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Exp Mol Pathol [journal]
- Prediction of common epitopes on hemagglutinin of the influenza A virus (H1 subtype). [JOURNAL ARTICLE]
- Exp Mol Pathol 2014 Dec 19.
Influenza A virus infection is a persistent threat to public health worldwide due to hemagglutinin (HA) variation. Current vaccines against influenza A virus provide immunity to viral isolates similar to vaccine strains. Antibodies against common epitopes provide immunity to diverse influenza virus strains and protect against future pandemic influenza. Therefore, it is vital to analyze common HA antigenic epitopes of influenza virus. In this study, 14 strains of monoclonal antibodies with high sensitivity to common epitopes of influenza virus antigens identified in our previous study were selected as the tool to predict common HA epitopes. The common HA antigenic epitopes were divided into four categories by ELISA blocking experiments, and separately, into three categories according to the preliminary results of computer simulation. Comparison between the results of computer simulations and ELISA blocking experiments indicated that at least two classes of common epitopes are present in influenza virus HA. This study provides experimental data for improving the prediction of HA epitopes of influenza virus (H1 subtype) and the development of a potential universal vaccine as well as novel approach for the prediction of epitopes on other pathogenic microorganisms.
- The importance of the interaction between hepatocyte and hepatic stellate cells in fibrogenesis induced by fatty accumulation. [JOURNAL ARTICLE]
- Exp Mol Pathol 2014 Dec 19.
Non-alcoholic fatty liver disease is characterized by an initial accumulation of triglycerides that can progress to non-alcoholic steatohepatitis, which can ultimately evolve to cirrhosis and hepatocellular carcinoma. Hepatic stellate cells play a key role in liver fibrogenesis by an increased activation and an altered profile of genes involved in the turnover of extracellular matrix components. To reproduce in-vitro the functional cell connections observed in vivo it is essential to consider cell-to-cell proximity and interaction. The aim of this study was to determine the response to free fatty acids in a simultaneous co-culture model of hepatocytes and hepatic stellate cells.Simultaneous co-culture model and monoculture of each cell type (control) were exposed to FFA for 24 up to 144h. Quantification of steatosis; stellate cells activation; assessment of fibrogenic response; expression and activity of metalloproteinases as well as collagen biosynthesis were evaluated.Free fatty acids induced comparable steatosis in simultaneous co-culture and monoculture. However, the activation of the stellate cells assessed by alpha-smooth muscle actin expression is greater when cells were in close contact. Furthermore, a time-dependent increment of tissue inhibitor metalloproteinase-2 protein was observed, which was inversely correlated with protein expression and activity of matrix-metalloproteinases, suggesting enhanced collagen biosynthesis. This behavior was absent in cell monoculture.These data indicate that cell-to-cell proximity between hepatocytes and stellate cells is necessary for the initiation of the fibrotic process.
- Evaluation of the effects of preconditioning regimens on hepatic veno-occlusive disease in mice after hematopoietic stem cell transplantation. [JOURNAL ARTICLE]
- Exp Mol Pathol 2014 Dec 20.
Pre-conditioning regimens before hematopoietic stem cell transplantation (HSCT), such as total body irradiation (TBI) or busulfan/cyclophosphamide (BU/CY), are associated with hepatic veno-occlusive disease (HVOD). However, the mechanism of these regimens on hepatic veno-occlusive disease remains unclear. The aim of this study is to evaluate the effect of TBI or BU/CY on HVOD in mice after HSCT. Mice received TBI or BU/CY followed by HSCT. Analysis of liver pathology and function, and platelet aggregation were performed. Both these regimens caused damage to liver sinusoid endothelial cells, leading to loss of normal structural integrity of liver sinusoid, abnormal liver function, fibrin deposition, inflammatory cells infiltration and platelet aggregation. No differences of liver function in these regimens were observed. Increased hepatic lipid droplets, mitochondrial swelling and higher incidence of HVOD were observed in BU/CY. In conclusion, both TBI and BU/CY caused damage to liver sinusoid endothelial cells and occurrence of HVOD with higher incidence for BU/CY. Meanwhile, inflammation and platelet activation was also observed, suggesting targeting them maybe beneficial in the prophylaxis of HVOD.
- Vacuolar protein sorting 4B regulates apoptosis of intestinal epithelial cells via p38 MAPK in Crohn's disease. [JOURNAL ARTICLE]
- Exp Mol Pathol 2014 Dec 20.
Vacuolar protein sorting 4B (VPS4B), a member of ATPase family proteins, reportedly possesses multiple biological functions, such as regulating the development of breast cancer and non-small-cell lung cancer, participating in Parkinson's disease, and modulating neuronal apoptosis after cerebral ischemia. However, its expression and potential functions in Crohn's disease (CD) has not been understood. In this study, we reported for the first time that VPS4B was over-expressed in intestinal epithelial cell (IECs) of patients with CD. In TNBS-induced mouse colitis models, we observed the up-regulation of VPS4B was accompanied with the elevated levels of IEC apoptotic markers (active caspase-3 and cleaved PARP) and phosphorylated p38 in colitis IECs. Co-localization of VPS4B and active caspase-3 in IECs of the TNBS group further indicated the possible involvement of VPS4B in IEC apoptosis. Employing the TNF-α-treated HT29 cells as an in vitro IEC apoptosis model, we confirmed the positive correlation of VPS4B with caspase-dependent cellular apoptosis. Knocking VPS4B down by siRNA significantly alleviated TNF-α-induced p38 phosphorylation and cellular apoptosis in HT29 cells. Taken together, our findings suggested that VPS4B may facilitate the IEC apoptosis in CD via p38 MAPK signaling pathway.
- Proinflammatory cytokines modulate the chemokine CCL2 (MCP-1) in human annulus cells in vitro: CCL2 expression and production. [JOURNAL ARTICLE]
- Exp Mol Pathol 2014 Dec 16.
Chemokines are important secondary inflammatory mediators released in response to stimuli which act as second-order cytokines with specialized functions in inflammation. The role of many of these specialized mediators is as yet poorly understood in the human intervertebral disk. Here we investigated CCL2 (chemokine (C-C motif) ligand 2, also known as monocyte chemotactic protein-1 (MCP-1)) in a study of its immunolocalization in disk tissue, and then hypothesized that exposure of cultured human annulus cells to proinflammatory cytokines might alter CCL2 gene expression and CCL2 production. CLL2 was localized to many disk cells in both herniated and non-herniated tissue specimens. Molecular analyses showed that cells exposed to IL-1β showed a 5.5 fold upregulation in CCL2 gene expression vs. controls, p=0.017. Cells exposed to TNF-α showed a 7.7 fold upregulation vs. controls, p=0.005. Cultured cells (grades II-V) showed increased MCP-1 production in IL1-β-treated cells vs. controls (p=0.016), with no significant difference in production in TNF-α-treated cells. Local production of CCL2 in vivo and vitro suggests that annulus cells may be primary effector cells (as well as target cells), with the ability to mediate physiological immune-related processes during disk degeneration by both autocrine and paracrine signaling.
- Characterization of the 19q12 amplification including CCNE1 and URI in different epithelial ovarian cancer subtypes. [JOURNAL ARTICLE]
- Exp Mol Pathol 2014 Dec 16.
CCNE1 is frequently amplified in high grade serous ovarian cancer and may serve as a target for ovarian cancer treatment. URI is closely related to CCNE1 at the 19q12 amplicon and may also contribute to the oncogenic effect. Our objective was to investigate the relevance of CCNE1 and URI gene amplification and protein expression in different histological subtypes of epithelial ovarian cancer (EOC).A novel dual-color 19q12 in situ hybridization (ISH), covering CCNE1 and URI, and chromosome 19 as a surrogate using Ventana BenchMark XT platform was developed and applied to 148 EOC. URI and CCNE1 amplification was separately assessed by fluorescence in situ hybridization (FISH). Immunohistochemistry using a Cyclin E1 and a novel URI monoclonal antibody was performed.Amplification of 19q12 was found in 36.6%, CCNE1 in 21.7%, URI in 9.9%, and both genes simultaneously in 9% of EOC cases. High Cyclin E1 and URI protein expression was observed in 52.2% and 26.1%, respectively. Amplification of 19q12 occurred in all EOC subtypes and was associated with amplification and expression of CCNE1/Cyclin E1, URI, TP53 mutation, and advanced stage.The novel 19q12 ISH probe reliably detects both CCNE1 and URI amplification as confirmed by FISH. The combination of 19q12 amplification with Cyclin E1 and URI protein expression may help to select patients more likely to benefit from CDK2 targeted therapies.
- Levels of metacaspase1 and chaperones related to protein quality control in alcoholic and nonalcoholic steatohepatitis. [JOURNAL ARTICLE]
- Exp Mol Pathol 2014 Dec 16.
Efficient management of misfolded or aggregated proteins in ASH and NASH is crucial for continued hepatic viability. Cellular protein quality control systems play an important role in the pathogenesis and progression of ASH and NASH. In a recent study, elevated Mca1 expression counteracted aggregation and accumulation of misfolded proteins and extended the life span of the yeast Saccharomyces cerevisiae (Hill et al, 2014). Mca1 may also associate with Ssa1 and Hsp104 in disaggregation and fragmentation of aggregated proteins and their subsequent degradation through the ER-associated degradation (ERAD) pathway. If degradation is not available, protection of the cellular environment from a misfolded protein is accomplished by its sequestration into two distinct inclusion bodies (Kaganovich et al., 2008) called the JUNQ (JUxta Nuclear Quality control compartment) and the IPOD (Insoluble Protein Deposit). Mca1, Hsp104, Hsp40, Ydj1, Ssa1, VCP/p97, and p62 all play important roles in protein quality control systems. This study aims to measure the expression of Mca1 and related chaperones involved in protein quality control in alcoholic steatohepatitis (ASH), and nonalcoholic steatohepatitis (NASH) compared with normal control livers. Mca1, Hsp104, Hsp40, Ydj1, Ssa1, VCP/p97, and p62 expressions were measured in three to six formalin-fixed paraffin embedded ASH and NASH liver biopsies and control normal liver specimens by immunofluorescence staining and quantified by immunofluorescence intensity. Mca1, Hsp104, Ydj1 and p62 were significantly up regulated compared to control (p<0.05) in ASH specimens. Ssa1, Hsp40 and VCP/p97 levels did not have significant differences with the control specimens. Although not significantly elevated compared to normal control, Hsp40 and VCP/p97 were significantly elevated in ASH compared to NASH (p<0.05). In NASH, the only significant difference was the increased expression of Hsp104 compared to control (p<0.05). The up regulation of Mca1, Hsp104, Ydj1 and p62 in ASH may be elicited as a response to the chronic exposure of the hepatocytes to the toxicity of alcohol. Recruitment of Mca1, Hsp104, Ydj1 and p62 may indicate that autophagy, ERAD, JUNQ, and IPOD systems are active in ASH. Whereas in NASH, only Hsp104 is significantly elevated compared to control. This may indicate that in NASH, IPOD may be the only active protein quality control system.
- Pancreatic cancer cells express CD44 variant 9 and multidrug resistance protein 1 during mitosis. [JOURNAL ARTICLE]
- Exp Mol Pathol 2014 Dec 3; 98(1):41-46.
Pancreatic cancer is one of the most lethal cancers with high metastatic potential and strong chemoresistance. Its intractable natures are attributed to high robustness in tumor cells for their survival. We demonstrate here that pancreatic cancer cells (PCCs) with an epithelial phenotype upregulate cell surface expression of CD44 variant 9 (CD44v9), an important cancer stem cell marker, during the mitotic phases of the cell cycle. Of five human CD44(+) PCC lines examined, three cell lines, PCI-24, PCI-43 and PCI-55, expressed E-cadherin and CD44 variants, suggesting that they have an epithelial phenotype. By contrast, PANC-1 and MIA PaCa-2 cells expressed vimentin and ZEB1, suggesting that they have a mesenchymal phenotype. PCCs with an epithelial phenotype upregulated cell surface expression of CD44v9 in prophase, metaphase, anaphase and telophase and downregulated CD44v9 expression in late-telophase, cytokinesis and interphase. Sorted CD44v9-negative PCI-55 cells resumed CD44v9 expression when they re-entered the mitotic stage. Interestingly, CD44v9(bright) mitotic cells expressed multidrug resistance protein 1 (MDR1) intracellularly. Upregulated expression of CD44v9 and MDR1 might contribute to the intractable nature of PCCs with high proliferative activity.
- Prolonged survival after neoadjuvant chemotherapy related with specific molecular alterations in the patients with nonsmall-cell lung carcinoma. [JOURNAL ARTICLE]
- Exp Mol Pathol 2014 Nov 20; 98(1):27-32.
Lung cancer is the most common cause of neoplasia-related death worldwide. Accounting for approximately 80% of all lung carcinomas, the non-small cell lung carcinoma (NSCLC) is the most common clinical form with its two predominant histological types, adenocarcinoma (ADC) and squamous cell carcinoma (SCC). Although surgical resection is the most favorable treatment for patients with NSCLC, relapse is still high, so neoadjuvant chemotherapy (NAC) is an accepted treatment modality. In this study we examined whether some of the key molecules associated with the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR signaling pathways could have predictive and prognostic value for the NAC application. To that end we examined the expression status of PTEN, pAKT, pERK and loss of heterozygosity (LOH) of PTEN in two groups of NSCLC patients, those who received and those who did not receive NAC. LOH PTEN and low pERK expression is shown to be correlated with the longest survival of patients with SCC and ADC, respectively, who received NAC. These results point that the application of NAC is beneficial in the NSCLC patients with specific molecular alterations which could further help to improve constant search for the druggable molecular targets used in personalized therapy.
- Pyrazine, 2-ethylpyridine, and 3-ethylpyridine are cigarette smoke components that alter the growth of normal and malignant human lung cells, and play a role in multidrug resistance development. [JOURNAL ARTICLE]
- Exp Mol Pathol 2014 Nov 18; 98(1):18-26.
Lung cancer is one of the few human diseases for which the primary etiological agent, cigarette smoke (CS), has been described; however, the precise role of individual cigarette smoke toxicant in tumor development and progression remains to be elusive. The purpose of this study was to assess in vitro the effects of previously identified cigarette smoke components, pyrazine, 2-ethylpyridine, and 3-ethylpyridine, on non-tumorigenic (MRC5) and adenocarcinomic (A549) human lung cell lines. Our data showed that the administration of three cigarette smoke components in combination perturbed the proliferation of both normal and adenocarcinomic cells. Study of malignant cells revealed that CS components were cytotoxic at high concentration (10(-6)M) and stimulatory in a dose-dependent manner at lower concentrations (10(-8)M to 10(-10)M). This adverse effect was enhanced when adenocarcinomic cells were maintained in hypoxia resembling intratumoral environment. Furthermore, exposure to pyrazine, 2-ethylpyridine, and 3-ethylpyridine induced oxidative stress in both normal and malignant cells. Finally, assessment of P-gp activity revealed that multidrug resistance was induced in CS component exposed adenocarcinomic lung cells and the induction was augmented in hypoxia. Taken together, pyrazine, 2-ethylpyridine, and 3-ethylpyridine adversely altered both normal and diseased lung cells in vitro and data collected from this study may help lung cancer patients to understand the importance of quitting smoking during lung cancer treatment.