A Novel Humanized Immune Stroma PDX Cancer Model for Therapeutic Studies.

Standard preclinical human tumor models lack a human tumor stroma. However, as stroma contributes to therapeutic resistance, the lack of human stroma may make current models less stringent for testing new therapies. To address this, using patient-derived tumor cells, patient derived cancer-associated mesenchymal stem/progenitor cells, and human endothelial cells, we created a Human Stroma-Patient Derived Xenograft (HS-PDX) tumor model. HS-PDX, compared to the standard PDX model, demonstrate greater resistance to targeted therapy and chemotherapy, and better reflect patient response to therapy. Furthermore, HS-PDX can be grown in mice with humanized bone marrow to create humanized immune stroma patient-derived xenograft (HIS-PDX) models. The HIS-PDX model contains human connective tissues, vascular and immune cell infiltrates. RNA sequencing analysis demonstrated a 94-96% correlation with primary human tumor. Using this model, we demonstrate the impact of human tumor stroma on response to CAR-T cell therapy and immune checkpoint inhibitor therapy. We show an immunosuppressive role for human tumor stroma and that this model can be used to identify immunotherapeutic combinations to overcome stromally mediated immunosuppression. Combined, our data confirm a critical role for human stoma in therapeutic response and indicate that HIS-PDX can be an important tool for preclinical drug testing. Statement of Significance: We developed a tumor model with human stromal, vascular, and immune cells. This model mirrors patient response to chemotherapy, targeted therapy, and immunotherapy, and can be used to study therapy resistance.

Co-exposure to aflatoxin B1 and therapeutic coartem worsens hepatic and renal function through enhanced oxido-inflammatory responses and apoptosis in rats.

Aflatoxin B1 (AFB1) is a mycotoxin synthesised as a secondary metabolite by members of the Aspergillus species contaminating agricultural produce. Aspergillus species thrive in tropical climes, endemic to malaria. Artemisinin-based combination therapies (ACTs) effectively treat and prevent malaria recrudescence; Coartem (COA) is an ACT whose toxicity is evident. Although there are scanty studies on COA toxicity, the scientific literature is replete on AFB1 toxic effects -including carcinogenicity. The current research investigates AFB1 and COA toxicity in experimental Wistar rats' hepatorenal systems. Thirty albino rats were randomly grouped into five cohorts (n = 6) and treated as follows: Group I: Untreated control (2 mL/kg of corn oil); group II: AFB1 alone (70 µg/kg); group III: COA alone (5 mg/kg); group IV: COA and a low dose of AFB11 (5 mg/kg & 35 µg/kg); while Group V: COA and a high dose AFB12 (5 mg/kg & 70 µg/kg) by gavage. Our results show that exposure to AFB1 and COA significantly (p < 0.05) reduced superoxide dismutase, catalase, glutathione peroxidase, and glutathione-S-transferase activities, besides reduced glutathione and total sulfhydryl groups level. Reactive oxygen and nitrogen species, lipid peroxidation, 8-hydroxy-2'-deoxyguanosine, nitric oxide, xanthine oxidase, and myeloperoxidase levels were increased (p < 0.05) in rats co-treated with COA and AFB1. Cell death was aggravated in COA and AFB1 groups, exemplified by increased Caspase-3 and 9 activities and alterations in the typical histological features of experimental rats' livers and kidneys. Finally, rats co-treated with AFB1 and COA experienced increased hepatorenal dysregulation, oxidative and inflammatory tissue damage, and apoptotic cell death. All the observed systemic perturbations occurred dose-dependently. It is crucial, therefore, to prevent AFB1 dietary contaminations during COA therapeutic regimen due to increased pathophysiological damage exerted on experimental rat liver and kidneys, as evidenced in this study.

A biochemical and histology experimental approach to investigate the adverse effect of chronic lead acetate and dietary furan on rat lungs.

Despite lead widespread environmental pollution, its effect on humans and livestock's respiratory systems remains inadequately investigated. Similarly, furan is industrially relevant with enormous environmental presence. Lead and furan can be ingested -via lead pipes contaminated water and heat-treated food respectively. Thus, humans are inadvertently exposed continuously. Lead toxicity is well studied, and furan have earned a position on the IARC's list of carcinogens. Here, we evaluate the effect of co-exposure to lead and furan on rat lungs. Thirty Wistar rats were grouped randomly into six cohorts (n = 6) consisting of a control group, furan alone group, lead acetate (PbAc) alone group and three other groups co-exposure to graded PbAc (1, 10 & 100 µg/L) alongside a constant furan (8 mg/kg) dose. After twenty-eight days, enzymatic and non-enzymatic antioxidant, oxidative stress and inflammatory biomarkers were biochemically evaluated. The ELISA-based technique was used to measure oxidative-DNA damage (8-OHG), tumour protein 53 (TP53) expressed and tumour necrotic factor-alpha (TNF-α) level. Dose-dependent increases (p < 0.05) in reactive oxygen and nitrogen species, malondialdehyde, nitric oxide, myeloperoxidase, TNF-α and TP53 level, with an associated decrease (p < 0.05) in enzymatic and non-enzymatic antioxidants were observed in the furan, PbAc and the co-treated rats relative to the control. In addition, PbAc and furan treatment impaired the histoarchitectural structures of rat lungs, exemplified by pro-inflammatory cell infiltration and trafficking into the bronchioles and alveolar spaces. Co-exposure to furan and PbAc may contribute to lung dysfunction via loss of redox balance, genomic damage/instability, inflammation and disrupted histoarchitectural features.

3-Indolepropionic acid prevented chlorpyrifos-induced hepatorenal toxicities in rats by improving anti-inflammatory, antioxidant, and pro-apoptotic responses and abating DNA damage.

The application of chlorpyrifos (CPF), an organophosphorus pesticide to control insects, is associated with oxidative stress and reduced quality of life in humans and animals. Indole-3-propionic acid (IPA) is a by-product of tryptophan metabolism with high antioxidant capacity and has the potential to curb CPF-mediated toxicities in the hepatorenal system of rats. It is against this background that we explored the subacute exposure of CPF and the effect of IPA in the liver and kidney of thirty rats using five cohort experimental designs (n = 6) consisting of control (corn oil 2 mL/kg body weight), CPF alone (5 mg/kg), IPA alone (50 mg/kg), CPF + IPA1 (5 mg/kg + 25 mg/kg), and CPF + IPA2 (5 mg/kg + 50 mg/kg). Subsequently, we evaluated biomarkers of hepatorenal damage, oxidative and nitrosative stress, inflammation, DNA damage, and apoptosis by spectrophotometric and enzyme-linked immunosorbent assay methods. Our results showed that co-treatment with IPA decreased CPF-upregulated serum hepatic transaminases, creatinine, and urea; reversed CPF downregulation of SOD, CAT, GPx, GST, GSH, Trx, TRx-R, and TSH; and abated CPF upregulation of XO, MPO, RONS, and LPO. Co-treatment with IPA decreased CPF-upregulated IL-1ß and 8-OHdG levels, caspase-9 and caspase-3 activities, and increased IL-10. In addition, IPA averts CPF-induced histological changes in the liver and kidney of rats. Our results demonstrate that co-dosing CPF-exposed rats with IPA can significantly decrease CPF-induced oxidative stress, pro-inflammatory responses, DNA damage, and subsequent pro-apoptotic responses in rats' liver and kidneys. Therefore, supplementing tryptophan-derived endogenous IPA from exogenous sources may help avert toxicity occasioned by inadvertent exposure to harmful chemicals, including CPF-induced systemic perturbation of liver and kidney function.

The BLT Humanized Mouse Model as a Tool for Studying Human Gamma Delta T Cell-HIV Interactions In Vivo.

Gamma-delta (γδ) T cells recognize antigens in a major histocompatibility complex (MHC) independent and have cytotoxic capability. Human immunodeficiency virus (HIV) infection reduces the proportion of the Vδ2 cell subset compared to the Vδ1 cell subset of γδ T cells in the blood in most infected individuals, except for elite controllers. The capacity of Vδ2 T cells to kill HIV-infected targets has been demonstrated in vitro, albeit in vivo confirmatory studies are lacking. Here, we provide the first characterization of γδ T cell-HIV interactions in bone marrow-liver-thymus (BLT) humanized mice and examined the immunotherapeutic potential of Vδ2 T cells in controlling HIV replication in vivo. We demonstrate a reduced proportion of Vδ2 T cells and an increased proportion of Vδ1 T cells in HIV-infected BLT humanized mice, like in HIV-positive individuals. HIV infection in BLT humanized mice also impaired the ex vivo expansion of Vδ2 T cells, like in HIV-positive individuals. Adoptive transfer of activated Vδ2 T cells did not control HIV replication during cell-associated HIV transmission in BLT humanized mice but instead exacerbated viremia, suggesting that Vδ2 T cells may serve as early targets for HIV replication. Our findings demonstrate that BLT humanized mice can model γδ T cell-HIV interactions in vivo.

3-Indolepropionic acid upturned male reproductive function by reducing oxido-inflammatory responses and apoptosis along the hypothalamic-pituitary-gonadal axis of adult rats exposed to chlorpyrifos.

We examined the effect of 3-Indolepropionic acid (3-IPA), an antioxidant on the organophosphorus pesticide chlorpyrifos (CPF)-induced reproductive toxicity in rats. The five experimental rat cohorts were treated per os for 14 consecutive days as follows: Control (Corn oil 2 mL/kg body weight), CPF alone (5 mg/kg), 3-IPA alone (40 mg/kg) and the co-treated rat cohorts (CPF:5 mg/kg + 3-IPA: 20 or 40 mg/kg). Biomarkers of testicular and epididymal function, oxidative stress, myeloperoxidase (MPO) activity and the levels of nitric oxide (NO), reactive oxygen and nitrogen (RONS) species and lipid peroxidation (LPO) were assessed. Also, tumour necrosis factor-alpha (TNF-α), Bcl-2-associated X (Bax) and B cell lymphoma 2 (Bcl-2) proteins were estimated, and tissue histology was microscopically examined. CPF alone significantly (p < 0.05) increased biomarkers of reproductive toxicities were averted in rats co-treated 3-IPA. Decreases in antioxidants and increases in lipid peroxidation and reactive oxygen and nitrogen species were lessened (p < 0.05) in CPF and 3-IPA co-treated rats. CPF mediated increases in TNF-α, NO, Bax, and MPO activity was reduced (p < 0.05) in the epididymis, testes, and hypothalamus of rats co-treated with 3-IPA. In addition, Bcl-2 expression was increased in rats co-treated with 3-IPA dose-dependently. Histopathological examination revealed severe lesions induced by CPF were prevented in rats co-treated with 3-IPA. Our findings demonstrate that exogenous 3-IPA reduced CPF-induced oxidative stress, inflammation, and apoptosis in the epididymis and testes of male rats.

In vitro antioxidant and antihypertensive properties of sesame seed enzymatic protein hydrolysate and ultrafiltration peptide fractions.

The objective of this study was to determine the in vitro antioxidant and antihypertensive potentials of sesame seed protein hydrolysate and its membrane ultrafiltration peptide fractions in comparison to the unhydrolyzed protein. Sesame seed protein isolate (SESPI) was prepared from the defatted sesame seed meal and then hydrolyzed using consecutive additions of pepsin and pancreatin to yield sesame protein hydrolysate (SESPH). The SESPH was subjected to membrane ultrafiltration consecutively to obtain fractions with peptide sizes of <1, 1-3, 3-5, and 5-10 kDa, respectively, which were then assayed for in vitro antioxidant and antihypertensive properties. The results showed that protein hydrolysis and fractionation led to significant (p < .05) increases in the content of hydrophobic amino acids. Radical scavenging and metal ion chelation were also significantly (p < .05) enhanced by these treatments. Inhibition of linoleic acid oxidation was stronger with the 1.0 mg/ml of sesame peptide samples in comparison to the mild inhibitory effect exhibited by the 0.5 mg/ml of samples. The <1 kDa peptide fraction was the most active inhibitor (81%) against angiotensin converting enzyme, whereas the bigger peptides (>3-5 and 5-10 kDa) were the most effective (75%-85% ) inhibitors against renin. These sesame products could be used as therapeutic agents in the development of health enhancing foods for the prevention and management of chronic diseases. PRACTICAL APPLICATIONS: Bioactive peptides have been produced from plant protein sources through in vitro enzymatic activities. Sesame seed peptides have demonstrated multifunctional potential to act as antioxidative and antihypertensive agents that could be utilized as ingredients for the development of novel functional foods and nutraceuticals.

Hyperglycemic memory in the rat bladder detrusor is associated with a persistent hypomethylated state.

Hyperglycemic memory is associated with several complications of diabetes. Although there is some physiological evidence that this phenomenon occurs with diabetic bladder dysfunction (DBD), there have been no studies in bladder that provide evidence of hyperglycemic memory at the molecular/biochemical level. In the present studies, we determined the effects of long-term diabetes on the metabolome of bladder detrusor in a rat model of streptozotocin-induced type-1-diabetes and the ability of insulin treatment to normalize metabolic changes. These studies demonstrated that although insulin reversed a majority of the metabolic changes caused by diabetes, with long-term diabetes there was a persistent decrease in the methylation index (indicated by a reduced ratio of S-adenosylmethionine to S-adenosyl homocysteine) after insulin treatment. We confirmed a "hypomethylated environment" develops in diabetic detrusor by demonstrating an overall reduction in methylated detrusor DNA that is only partially reversed with glycemic control. Furthermore, we confirmed that this hypomethylated environment is associated with epigenetic changes in the detrusor genome, which are again mostly, but not completely, reversed with glycemic control. Overall our studies provide strong molecular evidence for a mechanism by which diabetes alters methylation status and gene expression in the detrusor genome, and that these epigenetic modifications contribute to hyperglycemic memory. Our work suggests novel treatment strategies for diabetic patients who have attained glycemic control but continue to experience DBD. For example, epigenomic data can be used to identify "actionable gene targets" for its treatment and would also support a rationale for approaches that target the hypomethylation index.

Regulatory mechanisms mediated by peroxisome proliferator-activated receptor-ß/δ in skin cancer.

Considerable progress has been made during the past 20 years towards elucidating the role of peroxisome proliferator-activated receptor-ß/δ (PPARß/δ) in skin cancer. In 1999, the original notion that PPARß/δ was involved with epithelial cell function was postulated based on a correlation between PPARß/δ expression and the induction of messenger RNAs encoding proteins that mediate terminal differentiation in keratinocytes. Subsequent studies definitively revealed that PPARß/δ could induce terminal differentiation and inhibit proliferation of keratinocytes. Molecular mechanisms have since been discovered to explain how this nuclear receptor can be targeted for preventing and treating skin cancer. This includes the regulation of terminal differentiation, mitotic signaling, endoplasmic reticulum stress, and cellular senescence. Interestingly, the effects of activating PPARß/δ can preferentially target keratinocytes with genetic mutations associated with skin cancer. This review provides the history and current understanding of how PPARß/δ can be targeted for both nonmelanoma skin cancer and melanoma and postulates how future approaches that modulate PPARß/δ signaling may be developed for the prevention and treatment of these diseases.

Chronic hepatitis C infection-induced liver fibrogenesis is associated with M2 macrophage activation.

The immuno-pathogenic mechanisms of chronic hepatitis C virus (HCV) infection remain to be elucidated and pose a major hurdle in treating or preventing chronic HCV-induced advanced liver diseases such as cirrhosis. Macrophages are a major component of the inflammatory milieu in chronic HCV-induced liver disease, and are generally derived from circulating inflammatory monocytes; however very little is known about their role in liver diseases. To investigate the activation and role of macrophages in chronic HCV-induced liver fibrosis, we utilized a recently developed humanized mouse model with autologous human immune and liver cells, human liver and blood samples and cell culture models of monocyte/macrophage and/or hepatic stellate cell activation. We showed that M2 macrophage activation was associated with liver fibrosis during chronic HCV infection in the livers of both humanized mice and patients, and direct-acting antiviral therapy attenuated M2 macrophage activation and associated liver fibrosis. We demonstrated that supernatant from HCV-infected liver cells activated human monocytes/macrophages with M2-like phenotypes. Importantly, HCV-activated monocytes/macrophages promoted hepatic stellate cell activation. These results suggest a critical role for M2 macrophage induction in chronic HCV-associated immune dysregulation and liver fibrosis.

Factors predicting postoperative range of motion for anatomic total shoulder arthroplasty.

BACKGROUND: Total shoulder arthroplasty (TSA) has repeatedly been shown to be an effective and durable treatment option for end-stage arthritis with good long-term survivorship. Whereas pain relief is typically the primary goal, improvements in range of motion are typically expected as well. The factors that influence postoperative motion have not been well characterized. The purpose of the study was to examine the factors that influence ultimate postoperative motion after TSA. METHODS: A retrospective review was conducted of prospectively collected data of 230 patients with minimum 1-year follow-up after TSA for end-stage arthropathy with an intact rotator cuff. Analysis was focused on factors that may correlate with postoperative measured forward flexion, abduction, external rotation, and internal rotation. Included in this analysis was perception of motion, age, body mass index (BMI), comorbidities (smoking, diabetes, osteoporosis, hypercholesterolemia, inflammatory arthritis, and thyroid disease), and number of comorbidities. RESULTS: Preoperative motion in all directions was predictive of postoperative motion for forward flexion (R = 0.235; P < .001), abduction (R = 0.363; P < .001), external rotation (R = 0.325; P < .001), and internal rotation (R = 0.213; P = .002). BMI and diabetes both negatively correlated with internal rotation (R = -0.134, P = .40 and R = -0.196, P = .003, respectively). Individual and total number of comorbidities were not predictive of postoperative motion. The patient's perception of preoperative motion also did not correlate with postoperative motion. CONCLUSIONS: Preoperative range of motion before TSA is most predictive of final motion achieved. Individual and total number of comorbidities are not predictive of postoperative motion. Patients with high diabetes and increased BMI have limited postoperative internal rotation.

Chronic Opisthorchis viverrini infection and associated hepatobiliary disease is associated with iron loaded M2-like macrophages.

Chronic Opisthorchis viverrini-induced hepatobiliary disease is associated with significant leukocyte infiltration, including activated macrophages; however, the polarization of infiltrating macrophages remains to be fully characterized. In this study, we characterized macrophage polarization and phenotype in chronic O. viverrini-induced hepatobiliary disease in humans and hamsters using gene expression and histochemical analysis. Chronic O. viverrini infection and associated hepatobiliary diseases were associated with iron loaded M2-like macrophages in both humans and hamsters. This study provides suggestive evidence that iron loaded M2-like macrophages promote hepatobiliary disease in chronic O. viverrini infection.

A chimeric mouse model to study immunopathogenesis of HCV infection.

Several human hepatotropic pathogens including chronic hepatitis C virus (HCV) have narrow species restriction, thus hindering research and therapeutics development against these pathogens. Developing a rodent model that accurately recapitulates hepatotropic pathogens infection, human immune response, chronic hepatitis, and associated immunopathogenesis is essential for research and therapeutics development. Here, we describe the recently developed AFC8 humanized liver- and immune system-mouse model for studying chronic hepatitis C virus and associated human immune response, chronic hepatitis, and liver fibrosis.

Diabetes attenuates urothelial modulation of detrusor contractility and spontaneous activity.

OBJECTIVES: To investigate the effect of diabetes on urothelial modulation of bladder contractility. METHODS: Bladder strips (urothelium intact or denuded) were prepared from 8-week-old streptozotocin-induced diabetic (n = 19) and non-diabetic control rats (n = 10). The effect of modulators of MaxiK (iberiotoxin and tetraethylammonium) and Kv7 (XE991 and retigabine) potassium channel activity were investigated for their effects on both carbachol-induced force generation and spontaneous contractile activity. RESULTS: In bladder strips from non-diabetic animals, the presence of the urothelium resulted in marked sensitivity to carbachol-induced force generation by modulators of MaxiK and Kv7 channel activity, whereas in the diabetic animal urothelial sensitivity to these agents was significantly diminished. Urothelial-intact bladder strips from non-diabetic animals were more sensitive to modulators of Kv7 activity in reducing the amplitude of spontaneous phasic contractions than urothelial-denuded bladder strips, whereas in diabetic animals the presence or absence of the urothelium did not alter the sensitivity to modulators of Kv7 activity. Spontaneous activity in the presence of tetraethylammonium was not affected by the urothelium in bladder strips from either diabetic or non-diabetic animals. CONCLUSIONS: The presence of the urothelium in bladders from non-diabetic animals modulates the activity of potassium blockers to affect bladder contractility, whereas in the diabetic bladder this effect is attenuated. These findings could help to explain the lack of success of pharmaceutical treatments targeting potassium channels to treat bladder pathology in patients with diseases imparing urothelial function.

Modulation of aryl hydrocarbon receptor (AHR)-dependent signaling by peroxisome proliferator-activated receptor ß/δ (PPARß/δ) in keratinocytes.

Whether peroxisome proliferator-activated receptor ß/δ (PPARß/δ) reduces skin tumorigenesis by altering aryl hydrocarbon receptor (AHR)-dependent activities was examined. Polycyclic aromatic hydrocarbons (PAH) increased expression of cytochrome P4501A1 (CYP1A1), CYP1B1 and phase II xenobiotic metabolizing enzymes in wild-type skin and keratinocytes. Surprisingly, this effect was not found in Pparß/δ-null skin and keratinocytes. Pparß/δ-null keratinocytes exhibited decreased AHR occupancy and histone acetylation on the Cyp1a1 promoter in response to a PAH compared with wild-type keratinocytes. Bisulfite sequencing of the Cyp1a1 promoter and studies using a DNA methylation inhibitor suggest that PPARß/δ promotes demethylation of the Cyp1a1 promoter. Experiments with human HaCaT keratinocytes stably expressing shRNA against PPARß/δ also support this conclusion. Consistent with the lower AHR-dependent activities in Pparß/δ-null mice compared with wild-type mice, 7,12-dimethylbenz[a]anthracene (DMBA)-induced skin tumorigenesis was inhibited in Pparß/δ-null mice compared with wild-type. Results from these studies demonstrate that PPARß/δ is required to mediate complete carcinogenesis by DMBA. The mechanisms underlying this PPARß/δ-dependent reduction of AHR signaling by PAH are not due to alterations in the expression of AHR auxiliary proteins, ligand binding or AHR nuclear translocation between genotypes, but are likely influenced by PPARß/δ-dependent demethylation of AHR target gene promoters including Cyp1a1 that reduces AHR accessibility as shown by reduced promoter occupancy. This PPARß/δ/AHR crosstalk is unique to keratinocytes and conserved between mice and humans.

Hepatitis B virus infection and immunopathogenesis in a humanized mouse model: induction of human-specific liver fibrosis and M2-like macrophages.

The mechanisms of chronic HBV infection and immunopathogenesis are poorly understood due to a lack of a robust small animal model. Here we report the development of a humanized mouse model with both human immune system and human liver cells by reconstituting the immunodeficient A2/NSG (NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ mice with human HLA-A2 transgene) with human hematopoietic stem cells and liver progenitor cells (A2/NSG-hu HSC/Hep mice). The A2/NSG-hu HSC/Hep mouse supported HBV infection and approximately 75% of HBV infected mice established persistent infection for at least 4 months. We detected human immune responses, albeit impaired in the liver, chronic liver inflammation and liver fibrosis in infected animals. An HBV neutralizing antibody efficiently inhibited HBV infection and associated liver diseases in humanized mice. In addition, we found that the HBV mediated liver disease was associated with high level of infiltrated human macrophages with M2-like activation phenotype. Importantly, similar M2-like macrophage accumulation was confirmed in chronic hepatitis B patients with liver diseases. Furthermore, gene expression analysis showed that induction of M2-like macrophage in the liver is associated with accelerated liver fibrosis and necrosis in patients with acute HBV-induced liver failure. Lastly, we demonstrate that HBV promotes M2-like activation in both M1 and M2 macrophages in cell culture studies. Our study demonstrates that the A2/NSG-hu HSC/Hep mouse model is valuable in studying HBV infection, human immune responses and associated liver diseases. Furthermore, results from this study suggest a critical role for macrophage polarization in hepatitis B virus-induced immune impairment and liver pathology.

Liver immune-pathogenesis and therapy of human liver tropic virus infection in humanized mouse models.

Hepatitis B virus (HBV) and hepatitis C virus (HCV) infect and replicate primarily in human hepatocytes. Few reliable and easy accessible animal models are available for studying the immune system's contribution to the liver disease progression during hepatitis virus infection. Humanized mouse models reconstituted with human hematopoietic stem cells (HSCs) have been developed to study human immunology, human immunodeficiency virus 1 infection, and immunopathogenesis. However, a humanized mouse model engrafted with both human immune and human liver cells is needed to study infection and immunopathogenesis of HBV/HCV infection in vivo. We have recently developed the humanized mouse model with both human immune and human liver cells (AFC8-hu HSC/Hep) to study immunopathogenesis and therapy of HCV infection in vivo. In this review, we summarize the current models of HBV/HCV infection and their limitations in immunopathogenesis. We will then present our recent findings of HCV infection and immunopathogenesis in the AFC8-hu HSC/Hep mouse, which supports HCV infection, human T-cell response and associated liver pathogenesis. Inoculation of humanized mice with primary HCV isolates resulted in long-term HCV infection. HCV infection induced elevated infiltration of human immune cells in the livers of HCV-infected humanized mice. HCV infection also induced HCV-specific T-cell immune response in lymphoid tissues of humanized mice. Additionally, HCV infection induced liver fibrosis in humanized mice. Anti-human alpha smooth muscle actin (αSMA) staining showed elevated human hepatic stellate cell activation in HCV-infected humanized mice. We discuss the limitation and future improvements of the AFC8-hu HSC/Hep mouse model and its application in evaluating novel therapeutics, as well as studying both HCV and HBV infection, human immune responses, and associated human liver fibrosis and cancer.

Generation of a humanized mouse model with both human immune system and liver cells to model hepatitis C virus infection and liver immunopathogenesis.

Establishing a small animal model that accurately recapitulates hepatotropic pathogens, including hepatitis C virus (HCV) infection and immunopathogenesis, is essential for the study of hepatitis virus-induced liver disease and for therapeutics development. This protocol describes our recently developed humanized mouse model for studying HCV and other hepatotropic infections, human immune response and hepatitis and liver fibrosis. The first 5-h stage is the isolation of human liver progenitor and hematopoietic stem cells from fetal liver. Next, AFC8 immunodeficient mice are transplanted with the isolated progenitor/stem cells. This generally takes 2 h. The transplanted mice are then treated for a month with the mouse liver apoptosis-inducing AFC8 dimerizer and left for an additional 2-month period to permit human liver and immune cell growth as well as system reconstitution and development before inoculation with HCV clinical isolates. HCV infection, human immune response and liver disease are observed with high incidence from approximately 2 months after inoculation.

Peroxisome proliferator-activated receptor ß/δ cross talks with E2F and attenuates mitosis in HRAS-expressing cells.

The role of peroxisome proliferator-activated receptor ß/δ (PPARß/δ) in Harvey sarcoma ras (Hras)-expressing cells was examined. Ligand activation of PPARß/δ caused a negative selection with respect to cells expressing higher levels of the Hras oncogene by inducing a mitotic block. Mitosis-related genes that are predominantly regulated by E2F were induced to a higher level in HRAS-expressing Pparß/δ-null keratinocytes compared to HRAS-expressing wild-type keratinocytes. Ligand-activated PPARß/δ repressed expression of these genes by direct binding with p130/p107, facilitating nuclear translocation and increasing promoter recruitment of p130/p107. These results demonstrate a novel mechanism of PPARß/δ cross talk with E2F signaling. Since cotreatment with a PPARß/δ ligand and various mitosis inhibitors increases the efficacy of increasing G2/M arrest, targeting PPARß/δ in conjunction with mitosis inhibitors could become a suitable option for development of new multitarget strategies for inhibiting RAS-dependent tumorigenesis.