Lymphocytosis as a predictor of poor response to treatment of hepatitis C.

BACKGROUND/AIMS: Identification of factors predicting response to therapy is critical in the management of hepatitis C. This study assessed significance of lymphocytosis as a predictor of sustained virological response (SVR). METHODS: Retrospective analysis of lymphocytosis and its correlation with virologic response was performed in 110 subjects with chronic HCV infection, who underwent interferon based therapy. Lymphocytosis was defined as ratio of lymphocytes to neutrophils (L/N) above 0.6. L/N ratios were calculated to avoid the impact of hypersplenism and constitutional leukopenia seen in African Americans (AA). RESULTS: At baseline, L/N of HCV subjects (0.86) as compared to Hepatitis B controls (0.56) was significantly higher (P < 0.01). More AA HCV subjects (81.8%) had lymphocytosis at baseline when compared to Caucasian Americans subjects with HCV (37.9%) or AA controls (39.4%). Nonresponders had a higher frequency of lymphocytosis at baseline compared to subjects that achieved SVR (61.4% vs. 36.0%, p<0.05). More HCV subjects without lymphocytosis at baseline achieved SVR (33.3%) compared to HCV subjects with lymphocytosis (15%). At week 12 of therapy, nonresponders had higher L/N (1.02 vs. 0.86) and frequency of lymphocytosis (73% vs. 48%) compared to subjects that achieved SVR (p<0.05 for both). Only 17.2% of subjects with lymphocytosis at 12 weeks achieved SVR compared to 37.5% without lymphocytosis (p < 0.05). All responders exhibited significant normalization of lymphocytosis after treatment. CONCLUSIONS: HCV induces lymphocytosis, especially in AA, and is associated with lower rate of SVR. Furthermore, lymphocytosis may serve as an inexpensive pre-treatment tool to predict poor virologic response to HCV therapy.

Role of alpha-1-antichymotrypsin deficiency in promoting cirrhosis in two siblings with heterozygous alpha-1-antitrypsin deficiency phenotype SZ.

BACKGROUND: Alpha-1-antitrypsin (A1AT) deficiency is the most common inherited metabolic disorder with the potential to cause injury in the lung and liver. Recent reports suggested that alpha-1-antichymotrypsin (A1AC) deficiency may also be a possible cause of chronic liver disease. However, it has received little attention and is rarely investigated in the clinical setting. AIMS: To assess the role of A1AC deficiency in the pathogenesis of chronic liver disease in two siblings with heterozygous A1AT phenotype Pi SZ. PATIENTS: Two adult siblings with an A1AT Pi SZ phenotype and reduced levels of A1AC consistent with heterozygosity who developed cirrhosis and underwent liver transplantation. METHODS AND RESULTS: A1AT and A1AC levels in plasma measured by electroimmunoassay were 74 mg/dl and 90 mg/dl (140-470) and 0.12 mg/ml and 0.14 mg/ml (0.173-0.46), respectively. Immunohistochemistry revealed an apparent accumulation of both A1AT and A1AC in hepatocytes. A previously reported point mutation in exon III (Pro(229) to Ala substitution) of the A1AC gene was not detected by polymerase chain reaction amplification and a single strand conformation polymorphism analysis. CONCLUSIONS: Our report represents the first case of two siblings with A1CA phenotype Pi SZ who developed cirrhosis and underwent liver transplantation. Both siblings were heterozygous for A1AT and A1AC deficiency suggesting that combined deficiency of these two major serine protease inhibitors may enhance the risk of developing liver disease.

Microarray analysis of genes differentially expressed in HepG2 cells cultured in simulated microgravity: preliminary report.

Developed at NASA, the rotary cell culture system (RCCS) allows the creation of unique microgravity environment of low shear force, high-mass transfer, and enables three-dimensional (3D) cell culture of dissimilar cell types. Recently we demonstrated that a simulated microgravity is conducive for maintaining long-term cultures of functional hepatocytes and promote 3D cell assembly. Using deoxyribonucleic acid (DNA) microarray technology, it is now possible to measure the levels of thousands of different messenger ribonucleic acids (mRNAs) in a single hybridization step. This technique is particularly powerful for comparing gene expression in the same tissue under different environmental conditions. The aim of this research was to analyze gene expression of hepatoblastoma cell line (HepG2) during early stage of 3D-cell assembly in simulated microgravity. For this, mRNA from HepG2 cultured in the RCCS was analyzed by deoxyribonucleic acid microarray. Analyses of HepG2 mRNA by using 6K glass DNA microarray revealed changes in expression of 95 genes (overexpression of 85 genes and downregulation of 10 genes). Our preliminary results indicated that simulated microgravity modifies the expression of several genes and that microarray technology may provide new understanding of the fundamental biological questions of how gravity affects the development and function of individual cells.

Induction of hepatitis B virus gene expression at low temperature.

There is a limited understanding of the cellular regulation of HBV gene expression in differentiated hepatocytes. We previously demonstrated that HBV replication inversely correlates with cell proliferation and DNA synthesis. In this report, temperature-induced modulation of cell growth was used as a novel approach to study HBV gene expression in the absence of indirect effects from drugs or serum deprivation. We observed markedly elevated levels of hepatic HBV mRNA expression from integrated and episomal HBV DNA at 32 degrees C. Additionally, hepatoblastoma cells cultured at 32 degrees C expressed increased levels of albumin mRNA and decreased levels of c-myc mRNA, which demonstrates that liver-derived cells cultured at low temperature exhibit characteristics of functional and differentiated hepatocytes. In transiently transfected HepG2 cells cultured at 32 degrees C, the HBV enhancer 1 activated the X promoter and core/pregenomic promoter by 7.3- and 28-fold, respectively. In the absence of enhancer 1, core/pregenomic promoter activity was 2.4-fold higher than the X promoter in HepG2 cells at 32 degrees C. In contrast, enhancer 1 exclusively activated the X promoter in transfected non-liver cells at 32 degrees C. Therefore, the core/pregenomic promoter exhibits strict liver-specificity at low temperature. This work supports the hypothesis that HBV replication and gene expression are optimal in non-activated hepatocytes, and provides a novel system for delineating molecular aspects of the HBV replication process.

Induction of three-dimensional assembly of human liver cells by simulated microgravity.

The establishment of long-term cultures of functional primary human liver cells (PHLC) is formidable. Developed at NASA, the Rotary Cell Culture System (RCCS) allows the creation of the unique microgravity environment of low shear force, high-mass transfer, and 3-dimensional cell culture of dissimilar cell types. The aim of our study was to establish long-term hepatocyte cultures in simulated microgravity. PHLC were harvested from human livers by collagenase perfusion and were cultured in RCCS. PHLC aggregates were readily formed and increased up to 1 cm long. The expansion of PHLC in bioreactors was further evaluated with microcarriers and biodegradable scaffolds. While microcarriers were not conducive to formation of spheroids, PHLC cultured with biodegradable scaffolds formed aggregates up to 3 cm long. Analyses of PHLC spheroids revealed tissue-like structures composed of hepatocytes, biliary epithelial cells, and/or progenitor liver cells that were arranged as bile duct-like structures along nascent vascular sprouts. Electron microscopy revealed groups of cohesive hepatocytes surrounded by complex stromal structures and reticulin fibers, bile canaliculi with multiple microvilli, and tight cellular junctions. Albumin mRNA was expressed throughout the 60-d culture. A simulated microgravity environment is conducive to maintaining long-term cultures of functional hepatocytes. This model system will assist in developing improved protocols for autologous hepatocyte transplantation, gene therapy, and liver assist devices, and facilitate studies of liver regeneration and cell-to-cell interactions that occur in vivo.

Cultures of human liver cells in simulated microgravity environment.

We used microgravity-simulated bioreactors that create the unique environment of low shear force and high-mass transfer to establish long-term cultures of primary human liver cells (HLC). To assess the feasibility of establishing HLC cultures, human liver cells obtained either from cells dissociated by collagenase perfusion or minced tissues were cultured in rotating vessels. Formation of multidimensional tissue-like spheroids (up to 1.0 cm) comprised of hepatocytes and biliary epithelial cells that arranged as bile duct-like structures along newly formed vascular sprouts were observed. Electron microscopy revealed clusters of round hepatocytes and bile canaliculi with multiple microvilli and tight junctions. Scanning EM revealed rounded hepatocytes that were organized in tight clusters surrounded by a complex mesh of extracellular matrix. Also, we observed that co-culture of hepatocytes with endothelial cells stimulate albumin mRNA expression. In summary, a simulated microgravity environment is conducive for the establishment of long-term HLC cultures and allows the dissection of the mechanism of liver regeneration and cell-to-cell interactions that resembles in vivo conditions.

Microsatellite instability in human hepatocellular carcinoma: relationship to p53 abnormalities.

AIMS/BACKGROUND: Microsatellite instability was sought in 10 human hepatocellular carcinomas (HCCs) to determine whether defective DNA mismatch repair might be implicated in the multiple genetic alterations observed in the p53 tumor suppressor gene in some of these patients' tumors. METHODS: Genomic DNA from HCCs and adjacent nontumorous livers was subjected to PCR with primers for nine microsatellites, and PCR products were resolved in a denaturing gel. Microsatellite instability was defined as the presence of band shifts or additional bands for at least two microsatellite sequences in an HCC compared to the nontumorous liver tissue from the same patient. RESULTS: Microsatellite instability was detected in four of ten HCCs. Three of these four HCCs did not have p53 exon mutations. However, one HCC had microsatellite instability as well as multiple p53 exon mutations and multiple intron alterations. Four other patients with multiple p53 intron alterations in HCC (compared to their own nontumorous liver), three of whom also had a mutation in the exons, had no microsatellite instability. CONCLUSIONS: Defective DNA mismatch repair, as indicated by microsatellite instability, might have played a role in hepatocarcinogenesis in four of the ten patients, but in general it was not associated with p53 alterations. In one of the ten patients, defective DNA mismatch repair might have been the cause of multiple mutations in both the coding and intron sequences of the p53 gene.

Effect of hepatocyte proliferation and cellular DNA synthesis on hepatitis B virus replication.

BACKGROUND & AIMS: Interrelationship between hepatitis B virus (HBV) replication and the stage of hepatocyte proliferation and differentiation may play an important role in the pathogenesis of HBV infection. The aim of this study was to assess the effect of hepatocyte proliferation and/or cell arrest on HBV replication. METHODS: Hepatoblastoma cells transfected with HBV were subjected to serum deprivation or treatment with aphidicolin or camptothecin. Cell cycle analysis was performed using flow cytometry, and cellular DNA synthesis was analysed by assessing 5-bromo-2'-deoxyuridine incorporation. Distribution of episomal HBV DNA and proliferating cell nuclear antigen in liver specimens was assessed by simultaneous in situ hybridization and immunohistochemistry. RESULTS: Serum deprivation inhibited cellular DNA synthesis and increased levels of HBV messenger RNA (mRNA). Aphidicolin treatment resulted in cell arrest in C1, with concomitant increases in levels of HBV mRNA and viral DNA. Cell entry into S phase inhibited expression of HBV mRNA. Camptothecin induced G2 cell arrest and inhibited cellular DNA synthesis with increased amounts of viral replication and levels of HBV mRNA. In vivo studies showed an inverse correlation between expression of proliferating cell nuclear antigen and presence of episomal HBV DNA in individual hepatocytes. CONCLUSIONS: HBV replication is cell cycle dependent, supporting the concept of enhanced viral replication in quiescent hepatocytes. The results may explain the mechanism of viral elimination during cell regeneration.

Induction of senescent cell-derived inhibitor of DNA synthesis gene, SDI1, in hepatoblastoma (HepG2) cells arrested in the G2-phase of the cell cycle by 9-nitrocamptothecin.

BACKGROUND: Recent studies have demonstrated that the plant-derived alkaloid camptothecin (CPT) and its derivative, 9-nitro-CPT (9NC), are cytotoxic in tumorigenic cells but cytostatic in nontumorigenic cells in vitro and in vivo. Also, CPT induces differentiation of human leukemia cells in vitro along specific lineages. In this study, we have investigated the effects of 9NC on nontumorigenic HepG2 cells derived from human hepatoblastoma. A newly discovered senescent cell-derived inhibitor (SDI1) plays a critical role in the cell cycle, so we evaluated the effect of 9NC on the expression of the SDI1 gene. EXPERIMENTAL DESIGN: The effects of 9NC on HepG2 cells were evaluated by monitoring DNA synthesis, morphologic and ultrastructural changes of cells, and perturbation in the cell cycle and by assessing the levels of p53 protein and SDI1 mRNA. RESULTS: Treatment of HepG2 cells with 9NC results in a dose-dependent inhibition of cell proliferation and DNA synthesis. Flow cytometric analysis of DNA content showed that 9NC-treated HepG2 cells are arrested in the G2-phase of the cell cycle. Light and electron microscopic examination revealed that 9NC at low concentrations induces morphologic and growth features that resemble properties highly differentiated or senescent cells, i.e., increased cell size and decreased nuclear/cytoplasmic ratio, as well as enlarged numbers of lysosomes, mitochondria, and lipid in the cytoplasm. No significant alteration in the p53 protein level was noted in 9NC-treated cells. In contrast to untreated, logarithmically grown HepG2 cells, 9NC-treated cells arrested at the G2-phase of the cell cycle and contained increased levels of SDI1 mRNA. Kinetic studies revealed gradual increases in SDI1 mRNA synthesis. CONCLUSIONS: Induction of SDI1 mRNA by 9NC represents the first documentation that the SDI1 gene can be overexpressed in the G2-phase of the cell cycle and provides a valuable cell culture system to dissect the events controlling the G2 checkpoint. In addition, this finding corroborates the hypothesis that genes up-regulated in senescent cells can also be induced in tumor-derived immortalized cells.