Dysregulation of Liver Regeneration by Hepatitis B Virus Infection: Impact on Development of Hepatocellular Carcinoma.

The liver is unique in its ability to regenerate in response to damage. The complex process of liver regeneration consists of multiple interactive pathways. About 2 billion people worldwide have been infected with hepatitis B virus (HBV), and HBV causes 686,000 deaths each year due to its complications. Long-term infection with HBV, which causes chronic inflammation, leads to serious liver-related diseases, including cirrhosis and hepatocellular carcinoma. HBV infection has been reported to interfere with the critical mechanisms required for liver regeneration. In this review, the studies on liver tissue characteristics and liver regeneration mechanisms are summarized. Moreover, the inhibitory mechanisms of HBV infection in liver regeneration are investigated. Finally, the association between interrupted liver regeneration and hepatocarcinogenesis, which are both triggered by HBV infection, is outlined. Understanding the fundamental and complex liver regeneration process is expected to provide significant therapeutic advantages for HBV-associated hepatocellular carcinoma.

Spatial CRISPR genomics identifies regulators of the tumor microenvironment.

While CRISPR screens are helping uncover genes regulating many cell-intrinsic processes, existing approaches are suboptimal for identifying extracellular gene functions, particularly in the tissue context. Here, we developed an approach for spatial functional genomics called Perturb-map. We applied Perturb-map to knock out dozens of genes in parallel in a mouse model of lung cancer and simultaneously assessed how each knockout influenced tumor growth, histopathology, and immune composition. Moreover, we paired Perturb-map and spatial transcriptomics for unbiased analysis of CRISPR-edited tumors. We found that in Tgfbr2 knockout tumors, the tumor microenvironment (TME) was converted to a fibro-mucinous state, and T cells excluded, concomitant with upregulated TGFß and TGFß-mediated fibroblast activation, indicating that TGFß-receptor loss on cancer cells increased TGFß bioavailability and its immunosuppressive effects on the TME. These studies establish Perturb-map for functional genomics within the tissue at single-cell resolution with spatial architecture preserved and provide insight into how TGFß responsiveness of cancer cells can affect the TME.

Distinctive HBV Replication Capacity and Susceptibility to Tenofovir Induced by a Polymerase Point Mutation in Hepatoma Cell Lines and Primary Human Hepatocytes.

Tenofovir disoproxil fumarate (TDF) has been regarded as the most potent drug for treating patients with chronic hepatitis B (CHB). However recently, viral mutations associated with tenofovir have been reported. Here, we found a CHB patient with suboptimal response after more than 4 years of TDF treatment. Clonal analysis of hepatitis B virus (HBV) isolated from sequential sera of this patient identified the seven previously reported TDF-resistant mutations (CYELMVI). Interestingly, a threonine to alanine mutation at the 301 amino acid position of the reverse-transcriptase (RT) domain, (rtT301A), was commonly accompanied with CYELMVI at a high rate (72.7%). Since the rtT301A mutation has not been reported yet, we investigated the role of this naturally occurring mutation on the viral replication and susceptibility to tenofovir in various liver cells (hepatoma cells as well as primary human hepatocytes). A cell-based phenotypic assay revealed that the rtT301A mutation dramatically impaired the replication ability with meaningful reduction in sensitivity to tenofovir in hepatoma cell lines. However, attenuated viral replication by the rtT301A mutation was significantly restored in primary human hepatocytes (PHHs). Our findings suggest that the replication capability and drug sensitivity of HBV is different between hepatoma cell lines and PHHs. Therefore, our study emphasizes that validation studies should be performed not only in the liver cancer cell lines but also in the PHHs to understand the exact viral fitness under antiviral pressure in patients.

Suppression of Hepatocyte Nuclear Factor 4 α by Long-term Infection of Hepatitis B Virus Contributes to Tumor Cell Proliferation.

Hepatitis B virus (HBV) infection is a major factor in the development of various liver diseases such as hepatocellular carcinoma (HCC). Among HBV encoded proteins, HBV X protein (HBx) is known to play a key role in the development of HCC. Hepatocyte nuclear factor 4α (HNF4α) is a nuclear transcription factor which is critical for hepatocyte differentiation. However, the expression level as well as its regulatory mechanism in HBV infection have yet to be clarified. Here, we observed the suppression of HNF4α in cells which stably express HBV whole genome or HBx protein alone, while transient transfection of HBV replicon or HBx plasmid had no effect on the HNF4α level. Importantly, in the stable HBV- or HBx-expressing hepatocytes, the downregulated level of HNF4α was restored by inhibiting the ERK signaling pathway. Our data show that HNF4α was suppressed during long-term HBV infection in cultured HepG2-NTCP cells as well as in a mouse model following hydrodynamic injection of pAAV-HBV or in mice intravenously infected with rAAV-HBV. Importantly, HNF4α downregulation increased cell proliferation, which contributed to the formation and development of tumor in xenograft nude mice. The data presented here provide proof of the effect of HBV infection in manipulating the HNF4α regulatory pathway in HCC development.

Identification of a quadruple mutation that confers tenofovir resistance in chronic hepatitis B patients.

BACKGROUND & AIMS: Tenofovir disoproxil fumarate (TDF) is one the most potent nucleot(s)ide analogues for treating chronic hepatitis B virus (HBV) infection. Phenotypic resistance caused by genotypic resistance to TDF has not been reported. This study aimed to characterize HBV mutations that confer tenofovir resistance. METHODS: Two patients with viral breakthrough during treatment with TDF-containing regimens were prospectively enrolled. The gene encoding HBV reverse transcriptase was sequenced. Eleven HBV clones harboring a series of mutations in the reverse transcriptase gene were constructed by site-directed mutagenesis. Drug susceptibility of each clone was determined by Southern blot analysis and real-time PCR. The relative frequency of mutants was evaluated by ultra-deep sequencing and clonal analysis. RESULTS: Five mutations (rtS106C [C], rtH126Y [Y], rtD134E [E], rtM204I/V, and rtL269I [I]) were commonly found in viral isolates from 2 patients. The novel mutations C, Y, and E were associated with drug resistance. In assays for drug susceptibility, the IC50 value for wild-type HBV was 3.8 ±â€¯0.6 µM, whereas the IC50 values for CYE and CYEI mutants were 14.1 ±â€¯1.8 and 58.1 ±â€¯0.9 µM, respectively. The IC90 value for wild-type HBV was 30 ±â€¯0.5 µM, whereas the IC90 values for CYE and CYEI mutants were 185 ±â€¯0.5 and 790 ±â€¯0.2 µM, respectively. Both tenofovir-resistant mutants and wild-type HBV had similar susceptibility to the capsid assembly modulator NVR 3-778 (IC50 <0.4 µM vs. IC50 = 0.4 µM, respectively). CONCLUSIONS: Our study reveals that the quadruple (CYEI) mutation increases the amount of tenofovir required to inhibit HBV by 15.3-fold in IC50 and 26.3-fold in IC90. These results demonstrate that tenofovir-resistant HBV mutants can emerge, although the genetic barrier is high. LAY SUMMARY: Tenofovir is the most potent nucleotide analogue for the treatment of chronic hepatitis B virus infection and there has been no hepatitis B virus mutation that confers >10-fold resistance to tenofovir up to 8 years. Herein, we identified, for the first time, a quadruple mutation that conferred 15.3-fold (IC50) and 26.3-fold (IC90) resistance to tenofovir in 2 patients who experienced viral breakthrough during tenofovir treatment.

Protein Barcodes Enable High-Dimensional Single-Cell CRISPR Screens.

CRISPR pools are being widely employed to identify gene functions. However, current technology, which utilizes DNA as barcodes, permits limited phenotyping and bulk-cell resolution. To enable novel screening capabilities, we developed a barcoding system operating at the protein level. We synthesized modules encoding triplet combinations of linear epitopes to generate >100 unique protein barcodes (Pro-Codes). Pro-Code-expressing vectors were introduced into cells and analyzed by CyTOF mass cytometry. Using just 14 antibodies, we detected 364 Pro-Code populations; establishing the largest set of protein-based reporters. By pairing each Pro-Code with a different CRISPR, we simultaneously analyzed multiple phenotypic markers, including phospho-signaling, on dozens of knockouts. Pro-Code/CRISPR screens found two interferon-stimulated genes, the immunoproteasome component Psmb8 and a chaperone Rtp4, are important for antigen-dependent immune editing of cancer cells and identified Socs1 as a negative regulator of Pd-l1. The Pro-Code technology enables simultaneous high-dimensional protein-level phenotyping of 100s of genes with single-cell resolution.

Intracellular interleukin-32γ mediates antiviral activity of cytokines against hepatitis B virus.

Cytokines are involved in early host defense against pathogen infections. In particular, tumor necrosis factor (TNF) and interferon-gamma (IFN-γ) have critical functions in non-cytopathic elimination of hepatitis B virus (HBV) in hepatocytes. However, the molecular mechanisms and mediator molecules are largely unknown. Here we show that interleukin-32 (IL-32) is induced by TNF and IFN-γ in hepatocytes, and inhibits the replication of HBV by acting intracellularly to suppress HBV transcription and replication. The gamma isoform of IL-32 (IL-32γ) inhibits viral enhancer activities by downregulating liver-enriched transcription factors. Our data are validated in both an in vivo HBV mouse model and primary human hepatocytes. This study thus suggests that IL-32γ functions as intracellular effector in hepatocytes for suppressing HBV replication to implicate a possible mechanism of non-cytopathic viral clearance.

Quiescent Tissue Stem Cells Evade Immune Surveillance.

Stem cells are critical for the maintenance of many tissues, but whether their integrity is maintained in the face of immunity is unclear. Here we found that cycling epithelial stem cells, including Lgr5+ intestinal stem cells, as well as ovary and mammary stem cells, were eliminated by activated T cells, but quiescent stem cells in the hair follicle and muscle were resistant to T cell killing. Immune evasion was an intrinsic property of the quiescent stem cells resulting from systemic downregulation of the antigen presentation machinery, including MHC class I and TAP proteins, and is mediated by the transactivator NLRC5. This process was reversed upon stem cell entry into the cell cycle. These studies identify a link between stem cell quiescence, antigen presentation, and immune evasion. As cancer-initiating cells can derive from stem cells, these findings may help explain how the earliest cancer cells evade immune surveillance.

The Effects of the Severity of Periventricular Leukomalacia on the Neuropsychological Outcomes of Preterm Children.

This study investigates the developmental outcomes of preterm children according to severity of periventricular leukomalacia. One hundred preterm children with periventricular leukomalacia evident on brain magnetic resonance imaging and who had undergone neuropsychologic evaluation were selected. Intellectual disability was noted in 27.8% of the children with mild periventricular leukomalacia, 53.2% with moderate periventricular leukomalacia, and 77.1% with severe periventricular leukomalacia. The rates of major neurodevelopmental impairments such as cerebral palsy or intellectual disability were related to the severity of periventricular leukomalacia but not to gestational age or epilepsy. There were significant differences in the intelligence quotient (IQ) and social maturity quotient between 3 groups of periventricular leukomalacia. The performance IQ was significantly lower than the verbal IQ. Behavioral problems were noted in about one-third of the children but the rate was not related with the severity of periventricular leukomalacia. Our study revealed the significant associations between severity of periventricular leukomalacia and cognitive and social adaptive functions in the preterm children.

Cleaved c-FLIP mediates the antiviral effect of TNF-α against hepatitis B virus by dysregulating hepatocyte nuclear factors.

BACKGROUND & AIMS: Cytokines are key molecules implicated in the defense against virus infection. Tumor necrosis factor-alpha (TNF-α) is well known to block the replication of hepatitis B virus (HBV). However, the molecular mechanism and the downstream effector molecules remain largely unknown. METHODS: In this study, we investigated the antiviral effect and mechanism of p22-FLIP (FLICE-inhibitory protein) by ectopic expression in vitro and in vivo. In addition, to provide the biological relevance of our study, we examined that the p22-FLIP is involved in TNF-α-mediated suppression of HBV in primary human hepatocytes. RESULTS: We found that p22-FLIP, a newly discovered c-FLIP cleavage product, inhibited HBV replication at the transcriptional level in both hepatoma cells and primary human hepatocytes, and that c-FLIP conversion to p22-FLIP was stimulated by the TNF-α/NF-κB pathway. p22-FLIP inhibited HBV replication through the upregulation of HNF3ß but downregulation of HNF4α, thus inhibiting both HBV enhancer elements. Finally, p22-FLIP potently inhibited HBV DNA replication in a mouse model of HBV replication. CONCLUSIONS: Taken together, these findings suggest that the anti-apoptotic p22-FLIP serves a novel function of inhibiting HBV transcription, and mediates the antiviral effect of TNF-α against HBV replication.

GFP-specific CD8 T cells enable targeted cell depletion and visualization of T-cell interactions.

There are numerous cell types with scarcely understood functions, whose interactions with the immune system are not well characterized. To facilitate their study, we generated a mouse bearing enhanced green fluorescent protein (EGFP)-specific CD8+ T cells. Transfer of the T cells into EGFP reporter animals can be used to kill EGFP-expressing cells, allowing selective depletion of desired cell types, or to interrogate T-cell interactions with specific populations. Using this system, we eliminate a rare EGFP-expressing cell type in the heart and demonstrate its role in cardiac function. We also show that naive T cells are recruited into the mouse brain by antigen-expressing microglia, providing evidence of an immune surveillance pathway in the central nervous system. The just EGFP death-inducing (Jedi) T cells enable visualization of a T-cell antigen. They also make it possible to utilize hundreds of existing EGFP-expressing mice, tumors, pathogens and other tools, to study T-cell interactions with many different cell types, to model disease states and to determine the functions of poorly characterized cell populations.

Substitution at rt269 in Hepatitis B Virus Polymerase Is a Compensatory Mutation Associated with Multi-Drug Resistance.

The emergence of compensatory mutations in the polymerase gene of drug resistant hepatitis B virus (HBV) is associated with treatment failure. We previously identified a multi-drug resistant HBV mutant, which displayed resistance towards lamivudine (LMV), clevudine (CLV), and entecavir (ETV), along with a strong replication capacity. The aim of this study was to identify the previously unknown compensatory mutations, and to determine the clinical relevance of this mutation during antiviral therapy. In vitro mutagenesis, drug susceptibility assay, and molecular modeling studies were performed. The rtL269I substitution conferred 2- to 7-fold higher replication capacity in the wild-type (WT) or YMDD mutation backbone, regardless of drug treatment. The rtL269I substitution alone did not confer resistance to LMV, ETV, adefovir (ADV), or tenofovir (TDF). However, upon combination with YMDD mutation, the replication capacity under LMV or ETV treatment was enhanced by several folds. Molecular modeling studies suggested that the rtL269I substitution affects template binding, which may eventually lead to the enhanced activity of rtI269-HBV polymerase in both WT virus and YMDD mutant. The clinical relevance of the rtL269I substitution was validated by its emergence in association with YMDD mutation in chronic hepatitis B (CHB) patients with sub-optimal response or treatment failure to LMV or CLV. Our study suggests that substitution at rt269 in HBV polymerase is associated with multi-drug resistance, which may serve as a novel compensatory mutation for replication-defective multi-drug resistant HBV.

Anticancer effect of tectochrysin in colon cancer cell via suppression of NF-kappaB activity and enhancement of death receptor expression.

BACKGROUND: Flavonoids are a diverse family of natural phenolic compounds commonly found in fruits and vegetables. Epidemiologic studies showed that flavonoids also reduce the risk of colon cancer. Tectochrysin is one of the major flavonoids of Alpinia oxyphylla Miquel. However, the anti-cancer effects and the molecular mechanisms of tectochrysin in colon cancer cells have not yet been reported. We investigated whether tectochrysin could inhibit colon cancer cell growth at 1, 5, 10 µg/ml. In in vivo study, we injected a tectochrysin treatment dose of 5 mg/kg to each mouse. RESULTS: Tectochrysin suppressed the growth of SW480 and HCT116 human colon cancer cells. The expression of DR3, DR4 and Fas were significantly increased, and pro-apoptotic proteins were also increased. Tectochrysin treatment also inhibited activity of NF-κB. A docking model indicated that tectochrysin binds directly to the p50 unit. In in vivo, tumor weights and volumes in mice were reduced when treated with tectochrysin. Tectochrysin leads to apoptotic cell death in colon cancer cells through activation of death receptors expression via the inhibition of NF-κB. CONCLUSIONS: Tectochrysin can be a useful agent for the treatment of colon cancer cell growth as well as an adjuvant agent for chemo-resistant cancer cells growth.

Spontaneous HBsAg loss in Korean patients: relevance of viral genotypes, S gene mutations, and covalently closed circular DNA copy numbers.

BACKGROUND/AIMS: Occult HBV infection can persist following HBsAg loss and be transmitted, but the virological features are not well defined. METHODS: Here we investigated 25 Korean patients who lost HBsAg during follow up, either spontaneously or subsequent to therapy. RESULTS: Whereas subtype adr (genotype C) was found in 96% of HBsAg positive patients, 75 % of patients who lost HBsAg spontaneously were seemed to be infected with the ayw subtype with sequence similar to genotype D. Mutations in the major hydrophilic region (MHR) of HBsAg were found in 7 patients who lost HBsAg spontaneously. The mutations include T123S, M125I/N, C139R, D144E, V177A, L192F, and W196L, some of which have not been reported before. Functional analysis via transfection experiments indicate that the C139R and D144E mutations drastically reduced HBsAg antigenicity, while the Y225del mutation found in one interferon-treated patient impaired HBsAg secretion. CONCLUSIONS: Lack of detectable HBsAg in patient serum could be explained by low level of ccc DNA in liver tissue, low antigenicity of the surface protein, or its secretion defect.

Two-year outcomes of deep brain stimulation in adults with cerebral palsy.

OBJECTIVE: To investigate the effect of deep brain stimulation (DBS) on reducing dystonia and disability in adults with cerebral palsy (CP) and to compare the therapeutic outcomes between primary dystonia patients and CP patients over two years after bilateral pallidal DBS. METHODS: Five patients with primary dystonia and seven CP patients with dystonia were recruited. All subjects received DBS surgery in both globus pallidus. Burke-Fahn-Marsden dystonia rating scale consisting of dystonia movement score and disability score and subjective satisfaction scale were assessed after 1 month and every 6 months over two years following DBS treatment. RESULTS: On the dystonia movement scale, both groups of primary dystonia patients and CP patients showed a significant decrease over time following DBS. On the disability scale, patients with primary dystonia showed a significant decrease over time, whereas the disability score of CP patients did not change over the two years. Comparing the dystonia movement and disability scores of CP patients at each assessment, patients with primary dystonia showed a significant reduction after 6 months. Comparing the satisfaction scores of CP patients after DBS, patients with primary dystonia showed significantly higher subjective satisfaction. CONCLUSION: Whereas dystonia can be significantly reduced in patients with primary dystonia, CP patients showed a modest improvement on the dystonia movement scale, but not on the disability scale. Therefore, DBS may be considered with caution as a treatment modality of CP patients with dystonia.

Hepatocystin contributes to interferon-mediated antiviral response to hepatitis B virus by regulating hepatocyte nuclear factor 4α.

Hepatocystin/80K-H is known as a causative gene for autosomal dominant polycystic liver disease. However, the role of hepatocystin in hepatitis B virus-related liver disease remains unknown. Here, we investigated the role of hepatocystin on the cytokine-mediated antiviral response against hepatitis B virus infection. We investigated the antiviral effect and mechanism of hepatocystin by ectopic expression and RNAi knockdown in cell culture and mouse livers. Hepatocystin suppressed the replication of hepatitis B virus both in vitro and in vivo. This inhibitory effect was HBx-independent and mediated by the transcriptional regulation of viral genome via the activation of exogenous signal-regulated kinase 1/2 and the reduced expression of hepatocyte nuclear factor 4α, a transcription factor essential for hepatitis B virus replication. The amino-terminal region of hepatocystin was essential for regulation of this antiviral signaling pathway. We also found that hepatocystin acts as a critical component in interferon-mediated mitogen-activated protein kinase signaling pathway, and the interferon-induced antiviral activity against hepatitis B virus is associated with the expression levels of hepatocystin. We demonstrated that hepatocystin plays a critical role in modulating the susceptibility of hepatitis B virus to interferon, suggesting that the modulation of hepatocystin expression is important for cytokine-mediated viral clearance during hepatitis B virus infection.

The effect of obturator nerve block on hip lateralization in low functioning children with spastic cerebral palsy.

PURPOSE: Hip adductor spasticity has a great impact on developing hip displacement in children with cerebral palsy (CP). Obturator nerve (ON) block is less invasive intervention rather than soft tissue surgery for reduction of hip adductor spasticity. The aim of this study is to investigate the effect of ON block on hip lateralization in low functioning children with spastic CP. MATERIALS AND METHODS: The study was performed by retrospective investigation of the clinical and radiographic follow-up data of low functioning children [gross motor function classification system (GMFCS) level III to V] with spastic cerebral palsy whose hip was subluxated. Migration percentage (MP) was measured on hip radiographs and its annual change was calculated. In intervention group, ON block was done with 50% ethyl alcohol under the guidance of electrical stimulation. RESULTS: The data of 49 legs of 25 children for intervention group and the data of 41 legs of 23 children for nonintervention group were collected. In intervention group, the MP were significantly reduced at 1st follow-up and the MPs at 2nd and last follow-up did not show significant differences from initial MP. Whereas in nonintervention group, the MPs at 1st, 2nd and last follow-up were all significantly increased compared to initial MPs. CONCLUSION: ON block with ethyl alcohol is useful as an early effective procedure against progressive hip displacement in these children with spastic CP.

Alteration of synaptic activity-regulating genes underlying functional improvement by long-term exposure to an enriched environment in the adult brain.

BACKGROUND: Housing animals in an enriched environment (EE) enhances behavioral function. However, the mechanism underlying this EE-mediated functional improvement and the resultant changes in gene expression have yet to be elucidated. OBJECTIVES: We attempted to investigate the underlying mechanisms associated with long-term exposure to an EE by evaluating gene expression patterns. METHODS: We housed 6-week-old CD-1 (ICR) mice in standard cages or an EE comprising a running wheel, novel objects, and social interaction for 2 months. Motor and cognitive performances were evaluated using the rotarod test and passive avoidance test, and gene expression profile was investigated in the cerebral hemispheres using microarray and gene set enrichment analysis (GSEA). RESULTS: In behavioral assessment, an EE significantly enhanced rotarod performance and short-term working memory. Microarray analysis revealed that genes associated with neuronal activity were significantly altered by an EE. GSEA showed that genes involved in synaptic transmission and postsynaptic signal transduction were globally upregulated, whereas those associated with reuptake by presynaptic neurotransmitter transporters were downregulated. In particular, both microarray and GSEA demonstrated that EE exposure increased opioid signaling, acetylcholine release cycle, and postsynaptic neurotransmitter receptors but decreased Na+ / Cl- -dependent neurotransmitter transporters, including dopamine transporter Slc6a3 in the brain. Western blotting confirmed that SLC6A3, DARPP32 (PPP1R1B), and P2RY12 were largely altered in a region-specific manner. CONCLUSION: An EE enhanced motor and cognitive function through the alteration of synaptic activity-regulating genes, improving the efficient use of neurotransmitters and synaptic plasticity by the upregulation of genes associated with postsynaptic receptor activity and downregulation of presynaptic reuptake by neurotransmitter transporters.

HBx-induced NF-κB signaling in liver cells is potentially mediated by the ternary complex of HBx with p22-FLIP and NEMO.

Sustained activation of NF-κB is one of the causative factors for various liver diseases, including liver inflammation and hepatocellular carcinoma (HCC). It has been known that activating the NF-κB signal by hepatitis B virus X protein (HBx) is implicated in the development of HCC. However, despite numerous studies on HBx-induced NF-κB activation, the detailed mechanisms still remain unsolved. Recently, p22-FLIP, a cleavage product of c-FLIPL, has been reported to induce NF-κB activation through interaction with the IκB kinase (IKK) complex in primary immune cells. Since our previous report on the interaction of HBx with c-FLIPL, we explored whether p22-FLIP is involved in the modulation of HBx function. First, we identified the expression of endogenous p22-FLIP in liver cells. NF-κB reporter assay and electrophoretic mobility shift assay (EMSA) revealed that the expression of p22-FLIP synergistically enhances HBx-induced NF-κB activation. Moreover, we found that HBx physically interacts with p22-FLIP and NEMO and potentially forms a ternary complex. Knock-down of c-FLIP leading to the downregulation of p22-FLIP showed that endogenous p22-FLIP is involved in HBx-induced NF-κB activation, and the formation of a ternary complex is necessary to activate NF-κB signaling. In conclusion, we showed a novel mechanism of HBx-induced NF-κB activation in which ternary complex formation is involved among HBx, p22-FLIP and NEMO. Our findings will extend the understanding of HBx-induced NF-κB activation and provide a new target for intervention in HBV-associated liver diseases and in the development of HCC.

Environmental enrichment synergistically improves functional recovery by transplanted adipose stem cells in chronic hypoxic-ischemic brain injury.

We investigated the effects of environmental enrichment (EE) on the function of transplanted adipose stem cells (ASCs) and the combined effect of EE and ASC transplantation on neurobehavioral function in an animal model of chronic hypoxic-ischemic (HI) brain injury. HI brain damage was induced in 7-day-old mice by unilateral carotid artery ligation and exposure to hypoxia (8% O2 for 90 min). At 6 weeks of age, the mice were randomly injected with either ASCs or PBS into the striatum and were randomly assigned to either EE or standard cages (SC), comprising ASC-EE (n=18), ASC-SC (n=19), PBS-EE (n=12), PBS-SC (n=17), and untreated controls (n=23). Rotarod, forelimb-use asymmetry, and grip strength tests were performed to evaluate neurobehavioral function. The fate of transplanted cells and the levels of endogenous neurogenesis, astrocyte activation, and paracrine factors were also measured. As a result, EE and ASC transplantation synergistically improved rotarod latency, forelimb-use asymmetry, and grip strength compared to those of the other groups. The number of engrafted ASCs and ßIII-tubulin(+) neurons derived from the transplanted ASCs was significantly higher in mice in EE than those in SC. EE and ASC transplantation also synergistically increased BrdU(+)ßIII-tubulin(+) neurons, GFAP(+) astrocytic density, and fibroblast growth factor 2 (FGF2) level but not the level of CS-56(+) glial scarring in the striatum. In conclusion, EE and ASC transplantation synergistically improved neurobehavioral functions. The underlying mechanisms of this synergism included enhanced repair processes such as higher engraftment of the transplanted ASCs, increased endogenous neurogenesis and astrocytic activation coupled with upregulation of FGF2.