Danyun Zhao

The emerging role of T cell immunoglobulin mucin-1 in the mechanism of liver ischemia and reperfusion injury in the mouse.

The T cell immunoglobulin and mucin domain‐containing molecules (TIM) protein family, which is expressed by T cells, plays a crucial role in regulating host adaptive immunity and tolerance. However, its role in local inflammation, such as innate immunity‐dominated organ ischemia–reperfusion injury (IRI), remains unknown. Liver IRI occurs frequently after major hepatic resection or liver transplantation. Using an antagonistic anti–TIM‐1 antibody (Ab), we studied the role of TIM‐1 signaling in the model of partial warm liver ischemia followed by reperfusion. Anti–TIM‐1 Ab monotherapy ameliorated the hepatocellular damage and improved liver function due to IR, as compared with controls. Histological examination has revealed that anti–TIM‐1 Ab treatment decreased local neutrophil infiltration, inhibited sequestration of T lymphocytes, macrophages, TIM‐1 ligand–expressing TIM‐4+ cells, and reduced liver cell apoptosis. Intrahepatic neutrophil activity and induction of proinflammatory cytokines/chemokines were also reduced in the treatment group. In parallel in vitro studies, anti–TIM‐1 Ab suppressed interferon‐γ (IFN‐γ) production in concanavalin A (conA)–stimulated spleen T cells, and diminished tumor necrosis factor α (TNF‐α)/interleukin (IL)‐6 expression in a macrophage/spleen T cell coculture system. This is the first study to provide evidence for the novel role of TIM‐1 signaling in the mechanism of liver IRI. TIM‐1 regulates not only T for the role of cell activation but may also affect macrophage function in the local inflammation response. These results provide compelling data for further investigation of TIM‐1 pathway in the mechanism of IRI, to improve liver function, expand the organ donor pool, and improve the overall success of liver transplantation. (HEPATOLOGY 2010.)

The Inhibition of Neutrophil Elastase Ameliorates Mouse Liver Damage Due to Ischemia and Reperfusion

Neutrophils are considered crucial effector cells in the pathophysiology of organ ischemia/reperfusion injury (IRI). Although neutrophil elastase (NE) accounts for a substantial portion of the neutrophil activity, the function of NE in liver IRI remains unclear. This study focuses on the role of NE in the mechanism of liver IRI. Partial warm ischemia was produced in the left and middle hepatic lobes of C57BL/6 mice for 90 minutes, and this was followed by 6 to 24 hours of reperfusion. Mice were treated with neutrophil elastase inhibitor (NEI; 2 mg/kg per os) at 60 minutes prior to the ischemia insult. NEI treatment significantly reduced serum alanine aminotransferase levels in comparison with controls. Histological examination of liver sections revealed that unlike in controls, NEI treatment ameliorated hepatocellular damage and decreased local neutrophil infiltration, as assessed by myeloperoxidase assay, naphthol AS‐D chloroacetate esterase stains, and immunohistochemistry (anti–Ly‐6G). The expression of pro‐inflammatory cytokines (tumor necrosis factor alpha and interleukin 6) and chemokines [chemokine (C‐X‐C motif) ligand 1 (CXCL‐1), CXCL‐2, and CXCL‐10] was significantly reduced in the NEI treatment group, along with diminished apoptosis, according to terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labeling staining and caspase‐3 activity. In addition, toll‐like receptor 4 (TLR4) expression was diminished in NEI‐pretreated livers, and this implies a putative role of NE in the TLR4 signal transduction pathway. Thus, targeting NE represents a useful approach for preventing liver IRI and hence expanding the organ donor pool and improving the overall success of liver transplantation. Liver Transpl 15:939–947, 2009.

The Protective Function of Neutrophil Elastase Inhibitor in Liver Ischemia/Reperfusion Injury

Background. A neutrophil elastase (NE) inhibitor, Sivelestat, has been approved for the treatment of acute lung injury associated with systemic inflammation in humans. Some reports have also shown its protective effects in liver inflammatory states. We have recently documented the importance of NE in the pathophysiology of liver ischemia/reperfusion injury, a local Ag-independent inflammation response. This study was designed to explore putative cytoprotective functions of clinically available Sivelestat in liver ischemia/reperfusion injury. Methods. Partial warm ischemia was produced in the left and middle hepatic lobes of C57BL/6 mice for 90 min, followed by 6 or 24 hr of reperfusion. The mice were given Sivelestat (100 mg/kg, subcutaneous) at 10 min before ischemia, 10 min before reperfusion, and at 1 and 3 hr of reperfusion thereafter. Results. Sivelestat treatment significantly reduced serum alanine aminotransferase levels and NE activity, when compared with controls. Histological liver examination has revealed that unlike in controls, Sivelestat ameliorated the hepatocellular damage and decreased local neutrophil activity and infiltration. The expression of proinflammatory cytokines (tumor necrosis factor-&agr; and interleukin-6), chemokines (CXCL-1, CXCL-2, and CXCL-10), and toll-like receptor 4 was significantly reduced in the treatment group, along with diminished apoptosis through caspase-3 pathway. Moreover, in vitro studies confirmed downregulation of proinflammatory cytokine and chemokine programs in mouse macrophage cell cultures, along with depression of innate toll-like receptor 4 signaling. Conclusion. Sivelestat-mediated NE inhibition may represent an effective therapeutic option in liver transplantation and other inflammation disease states.

Molecular subtype and response to dasatinib, an Src/Abl small molecule kinase inhibitor, in hepatocellular carcinoma cell lines in vitro

Hepatocellular carcinoma (HCC) is the fifth most common malignancy and is the third leading cause of cancer death worldwide. Recently, the multitargeted kinase inhibitor sorafenib was shown to be the first systemic agent to improve survival in advanced HCC. Unlike other malignancies such as breast cancer, in which molecular subtypes have been clearly defined (i.e., luminal, HER2 amplified, basal, etc.) and tied to effective molecular therapeutics (hormone blockade and trastuzumab, respectively), in HCC this translational link does not exist. Molecular profiling studies of human HCC have identified unique molecular subtypes of the disease. We hypothesized that a panel of human HCC cell lines would maintain molecular characteristics of the clinical disease and could then be used as a model for novel therapeutics. Twenty human HCC cell lines were collected and RNA was analyzed using the Agilent microarray platform. Profiles from the cell lines in vitro recapitulate previously described subgroups from clinical material. Next, we evaluated whether molecular subgroup would have predictive value for response to the Src/Abl inhibitor dasatinib. The results demonstrate that sensitivity to dasatinib was associated with a progenitor subtype. Dasatinib was effective at inducing cell cycle arrest and apoptosis in “progenitor‐like” cell lines but not in resistant lines. Conclusion: These findings suggest that cell line models maintain the molecular background of HCC and that subtype may be important for selecting patients for response to novel therapies. In addition, it highlights a potential role for Src family signaling in this progenitor subtype of HCC. (HEPATOLOGY 2013)

Allochimeric class I MHC molecules prevent chronic rejection and attenuate alloantibody responses

BACKGROUND We have shown that treatment with molecularly engineered, allochimeric [alpha1 hl/u]-RT1.Aa class I MHC antigens bearing donor-type Wistar-Furth (WF, RT1.Au) amino acid substitutions for host-type ACI (RTI.Aa) sequences in the alpha1-helical region induces donor-specific tolerance to cardiac allografts in rat recipients. This study examined the effect of allochimeric molecules on the development of chronic rejection. METHODS Allochimeric [alpha1 hl/u]-RT1.Aa class I MHC antigenic extracts (1 mg) were administered via the portal vein into ACI recipients of WF hearts on the day of transplantation in conjunction with subtherapeutic oral cyclosporine (CsA, 10 mg/kg/day, days 0-2). Control groups included recipients of syngeneic grafts and ACI recipients of WF heart allografts treated with high-dose CsA (10 mg/kg/day, days 0-6). RESULTS WF hearts in ACI rats receiving 7 days of CsA exhibited myocardial fibrosis, perivascular inflammation, and intimal hyperplasia at day 80. At day 120, these grafts displayed severe chronic rejection with global architectural disorganization, ventricular fibrosis, intimal hyperplasia, and progressive luminal narrowing. In contrast, WF hearts in rats treated with [alpha1 hl/u]-RT1.Aa molecules revealed only mild perivascular fibrosis, minimal intimal thickening, and preserved myocardial architecture. Alloantibody analysis demonstrated no IgM alloantibodies in all groups. An attenuated, but detectable, anti-WF IgG response was present in recipients receiving allochimeric molecules, with IgG1 and IgG2a subclasses predominating. Immunohistochemical analysis of allografts demonstrated minimal T cell infiltration and IgG binding to vascular endothelium. CONCLUSION Treatment with allochimeric molecules prevents the development of chronic rejection. Such effect may be in part caused by deviation of host alloantibody responses.

Abstract 3858: Gains in FGF19 are predictive of response to the fibroblast growth factor receptor (FGFR) small molecule tyrosine kinase inhibitor BGJ 398 in vitro

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Hepatocellular carcinoma (HCC) is a leading cause of cancer death world-wide. Sorafenib, a multi-targeted small molecule inhibitor of the vascular endothelial growth factor (VEGF) receptor and other kinases is the only approved systemic agent in advanced HCC. Despite multiple studies defining the molecular heterogeneity of HCC, clinical trials of new agents do not pre-select for predictive markers. The fibroblast growth factor (FGF) receptor consists of 4 receptor tyrosine kinases and over 20 ligands. Differential expression and activation of these receptors and ligands have been implicated in oncogenesis and the pathogenesis of HCC (Sawey 2011). BGJ398 is a selective small molecule tyrosine kinase inhibitor of the FGFR family of receptors 1-4. Given the molecular diversity of HCC, we hypothesized that there would be a subset of HCC that would be more dependent on FGFR inhibition and used cell line models to identify predictive markers of response to BGK 398. Methods: 20 human HCC cell lines were used. In vitro dose response curves were generated using direct cell count assays over 6 days. IC 50 values were calculated from the dose response curves. Genomic data on the cell lines was derived using the Agilent 105K CGH platform. These data were analyzed in the context of the sensitivity data to BGJ 398. Western blot analysis was performed for effects of BGJ398 on phospho-FRS2Δ (tyr-196), a downstream signaling molecule for FGFR activation. Flow cytometry was performed for effects of BGJ 398 on the cell cycle and apoptosis. Results: There was differential sensitivity to BGJ 398 across the HCC cell lines. 3 cell lines were identified as having IC 50 values less than 1 μM versus the remaining cell lines which were all >1 uM. When analyzed in the context of the CGH data, all 3 cell lines had gains in FGF 19 of log2 ratio of 0.9 to 2.6. BGJ inhibited FRS2α activation in all cell lines tested. Flow cytometry revealed the BGJ induced cell cycle arrest in sensitive lines without significant apoptosis. Conclusions: Gains in FGF19 are predictive for growth inhibitory effects of BGJ398 in human HCC cell lines in vitro and provide rationale for patient selection in clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3858. doi:1538-7445.AM2012-3858