and Escherichia coli. In addition, sequences from these clones were not detected in plasma or liver tissue of tamarins prior to their inoculation with the GB agent. These sequences were detected by reverse transcription-PCR in acute-phase plasma of tamarins inoculated with the GB agent. Probes derived from two of the seven clones detected an RNA species of > or = 8.3 kb in the liver of a GB-agent-infected tamarin by Northern blot hybridization. Sequence analysis indicated that five of the seven clones encode polypeptides that possess limited amino acid identity with the nonstructural proteins of hepatitis C virus. Extension of the sequences found in the seven clones revealed that plasma from an infected tamarin contained two RNA molecules > 9 kb long. Limited sequence identity with various isolates of hepatitis C virus and the relative positions of putative RNA helicases and RNA-dependent RNA polymerases in the predicted protein products of these molecules suggested that the GB agent contains two unique flavivirus-like genomes.

Avaliação físico-química de óleos da amêndoa de pequi (Caryocar brasiliense Camb.) obtidos artesanalmente e por prensagem a frio e sua influência sobre marcadores bioquímicos, oxidativos e inflamatórios de ratos submetidos à toxicidade aguda por tetracloreto de carbono

O óleo da amêndoa de pequi (Caryocar brasiliense Camb.) é considerado um coproduto do fruto, consumido na região do Cerrado, bioma brasileiro. Ele é fundamental para agregar valor e ampliar a utilização deste fruto regional a outros setores produtivos comerciais. Relatos na literatura apontam que o óleo possui capacidade antioxidante e efeitos benéficos sobre doenças inflamatórias, que estão associados à presença de ácidos graxos insaturados e fitoquímicos em sua composição química. Por outro lado, o tetracloreto de carbono (CCl4) é uma potente hepatotoxina, capaz de gerar radicais livres que levam ao estresse oxidativo e à inflamação. Nesse contexto, o objetivo do presente estudo foi caracterizar os óleos da amêndoa de pequi obtidos artesanalmente e por prensagem a frio e verificar o efeito de seus constituintes graxos e bioativos sobre parâmetros oxidativos e inflamatórios de ratos submetidos à toxicidade aguda induzida por tetracloreto de carbono. Inicialmente, foram investigados os parâmetros de qualidade dos óleos, bem como o perfil de ácidos graxos, teores de compostos bioativos, capacidade antioxidante e estabilidade termo-oxidativa. Os óleos da amêndoa de pequi apresentaram boa qualidade e resistência termo-oxidativa e mostraram-se ricos em ácido graxo oleico, além de possuírem compostos com propriedades antioxidantes, como fenólicos, carotenoides, tocoferóis e fitosteróis. Posteriormente, o efeito do tratamento por 22 dias com óleos da amêndoa de pequi artesanal ou prensado a frio (3 mL/kg) sobre a toxicidade aguda induzida pelo CCl4 em ratos \"Wistar\" machos foi avaliado. Para tal, foram determinados marcadores bioquímicos séricos, perfil lipídico, peroxidação lipídica, marcadores do sistema de defesa antioxidante e detoxificante, além de parâmetros inflamatórios do tecido hepático. De maneira geral, verificou-se que os óleos da amêndoa de pequi não minimizaram as alterações hepáticas induzidas pelo CCl4, evidenciadas pelas enzimas marcadoras do dano hepático e por parâmetros inflamatórios, no entanto os animais tratados com o óleo prensado a frio aumentaram sua capacidade antioxidante.

Function and Regulation of Cytochrome P450 4V2 and the Implications in Bietti’s Crystalline Dystrophy

Thesis (Ph.D.)--University of Washington, 2016-06

Steatosis in chronic hepatitis C: Association with increased messenger RNA expression of collagen I, tumor necrosis factor-α and cytochrome P450 2E1

Background: Increased levels of tumor necrosis factor (TNF)-alpha and oxidative stress have been implicated as factors contributing to hepatic injury in fatty liver diseases. As steatosis is associated with an accelerated progression of fibrosis in chronic hepatitis C (HCV), we hypothesized that the messenger (m)RNA expression of genes involved with the production of reactive oxygen species, inflammation and cellular injury would be increased in liver tissue from subjects with steatosis and chronic HCV. Methods: Real-time polymerase chain reaction was performed to determine relative mRNA expression levels of collagen I, TNF-alpha, cytochrome P450 2E1 (CYP 2E1), transforming growth factor-beta1 and CD14 in liver biopsies from 38 patients with chronic HCV. The mRNA expression levels were compared between subjects with and without steatosis, fibrosis, and inflammation. Results: Multivariate analysis demonstrated that collagen I mRNA expression was increased by 199% in steatosis (P = 0.02), 85% in moderate to severe fibrosis (P = 0.02) and 157% in inflammation (P = 0.03). Livers of patients with steatosis also had an increase in TNF-alpha mRNA expression by 50% (P = 0.03) and CYP 2E1 expression by 37% (P = 0.04) compared with non-steatotic livers. Tumor necrosis factor-alpha protein was localized to Kupffer cells, bile ducts and portal inflammatory cells by immunohistochemistry. Conclusion: Increased expression of TNF-alpha may be involved in the pathogenesis of liver injury and progression of fibrosis in individuals who have steatosis in association with chronic HCV. (C) 2003 Blackwell Publishing Asia Pty Ltd.