Daniel J. Chin

A role for receptor tyrosine phosphatase zeta in glioma cell migration

Glioblastomas (GBM) are the most frequent and malignant human brain tumor type. Typically striking in adulthood, tumor progression is rapid, relentless, and ultimately leads to the patients death within a year of diagnosis. The identification of transcriptionally regulated genes can lead to the discovery of targets for antibody or small-molecule-mediated therapy, as well as diagnostic markers. We prepared cDNA arrays that are specifically enriched for genes expressed in human brain tumors and profiled gene expression patterns in 14 individual tumor samples. Out of 25 000 clones arrayed, greater than 200 genes were found transcriptionally induced in glioblastomas compared to normal human brain tissue including the receptor tyrosine phosphataseζ (RPTPζ) and one of its ligands, pleiotrophin (Ptn). We confirmed by Northern blot analysis and immunohistochemistry that RPTPζ is enriched in tumor samples. Knockdown of RPTPζ by RNA interference studies established a functional role of RPTPζ in cell migration. Our results suggest a novel function for RPTPζ in regulating glioblastoma cell motility and point to the therapeutic utility of RPTPζ as a target for antibody-mediated therapy of brain tumors.

Comprehensive regional and temporal gene expression profiling of the rat brain during the first 24 h after experimental stroke identifies dynamic ischemia-induced gene expression patterns, and reveals a biphasic activation of genes in surviving tissue.

In order to identify biological processes relevant for cell death and survival in the brain following stroke, the postischemic brain transcriptome was studied by a large‐scale cDNA array analysis of three peri‐infarct brain regions at eight time points during the first 24 h of reperfusion following middle cerebral artery occlusion in the rat. K‐means cluster analysis revealed two distinct biphasic gene expression patterns that contained 44 genes (including 18 immediate early genes), involved in cell signaling and plasticity (i.e. MAP2K7, Sprouty2, Irs‐2, Homer1, GPRC5B, Grasp). The first gene induction phase occurred at 0–3 h of reperfusion, and the second at 9–15 h, and was validated by in situ hybridization. Four gene clusters displayed a progressive increase in expression over time and included 50 genes linked to cell motility, lipid synthesis and trafficking (i.e. ApoD, NPC1, G3P‐dehydrogenase1, and Choline kinase) or cell death‐regulating genes such as mitochondrial CLIC. We conclude that a biphasic transcriptional up‐regulation of the brain‐derived neurotrophic factor (BDNF)–G‐protein coupled receptor (GPCR)–mitogen‐activated protein (MAP) kinase signaling pathways occurs in surviving tissue, concomitant with a progressive and persistent activation of cell proliferation signifying tissue regeneration, which provide the means for cell survival and postischemic brain plasticity.

Transcriptomic analysis of the woodchuck model of chronic hepatitis B

The Eastern woodchuck (Marmota monax) is naturally infected with woodchuck hepatitis virus (WHV), a hepadnavirus closely related to the human hepatitis B virus (HBV). The woodchuck is used as an animal model for studying chronic hepatitis B (CHB) and HBV‐associated hepatocellular carcinoma (HCC) in humans, but the lack of sequence information has hitherto precluded functional genomics analysis. To address this major limitation of the model, we report here the sequencing, assembly, and annotation of the woodchuck transcriptome, together with the generation of custom woodchuck microarrays. Using this new platform, we characterized the transcriptional response to persistent WHV infection and WHV‐induced HCC. This revealed that chronic WHV infection, like HBV, is associated with (1) a limited intrahepatic type I interferon response; (2) intrahepatic induction of markers associated with T cell exhaustion; (3) elevated levels of suppressor of cytokine signaling 3 (SOCS3) in the liver; and (4) intrahepatic accumulation of neutrophils. Underscoring the translational value of the woodchuck model, this study also determined that WHV‐induced HCC shares molecular characteristics with a subtype of human HCC with poor prognosis. Conclusion: Our data establish the translational value of the woodchuck model and provide new insight into immune pathways which may play a role either in the persistence of HBV infection or the sequelae of CHB. (HEPATOLOGY 2012;56:820–830)

Npas4, a novel helix–loop–helix PAS domain protein, is regulated in response to cerebral ischemia

Basic helix–loop–helix PAS domain proteins form a growing family of transcription factors. These proteins are involved in the process of adaptation to cellular stresses and environmental factors such as a change in oxygen concentration. We describe the identification and characterization of a recently cloned PAS domain protein termed Npas4 in ischemic rat brain. Using gene expression profiling following middle cerebral artery occlusion, we showed that the Npas4 mRNA is differentially expressed in ischemic tissue. The full‐length gene was cloned from rat brain and its spatial and temporal expression characterized with in situ hybridization and Northern blotting. The Npas4 mRNA is specifically expressed in the brain and is highly up‐regulated in ischemic tissues following both focal and global cerebral ischemic insults. Immunohistochemistry revealed a strong expression in the limbic system and thalamus, as well as in layers 3 and 5 in the cortex of the unchallenged brain. When overexpressed in HEK 293 cells, Npas4 appears as a protein of ∼ 100 kDa. In brain samples, however, in addition to the 100 kDa band a specific 200 kDa immunoreactive band was also detected. Ischemic challenge lead to a decrease in the 200 kDa form and a simultaneous increase in the 100 kDa immunoreactivity. This could indicate a novel regulatory mechanism for activation and/or deactivation of this protein in response to ischemic brain injury.

Identification of an intrahepatic transcriptional signature associated with self‐limiting infection in the woodchuck model of hepatitis B

The woodchuck model of hepatitis B virus (HBV) infection displays many characteristics of human infection and has particular value for characterizing the host immune responses during the development of chronic infection. Using the newly developed custom woodchuck microarray platform, we compared the intrahepatic transcriptional profiles of neonatal woodchucks with self‐limiting woodchuck hepatitis virus (WHV) infection to those woodchucks progressing to persistent WHV infection. This revealed that WHV does not induce significant intrahepatic gene expression changes during the early‐acute stage of infection (8 weeks), suggesting it is a stealth virus. At the mid‐acute phase of infection (14 weeks), resolution was associated with induction of a prominent cytotoxic T‐cell signature. Strikingly, this was accompanied by high‐level expression of PD‐1 and various other inhibitory T‐cell receptors, which likely act to minimize liver damage by cytotoxic T cells during viral clearance. In contrast to the expression of perforin and other cytotoxic effector genes, the interferon‐γ (IFN‐γ) signaling response in the mid‐acute phase was comparable to that in chronically infected adult animals. The absence of a strong IFN‐α/β transcriptional response indicated that type I IFN is not a critical mediator of self‐limiting infection. Nevertheless, a number of antiviral genes, including viperin, were differentially expressed during resolving infection, suggesting that a subset of IFN‐stimulated genes (ISG) may play a role in the control of WHV replication. Conclusion: We identified new immune pathways associated with the clearance of hepadnavirus infection revealing novel molecular targets with potential for the therapeutic treatment of chronic hepatitis B. (HEPATOLOGY 2013)

Intrahepatic Transcriptional Signature Associated with Response to Interferon-α Treatment in the Woodchuck Model of Chronic Hepatitis B

Recombinant interferon-alpha (IFN-α) is an approved therapy for chronic hepatitis B (CHB), but the molecular basis of treatment response remains to be determined. The woodchuck model of chronic hepatitis B virus (HBV) infection displays many characteristics of human disease and has been extensively used to evaluate antiviral therapeutics. In this study, woodchucks with chronic woodchuck hepatitis virus (WHV) infection were treated with recombinant woodchuck IFN-α (wIFN-α) or placebo (n = 12/group) for 15 weeks. Treatment with wIFN-α strongly reduced viral markers in the serum and liver in a subset of animals, with viral rebound typically being observed following cessation of treatment. To define the intrahepatic cellular and molecular characteristics of the antiviral response to wIFN-α, we characterized the transcriptional profiles of liver biopsies taken from animals (n = 8–12/group) at various times during the study. Unexpectedly, this revealed that the antiviral response to treatment did not correlate with intrahepatic induction of the majority of IFN-stimulated genes (ISGs) by wIFN-α. Instead, treatment response was associated with the induction of an NK/T cell signature in the liver, as well as an intrahepatic IFN-γ transcriptional response and elevation of liver injury biomarkers. Collectively, these data suggest that NK/T cell cytolytic and non-cytolytic mechanisms mediate the antiviral response to wIFN-α treatment. In summary, by studying recombinant IFN-α in a fully immunocompetent animal model of CHB, we determined that the immunomodulatory effects, but not the direct antiviral activity, of this pleiotropic cytokine are most closely correlated with treatment response. This has important implications for the rational design of new therapeutics for the treatment of CHB.

HBsAg loss in patients treated with peginterferon alfa-2a and adefovir is associated with SLC16A9 gene variation and lower plasma carnitine levels

BACKGROUND & AIMS Achievement of HBsAg loss remains the hallmark of chronic hepatitis B treatment. In order to identify host factors contributing to treatment-induced HBsAg loss, we performed a genome-wide screen of single nucleotide polymorphisms (SNPs) and studied its immunological consequence. METHODS Chronic hepatitis B patients (40 HBeAg-positive and 44 HBeAg-negative) treated with peginterferon alfa-2a and adefovir were genotyped for 999,091 SNPs, which were associated with HBsAg loss at week 96 (n = 9). Plasma carnitine levels were measured by tandem-mass spectrometry, and the effect of carnitine on the proliferative capacity of hepatitis B virus (HBV)-specific and non-specific CD8 T cells was studied in vitro. RESULTS One polymorphism, rs12356193 located in the SLC16A9 gene, was genome-wide significantly associated with HBsAg loss at week 96 (p = 1.84 × 10(-8)). The previously reported association of rs12356193 with lower carnitine levels was confirmed in our cohort, and baseline carnitine levels were lower in patients with HBsAg loss compared to patients with HBsAg persistence (p = 0.02). Furthermore, we demonstrated that carnitine suppressed HBV-specific CD8 T cell proliferation. CONCLUSIONS In chronic hepatitis B patients treated with peginterferon and adefovir, we identified strong associations of SLC16A9 gene variation and carnitine levels with HBsAg loss. Our results further suggest that a lower baseline plasma carnitine level increases the proliferative capacity of CD8 T cells, making patients more susceptible to the immunological effect of this treatment. These novel findings may provide new insight into factors involved in treatment-induced HBsAg loss, and play a role in the prediction of treatment outcome.