A phase I oncolytic virus trial with vesicular stomatitis virus expressing human interferon beta and tyrosinase related protein 1 administered intratumorally and intravenously in uveal melanoma: safety, efficacy, and T cell responses.

Introduction: Metastatic uveal melanoma (MUM) has a poor prognosis and treatment options are limited. These patients do not typically experience durable responses to immune checkpoint inhibitors (ICIs). Oncolytic viruses (OV) represent a novel approach to immunotherapy for patients with MUM. Methods: We developed an OV with a Vesicular Stomatitis Virus (VSV) vector modified to express interferon-beta (IFN-ß) and Tyrosinase Related Protein 1 (TYRP1) (VSV-IFNß-TYRP1), and conducted a Phase 1 clinical trial with a 3 + 3 design in patients with MUM. VSV-IFNß-TYRP1 was injected into a liver metastasis, then administered on the same day as a single intravenous (IV) infusion. The primary objective was safety. Efficacy was a secondary objective. Results: 12 patients with previously treated MUM were enrolled. Median follow up was 19.1 months. 4 dose levels (DLs) were evaluated. One patient at DL4 experienced dose limiting toxicities (DLTs), including decreased platelet count (grade 3), increased aspartate aminotransferase (AST), and cytokine release syndrome (CRS). 4 patients had stable disease (SD) and 8 patients had progressive disease (PD). Interferon gamma (IFNγ) ELIspot data showed that more patients developed a T cell response to virus encoded TYRP1 at higher DLs, and a subset of patients also had a response to other melanoma antigens, including gp100, suggesting epitope spreading. 3 of the patients who responded to additional melanoma antigens were next treated with ICIs, and 2 of these patients experienced durable responses. Discussion: Our study found that VSV-IFNß -TYRP1 can be safely administered via intratumoral (IT) and IV routes in a previously treated population of patients with MUM. Although there were no clear objective radiographic responses to VSV-IFNß-TYRP1, dose-dependent immunogenicity to TYRP1 and other melanoma antigens was seen.

APOBEC3B-mediated corruption of the tumor cell immunopeptidome induces heteroclitic neoepitopes for cancer immunotherapy.

APOBEC3B, an anti-viral cytidine deaminase which induces DNA mutations, has been implicated as a mediator of cancer evolution and therapeutic resistance. Mutational plasticity also drives generation of neoepitopes, which prime anti-tumor T cells. Here, we show that overexpression of APOBEC3B in tumors increases resistance to chemotherapy, but simultaneously heightens sensitivity to immune checkpoint blockade in a murine model of melanoma. However, in the vaccine setting, APOBEC3B-mediated mutations reproducibly generate heteroclitic neoepitopes in vaccine cells which activate de novo T cell responses. These cross react against parental, unmodified tumors and lead to a high rate of cures in both subcutaneous and intra-cranial tumor models. Heteroclitic Epitope Activated Therapy (HEAT) dispenses with the need to identify patient specific neoepitopes and tumor reactive T cells ex vivo. Thus, actively driving a high mutational load in tumor cell vaccines increases their immunogenicity to drive anti-tumor therapy in combination with immune checkpoint blockade.

Generation of a Tumor-Specific Chemokine Gradient Using Oncolytic Vesicular Stomatitis Virus Encoding CXCL9.

Genetically modified vesicular stomatitis virus (VSV) is an attractive agent for cancer treatment due to rapid intratumoral replication and observed clinical responses. Although VSV selectively kills malignant cells and can boost antitumor immunity, limited induction of intratumoral immune infiltration remains a barrier to efficacy in some cancer models. Here we engineered the oncolytic VSV platform to encode the T cell chemokine CXCL9, which is known to mediate the recruitment of activated CD8+ cytotoxic T cells and CD4+ T helper cells, and demonstrates conserved protein function between mice and humans. Chemotactic activity of the virally encoded chemokine was confirmed in vitro. Intratumoral concentration of CXCL9 was shown to increase after VSV therapy in three different cancer models, but to a much greater degree after VSV-CXCL9 therapy as compared with VSV control viruses. Despite a steep chemokine gradient from the tumor to the bloodstream, tumor trafficking of adoptively transferred and endogenous T cells was not measurably increased following VSV-CXCL9 therapy. Our results indicate that oncolytic VSV infection promotes release of CXCL9 in the tumor microenvironment, but further boosting of the functional chemokine gradient through virus engineering has little incremental impact on intratumoral immune cell infiltration in mouse and human tumor models.

Targeted Derivation of Organotypic Glucose- and GLP-1-Responsive ß Cells Prior to Transplantation into Diabetic Recipients.

Generation of functional ß cells from pluripotent sources would accelerate diagnostic and therapeutic applications for diabetes research and therapy. However, it has been challenging to generate competent ß cells with dynamic insulin-secretory capacity to glucose and incretin stimulations. We introduced transcription factors, critical for ß-cell development and function, in differentiating human induced pluripotent stem cells (PSCs) and assessed the impact on the functionality of derived ß-cell (psBC) progeny. A perifusion system revealed stepwise transduction of the PDX1, NEUROG3, and MAFA triad (PNM) enabled in vitro generation of psBCs with glucose and GLP-1 responsiveness within 3 weeks. PNM transduction upregulated genes associated with glucose sensing, insulin secretion, and ß-cell maturation. In recipient diabetic mice, PNM-transduced psBCs showed glucose-responsive insulin secretion as early as 1 week post transplantation. Thus, enhanced pre-emptive ß-cell specification of PSCs by PNM drives generation of glucose- and incretin-responsive psBCs in vitro, offering a competent tissue-primed biotherapy.

Hyperinsulinemic hypoglycemia subtype glucokinase V91L mutant induces necrosis in ß-cells via ATP depletion.

Hyperinsulinemic hypoglycemia subtype glucokinase (GCK-HH) is caused by an activating mutation in glucokinase (GCK) and has been shown to increase ß-cell death. However, the mechanism of ß-cell death in GCK-HH remains poorly understood. Here, we expressed the GCK-HH V91L GCK mutant in INS-1 832/13 cells to determine the effect of the mutation on ß-cell viability and the mechanisms of ß-cell death. We showed that expression of the V91L GCK mutant in INS-1 832/13 cells resulted in a rapid glucose concentration-dependent loss of cell viability. At 11 mM D-glucose, INS-1 832/13 cells expressing V91L GCK showed increased cell permeability without significant increases in Annexin V staining or caspase 3/7 activation, indicating that these cells are primarily undergoing cell death via necrosis. Over-expression of SV40 large T antigen, which inhibits the p53 pathway, did not affect the V91L GCK-induced cell death. We also found that non-phosphorylatable L-glucose did not induce rapid cell death. Of note, glucose phosphorylation coincided with a 90% loss of intracellular ATP content. Thus, our data suggest that the GCK V91L mutant induces rapid necrosis in INS-1 cells through accelerated glucose phosphorylation, ATP depletion, and increased cell permeability.

Impaired ß-cell glucokinase as an underlying mechanism in diet-induced diabetes.

High-fat diet (HFD)-fed mouse models have been widely used to study early type 2 diabetes. Decreased ß-cell glucokinase (GCK) expression has been observed in HFD-induced diabetes. However, owing to its crucial roles in glucose metabolism in the liver and in islet ß-cells, the contribution of decreased GCK expression to the development of HFD-induced diabetes is unclear. Here, we employed a ß-cell-targeted gene transfer vector and determined the impact of ß-cell-specific increase in GCK expression on ß-cell function and glucose handling in vitro and in vivo Overexpression of GCK enhanced glycolytic flux, ATP-sensitive potassium channel activation and membrane depolarization, and increased proliferation in Min6 cells. ß-cell-targeted GCK transduction did not change glucose handling in chow-fed C57BL/6 mice. Although adult mice fed a HFD showed reduced islet GCK expression, impaired glucose tolerance and decreased glucose-stimulated insulin secretion (GSIS), ß-cell-targeted GCK transduction improved glucose tolerance and restored GSIS. Islet perifusion experiments verified restored GSIS in isolated HFD islets by GCK transduction. Thus, our data identify impaired ß-cell GCK expression as an underlying mechanism for dysregulated ß-cell function and glycemic control in HFD-induced diabetes. Our data also imply an etiological role of GCK in diet-induced diabetes.This article has an associated First Person interview with the first author of the paper.

Divergent susceptibilities to AAV-SaCas9-gRNA vector-mediated genome-editing in a single-cell-derived cell population.

OBJECTIVE: Recombinant adeno-associated virus (AAV)-based vectors are characterized by their robust and safe transgene delivery. The CRISPR/Cas9 and guide RNA (gRNA) system present a promising genome-editing platform, and a recent development of a shorter Cas9 enzyme from Staphylococcus aureus (SaCas9) allows generation of high titer single AAV vectors which carry both saCas9- and gRNA-expression cassettes. Here, we used two AAV-SaCas9 vectors with distinct GFP-targeted gRNA sequences and determined the impact of AAV-SaCas9-gRNA vector treatment in a single cell clone carrying a GFP-expression cassette. RESULTS: Our results showed comparable GFP knockout efficiencies (40-50%) upon a single low-dose infection. Three consecutive transductions of 25-fold higher doses of vectors showed 80% GFP knockout efficiency. To analyze the "AAV-SaCas9-resistant cell population", we sorted the residual GFP-positive cells and assessed their permissiveness to super-infection with two AAV-Cas9-GFP vectors. We found the sorted cells were significantly more resistant to the GFP knockout mediated by the same AAV vector, but not by the other GFP-targeted AAV vector. Our data therefore demonstrate highly efficient genome-editing by the AAV-SaCas9-gRNA vector system. Differential susceptibilities of single cell-derived cells to the AAV-SaCas9-gRNA-mediated genome editing may represent a formidable barrier to achieve 100% genome editing efficiency by this vector system.

Tumor-Free Transplantation of Patient-Derived Induced Pluripotent Stem Cell Progeny for Customized Islet Regeneration.

UNLABELLED: Human induced pluripotent stem cells (iPSCs) and derived progeny provide invaluable regenerative platforms, yet their clinical translation has been compromised by their biosafety concern. Here, we assessed the safety of transplanting patient-derived iPSC-generated pancreatic endoderm/progenitor cells. Transplantation of progenitors from iPSCs reprogrammed by lentiviral vectors (LV-iPSCs) led to the formation of invasive teratocarcinoma-like tumors in more than 90% of immunodeficient mice. Moreover, removal of primary tumors from LV-iPSC progeny-transplanted hosts generated secondary and metastatic tumors. Combined transgene-free (TGF) reprogramming and elimination of residual pluripotent cells by enzymatic dissociation ensured tumor-free transplantation, ultimately enabling regeneration of type 1 diabetes-specific human islet structures in vivo. The incidence of tumor formation in TGF-iPSCs was titratable, depending on the oncogenic load, with reintegration of the cMYC expressing vector abolishing tumor-free transplantation. Thus, transgene-free cMYC-independent reprogramming and elimination of residual pluripotent cells are mandatory steps in achieving transplantation of iPSC progeny for customized and safe islet regeneration in vivo. SIGNIFICANCE: Pluripotent stem cell therapy for diabetes relies on the safety as well as the quality of derived insulin-producing cells. Data from this study highlight prominent tumorigenic risks of induced pluripotent stem cell (iPSC) products, especially when reprogrammed with integrating vectors. Two major underlying mechanisms in iPSC tumorigenicity are residual pluripotent cells and cMYC overload by vector integration. This study also demonstrated that combined transgene-free reprogramming and enzymatic dissociation allows teratoma-free transplantation of iPSC progeny in the mouse model in testing the tumorigenicity of iPSC products. Further safety assessment and improvement in iPSC specification into a mature ß cell phenotype would lead to safe islet replacement therapy for diabetes.

Exercise Prevents Diet-Induced Cellular Senescence in Adipose Tissue.

Considerable evidence implicates cellular senescence in the biology of aging and chronic disease. Diet and exercise are determinants of healthy aging; however, the extent to which they affect the behavior and accretion of senescent cells within distinct tissues is not clear. Here we tested the hypothesis that exercise prevents premature senescent cell accumulation and systemic metabolic dysfunction induced by a fast-food diet (FFD). Using transgenic mice that express EGFP in response to activation of the senescence-associated p16(INK4a) promoter, we demonstrate that FFD consumption causes deleterious changes in body weight and composition as well as in measures of physical, cardiac, and metabolic health. The harmful effects of the FFD were associated with dramatic increases in several markers of senescence, including p16, EGFP, senescence-associated ß-galactosidase, and the senescence-associated secretory phenotype (SASP) specifically in visceral adipose tissue. We show that exercise prevents the accumulation of senescent cells and the expression of the SASP while nullifying the damaging effects of the FFD on parameters of health. We also demonstrate that exercise initiated after long-term FFD feeding reduces senescent phenotype markers in visceral adipose tissue while attenuating physical impairments, suggesting that exercise may provide restorative benefit by mitigating accrued senescent burden. These findings highlight a novel mechanism by which exercise mediates its beneficial effects and reinforces the effect of modifiable lifestyle choices on health span.

B-Type Natriuretic Peptide Deletion Leads to Progressive Hypertension, Associated Organ Damage, and Reduced Survival: Novel Model for Human Hypertension.

Altered myocardial structure and function, secondary to chronically elevated blood pressure, are leading causes of heart failure and death. B-type natriuretic peptide (BNP), a guanylyl cyclase A agonist, is a cardiac hormone integral to cardiovascular regulation. Studies have demonstrated a causal relationship between reduced production or impaired BNP release and the development of human hypertension. However, the consequences of BNP insufficiency on blood pressure and hypertension-associated complications remain poorly understood. Therefore, the goal of this study was to create and characterize a novel model of BNP deficiency to investigate the effects of BNP absence on cardiac and renal structure, function, and survival. Genetic BNP deletion was generated in Dahl salt-sensitive rats. Compared with age-matched controls, BNP knockout rats demonstrated adult-onset hypertension. Increased left ventricular mass with hypertrophy and substantially augmented hypertrophy signaling pathway genes, developed in young adult knockout rats, which preceded hypertension. Prolonged hypertension led to increased cardiac stiffness, cardiac fibrosis, and thrombi formation. Significant elongation of the QT interval was detected at 9 months in knockout rats. Progressive nephropathy was also noted with proteinuria, fibrosis, and glomerular alterations in BNP knockout rats. End-organ damage contributed to a significant decline in overall survival. Systemic BNP overexpression reversed the phenotype of genetic BNP deletion. Our results demonstrate the critical role of BNP defect in the development of systemic hypertension and associated end-organ damage in adulthood.

Nuclear reprogramming with a non-integrating human RNA virus.

INTRODUCTION: Advances in the field of stem cells have led to novel avenues for generating induced pluripotent stem cells (iPSCs) from differentiated somatic cells. iPSCs are typically obtained by the introduction of four factors--OCT4, SOX2, KLF4, and cMYC--via integrating vectors. Here, we report the feasibility of a novel reprogramming process based on vectors derived from the non-integrating vaccine strain of measles virus (MV). METHODS: We produced a one-cycle MV vector by substituting the viral attachment protein gene with the green fluorescent protein (GFP) gene. This vector was further engineered to encode for OCT4 in an additional transcription unit. RESULTS: After verification of OCT4 expression, we assessed the ability of iPSC reprogramming. The reprogramming vector cocktail with the OCT4-expressing MV vector and SOX2-, KLF4-, and cMYC-expressing lentiviral vectors efficiently transduced human skin fibroblasts and formed iPSC colonies. Reverse transcription-polymerase chain reaction and immunostaining confirmed induction of endogenous pluripotency-associated marker genes, such as SSEA-4, TRA-1-60, and Nanog. Pluripotency of derived clones was confirmed by spontaneous differentiation into three germ layers, teratoma formation, and guided differentiation into beating cardiomyocytes. CONCLUSIONS: MV vectors can induce efficient nuclear reprogramming. Given the excellent safety record of MV vaccines and the translational capabilities recently developed to produce MV-based vectors now used for cancer clinical trials, our MV vector system provides an RNA-based, non-integrating gene transfer platform for nuclear reprogramming that is amenable for immediate clinical translation.

Beta cell regeneration after single-round immunological destruction in a mouse model.

AIMS/HYPOTHESIS: Achieving a better understanding of beta cell regeneration after immunological destruction is crucial for the development of immunotherapy approaches for type 1 diabetes. In previous type 1 diabetes models, sustained immune activation eliminates regenerating beta cells, thus limiting the study of the regenerative capacity of beta cells upon immunological destruction. Here, we employed an adeno-associated virus 8 (AAV8) vector for beta cell-targeted overexpression of a foreign antigen to induce single-round immunological destruction of existing beta cells. METHODS: Young and aged C57BL/6J mice were treated with AAV8 vectors expressing the foreign antigen luciferase. Islet inflammation and regeneration was observed at 3, 6, 10 and 22 weeks post-AAV delivery. RESULTS: In young C57BL/6J mice, robust humoral and cellular immune responses were developed towards antigen-expressing beta cells, leading to decreased beta cell mass. This was followed by beta cell mass replenishment, along with enhanced proliferation of insulin-positive cells, recruitment of nestin/CD34-positive endothelial cells, displacement of alpha cells and mobilisation of cytoplasmic neurogenin 3-positive cells. Mice with recovering beta cells showed normal or reduced fasting blood glucose levels and faster glucose clearance than controls. Although aged mice demonstrated similar responses to the treatment, they initially exhibited notable islet scarring and fluctuations in blood glucose levels, indicating that beta cell regeneration is slower in aged mice. CONCLUSIONS/INTERPRETATION: Our hit-and-run, beta cell-targeted antigen expression system provides an opportunity to monitor the impact of single-round immunological beta cell destruction in animals with diverse genetic backgrounds or ageing status.

Cardiac BNP gene delivery prolongs survival in aged spontaneously hypertensive rats with overt hypertensive heart disease.

BACKGROUND: Hypertension is a highly prevalent disease associated with cardiovascular morbidity and mortality. Recent studies suggest that patients with hypertension also have a deficiency of certain cardiac peptides. Previously we demonstrated that a single intravenous injection of the myocardium-tropic adeno-associated virus (AAV) 9-based vector encoding for proBNP prevented the development of hypertensive heart disease (HHD) in spontaneously hypertensive rats (SHRs). The current study was designed to determine the duration of cardiac transduction after a single AAV9 injection and to determine whether cardiac BNP overexpression can delay the progression of previously established HHD, and improve survival in aged SHRs with overt HHD. METHODS AND RESULTS: To evaluate the duration of cardiac transduction induced by the AAV9 vector, we used four week old SHRs. Effective long-term selective cardiac transduction was determined by luciferase expression. A single intravenous administration of a luciferase-expressing AAV9 vector resulted in efficient cardiac gene delivery for up to 18-months. In aged SHRs (9-months of age), echocardiographic studies demonstrated progression of HHD in untreated controls, while AAV9-BNP vector treatment arrested the deterioration of cardiac function at six months post-injection (15-months of age). Aged SHRs with established overt HHD were further monitored to investigate survival. A single intravenous injection of the AAV9-vector encoding rat proBNP was associated with significantly prolonged survival in the treated SHRs (613?38 days, up to 669 days) compared to the untreated rats (480±69 days, up to 545 days)(p<0.05). CONCLUSIONS: A single intravenous injection of AAV9 vector elicited prolonged cardiac transduction (up to 18 months post-injection). AAV9 induced cardiac BNP overexpression prevented development of congestive heart failure, and significantly prolonged the survival of aged SHRs with previously established overt HHD. These findings support the beneficial effects of chronic supplementation of BNP in a frequent and highly morbid condition such as HHD.

Murine leukemia virus uses TREX components for efficient nuclear export of unspliced viral transcripts.

Previously we reported that nuclear export of both unspliced and spliced murine leukemia virus (MLV) transcripts depends on the nuclear export factor (NXF1) pathway. Although the mRNA export complex TREX, which contains Aly/REF, UAP56, and the THO complex, is involved in the NXF1-mediated nuclear export of cellular mRNAs, its contribution to the export of MLV mRNA transcripts remains poorly understood. Here, we studied the involvement of TREX components in the export of MLV transcripts. Depletion of UAP56, but not Aly/REF, reduced the level of both unspliced and spliced viral transcripts in the cytoplasm. Interestingly, depletion of THO components, including THOC5 and THOC7, affected only unspliced viral transcripts in the cytoplasm. Moreover, the RNA immunoprecipitation assay showed that only the unspliced viral transcript interacted with THOC5. These results imply that MLV requires UAP56, THOC5 and THOC7, in addition to NXF1, for nuclear export of viral transcripts. Given that naturally intronless mRNAs, but not bulk mRNAs, require THOC5 for nuclear export, it is plausible that THOC5 plays a key role in the export of unspliced MLV transcripts.

Murine leukemia virus uses NXF1 for nuclear export of spliced and unspliced viral transcripts.

UNLABELLED: Intron-containing mRNAs are subject to restricted nuclear export in higher eukaryotes. Retroviral replication requires the nucleocytoplasmic transport of both spliced and unspliced RNA transcripts, and RNA export mechanisms of gammaretroviruses are poorly characterized. Here, we report the involvement of the nuclear export receptor NXF1/TAP in the nuclear export of gammaretroviral RNA transcripts. We identified a conserved cis-acting element in the pol gene of gammaretroviruses, including murine leukemia virus (MLV) and xenotropic murine leukemia virus (XMRV), named the CAE (cytoplasmic accumulation element). The CAE enhanced the cytoplasmic accumulation of viral RNA transcripts and the expression of viral proteins without significantly affecting the stability, splicing, or translation efficiency of the transcripts. Insertion of the CAE sequence also facilitated Rev-independent HIV Gag expression. We found that the CAE sequence interacted with NXF1, whereas disruption of NXF1 ablated CAE function. Thus, the CAE sequence mediates the cytoplasmic accumulation of gammaretroviral transcripts in an NXF1-dependent manner. Disruption of NXF1 expression impaired cytoplasmic accumulations of both spliced and unspliced RNA transcripts of XMRV and MLV, resulting in their nuclear retention or degradation. Thus, our results demonstrate that gammaretroviruses use NXF1 for the cytoplasmic accumulation of both spliced and nonspliced viral RNA transcripts. IMPORTANCE: Murine leukemia virus (MLV) has been studied as one of the classic models of retrovirology. Although unspliced host messenger RNAs are rarely exported from the nucleus, MLV actively exports unspliced viral RNAs to the cytoplasm. Despite extensive studies, how MLV achieves this difficult task has remained a mystery. Here, we have studied the RNA export mechanism of MLV and found that (i) the genome contains a sequence which supports the efficient nuclear export of viral RNAs, (ii) the cellular factor NXF1 is involved in the nuclear export of both spliced and unspliced viral RNAs, and, finally, (iii) depletion of NXF1 results in nuclear retention or degradation of viral RNAs. Our study provides a novel insight into MLV nuclear export.

Global gene expression profiling of pancreatic islets in mice during streptozotocin-induced ß-cell damage and pancreatic Glp-1 gene therapy.

Streptozotocin (STZ), a glucosamine-nitrosourea compound, has potent genotoxic effects on pancreatic ß-cells and is frequently used to induce diabetes in experimental animals. Glucagon-like peptide-1 (GLP-1) has ß-cell protective effects and is known to preserve ß-cells from STZ treatment. In this study, we analyzed the mechanisms of STZ-induced diabetes and GLP-1-mediated ß-cell protection in STZ-treated mice. At 1 week after multiple low-dose STZ administrations, pancreatic ß-cells showed impaired insulin expression, while maintaining expression of nuclear Nkx6.1. This was accompanied by significant upregulation of p53-responsive genes in islets, including a mediator of cell cycle arrest, p21 (also known as Waf1 and Cip1). STZ treatment also suppressed expression of a wide range of genes linked with key ß-cell functions or diabetes development, such as G6pc2, Slc2a2 (Glut2), Slc30a8, Neurod1, Ucn3, Gad1, Isl1, Foxa2, Vdr, Pdx1, Fkbp1b and Abcc8, suggesting global ß-cell defects in STZ-treated islets. The Tmem229B, Prss53 and Ttc28 genes were highly expressed in untreated islets and strongly suppressed by STZ, suggesting their potential roles in ß-cell function. When a pancreas-targeted adeno-associated virus (AAV) vector was employed for long-term Glp-1 gene delivery, pancreatic GLP-1 expression protected mice from STZ-induced diabetes through preservation of the ß-cell mass. Despite its potent ß-cell protective effects, however, pancreatic GLP-1 overexpression showed limited effects on the global gene expression profiles in the islets. Network analysis identified the programmed-cell-death-associated pathways as the most relevant network in Glp-1 gene therapy. Upon pancreatic GLP-1 expression, upregulation of Cxcl13 and Nptx2 was observed in STZ-damaged islets, but not in untreated normal islets. Given the pro-ß-cell-survival effects of Cxcl12 (Sdf-1) in inducing GLP-1 production in α-cells, pancreatic GLP-1-mediated Cxcl13 induction might also play a crucial role in maintaining the integrity of ß-cells in damaged islets.

Long-term infection and vertical transmission of a gammaretrovirus in a foreign host species.

Increasing evidence has indicated natural transspecies transmission of gammaretroviruses; however, viral-host interactions after initial xeno-exposure remain poorly understood. Potential association of xenotropic murine leukemia virus-related virus (XMRV) in patients with prostate cancer and chronic fatigue syndrome has attracted broad interests in this topic. Although recent studies have indicated that XMRV is unlikely a human pathogen, further understanding of XMRV xenoinfection would allow in vivo modeling of the initial steps of gammaretroviral interspecies transmission, evolution and dissemination in a new host population. In this study, we monitored the long-term consequences of XMRV infection and its possible vertical transmission in a permissive foreign host, wild-derived Mus pahari mice. One year post-infection, XMRV-infected mice showed no notable pathological changes, while proviral DNA was detected in three out of eight mice. XMRV-infected mice remained seropositive throughout the study although the levels of gp70 Env- and p30 capsid-specific antibodies gradually decreased. When vertical XMRV transmission was assessed, no viremia, humoral immune responses nor endogenization were observed in nine offspring from infected mothers, yet one offspring was found PCR-positive for XMRV-specific sequences. Amplified viral sequences from the offspring showed several mutations, including one amino acid deletion in the receptor binding domain of Env SU. Our results therefore demonstrate long-term asymptomatic infection, low incidence of vertical transmission and limited evolution of XMRV upon transspecies infection of a permissive new host, Mus pahari.

Secretion of glycosylated pro-B-type natriuretic peptide from normal cardiomyocytes.

BACKGROUND: B-type natriuretic peptide (BNP), a key cardiac hormone in cardiorenal homeostasis, is produced as a 108 amino acid prohormone, proBNP1-108, which is converted to a biologically active peptide BNP1-32 and an inactive N-terminal (NT)-proBNP1-76. The widely accepted model is that the normal heart releases a proteolytically processed BNP1-32 and NT-proBNP, whereas the diseased heart secretes high amounts of unprocessed/glycosylated proBNP1-108 or inappropriately processed BNPs. In contrast, circulating proBNP1-108 has recently been identified in healthy individuals, indicating that the normal heart also secretes unprocessed proBNP1-108. However, the mechanism of proBNP1-108 secretion from the normal heart remains elusive. Our goal was to determine the molecular mechanisms underlying proBNP1-108 intracellular trafficking and secretion from the normal heart. METHODS: We expressed preproBNP in cardiomyocytes, and determined the subcellular localization and dominant intracellular and extracellular forms of BNP. RESULTS: Intracellular immunoreactive BNPs were first accumulated in the Golgi apparatus, and then distributed throughout the cytoplasm as secretory vesicles. The predominant intracellular form of BNP was nonglycosylated proBNP1-108, rather than BNP1-32. Glycosylated proBNP1-108, but not nonglycosylated proBNP1-108, was detected as the major extracellular form in the culture supernatants of preproBNP-expressing cell lines and primary human cardiomyocytes. Ablation of O-glycosylation of proBNP1-108 at T71 residue, near the convertase recognition site, reduced the extracellular proBNP1-108 and increased extracellular BNP1-32. CONCLUSIONS: Intracellular proBNP trafficking occurs through a conventional Golgi-endoplasmic reticulum pathway. Glycosylation of proBNP1-108 controls the stability and processing of extracellular proBNP1-108. Our data establish a new BNP secretion model in which the normal cardiac cells secrete glycosylated proBNP1-108.

Long-term cardiac pro-B-type natriuretic peptide gene delivery prevents the development of hypertensive heart disease in spontaneously hypertensive rats.

BACKGROUND: Diastolic dysfunction associated with high blood pressure (BP) leads to cardiac remodeling and fibrosis and progression to congestive heart failure. B-type natriuretic peptide (BNP) has BP-lowering, antifibrotic, and antihypertrophic properties, which makes BNP an attractive agent for attenuating the adverse cardiac remodeling associated with hypertension. In the current study, we tested the effects of sustained cardiac proBNP gene delivery on BP, cardiac function, and remodeling in spontaneously hypertensive rats (SHR). METHODS AND RESULTS: We used the myocardium-tropic adeno-associated virus serotype 9 (AAV9) vector to achieve continuously enhanced cardiac rat proBNP expression. In SHR, a single systemic administration of AAV9 vector allowed long-term cardiac BNP overexpression, resulting in reductions in systolic and diastolic BP for 9 months after injection. Left ventricular (LV) thickness, LV end-systolic dimensions, and LV mass were reduced, whereas ejection fraction was significantly increased, in BNP-treated compared with untreated SHR. Circumferential systolic strain and strain rate of the early phase of diastole were improved in BNP-treated compared with untreated SHR. Noncardiac overexpression of BNP via AAV2 vector was not associated with changes in BP and plasma BNP in SHR. Furthermore, normal Wistar rats injected with AAV9 proBNP vector showed significantly reduced heart weights 4 weeks after injection without BP reduction. CONCLUSIONS: AAV9 vector facilitates sustained cardiac proBNP overexpression and improves LV function in hypertensive heart disease. Long-term proBNP delivery improved both systolic and diastolic function. The effects on cardiac structure and function occurred independently of BP-lowering effects in normal Wistar rats.

No evidence of XMRV in prostate cancer cohorts in the Midwestern United States.

BACKGROUND: Xenotropic murine leukemia virus (MLV)-related virus (XMRV) was initially identified in prostate cancer (PCa) tissue, particularly in the prostatic stromal fibroblasts, of patients homozygous for the RNASEL R462Q mutation. A subsequent study reported XMRV antigens in malignant prostatic epithelium and association of XMRV infection with PCa, especially higher-grade tumors, independently of the RNASEL polymorphism. Further studies showed high prevalence of XMRV or related MLV sequences in chronic fatigue syndrome patients (CFS), while others found no, or low, prevalence of XMRV in a variety of diseases including PCa or CFS. Thus, the etiological link between XMRV and human disease remains elusive. To address the association between XMRV infection and PCa, we have tested prostate tissues and human sera for the presence of viral DNA, viral antigens and anti-XMRV antibodies. RESULTS: Real-time PCR analysis of 110 PCa (Gleason scores >4) and 40 benign and normal prostate tissues identified six positive samples (5 PCa and 1 non-PCa). No statistical link was observed between the presence of proviral DNA and PCa, PCa grades, and the RNASEL R462Q mutation. The amplified viral sequences were distantly related to XMRV, but nearly identical to endogenous MLV sequences in mice. The PCR positive samples were also positive for mouse mitochondrial DNA by nested PCR, suggesting contamination of the samples with mouse DNA. Immuno-histochemistry (IHC) with an anti-XMRV antibody, but not an anti-MLV antibody that recognizes XMRV, sporadically identified antigen-positive cells in prostatic epithelium, irrespectively of the status of viral DNA detection. No serum (159 PCa and 201 age-matched controls) showed strong neutralization of XMRV infection at 1:10 dilution. CONCLUSION: The lack of XMRV sequences or strong anti-XMRV neutralizing antibodies indicates no or very low prevalence of XMRV in our cohorts. We conclude that real-time PCR- and IHC-positive samples were due to laboratory contamination and non-specific immune reactions, respectively.