Chronic viral infection alters PD-1 locus subnuclear localization in cytotoxic CD8+ T cells.

During chronic infection, virus-specific CD8+ cytotoxic T lymphocytes (CTLs) progressively lose their ability to mount effective antiviral responses. This "exhaustion" is coupled to persistent upregulation of inhibitory receptor programmed death-1 (PD-1) (Pdcd1)-key in suppressing antiviral CTL responses. Here, we investigate allelic Pdcd1 subnuclear localization and transcription during acute and chronic lymphocytic choriomeningitis virus (LCMV) infection in mice. Pdcd1 alleles dissociate from transcriptionally repressive chromatin domains (lamin B) in virus-specific exhausted CTLs but not in naive or effector CTLs. Relative to naive CTLs, nuclear positioning and Pdcd1-lamina dissociation in exhausted CTLs reflect loss of Pdcd1 promoter methylation and greater PD-1 upregulation, although a direct correlation is not observed in effector cells, 8 days post-infection. Genetic deletion of B lymphocyte-induced maturation protein 1 (Blimp-1) enhances Pdcd1-lamina dissociation in effector CTLs, suggesting that Blimp-1 contributes to maintaining Pdcd1 localization to repressive lamina. Our results identify mechanisms governing Pdcd1 subnuclear localization and the broader role of chromatin dynamics in T cell exhaustion.

Web-Based Cognitive Remediation Improves Supported Employment Outcomes in Severe Mental Illness: Randomized Controlled Trial.

BACKGROUND: Finding work is a top priority for most people; however, this goal remains out of reach for the majority of individuals with a severe mental illness (SMI) who remain on benefits or are unemployed. Supported employment (SE) programs aimed at returning people with a severe mental illness to work are successful; however, they still leave a significant number of people with severe mental illness unemployed. Cognitive deficits are commonly found in SMI and are a powerful predictor of poor outcome. Fortunately, these deficits are amenable to treatment with cognitive remediation therapy (CRT) that significantly improves cognition in SMI. CRT combined with SE significantly increases the likelihood of individuals with severe mental illness obtaining and staying in work. However, the availability of CRT is limited in many settings. OBJECTIVE: The aim of this study was to examine whether Web-based CRT combined with a SE program can improve the rate return to work of people with severe mental illness. METHODS: A total of 86 people with severe mental illness (mean age 39.6 years; male: n=55) who were unemployed and who had joined a SE program were randomized to either a Web-based CRT program (CogRem) or an Internet-based control condition (WebInfo). Primary outcome measured was hours worked over 6 months post treatment. RESULTS: At 6 months, those participants randomized to CogRem had worked significantly more hours (P=.01) and had earned significantly more money (P=.03) than those participants randomized to the WebInfo control condition. No change was observed in cognition. CONCLUSIONS: This study corroborates other work that has found a synergistic effect of combining CRT with a SE program and extends this to the use of Web-based CRT. The lack of any improvement in cognition obscures the mechanism by which an improved wage outcome for participants randomized to the active treatment was achieved. However, the study substantially lowers the barrier to the deployment of CRT with other psychosocial interventions for severe mental illness. TRIAL REGISTRATION: Australian and New Zealand Clinical Trials Registry (ANZCTR) 12611000849998; http://www.anzctr.org.au/TrialSearch.aspx?searchTxt=12611000849998&isBasic=True (Archived by WebCite at http://www.webcitation.org/6sMKwpeos).

Enrichment of human embryonic stem cell-derived NKX6.1-expressing pancreatic progenitor cells accelerates the maturation of insulin-secreting cells in vivo.

Human embryonic stem cells (hESCs) are considered a potential alternative to cadaveric islets as a source of transplantable cells for treating patients with diabetes. We previously described a differentiation protocol to generate pancreatic progenitor cells from hESCs, composed of mainly pancreatic endoderm (PDX1/NKX6.1-positive), endocrine precursors (NKX2.2/synaptophysin-positive, hormone/NKX6.1-negative), and polyhormonal cells (insulin/glucagon-positive, NKX6.1-negative). However, the relative contributions of NKX6.1-negative versus NKX6.1-positive cell fractions to the maturation of functional ß-cells remained unclear. To address this question, we generated two distinct pancreatic progenitor cell populations using modified differentiation protocols. Prior to transplant, both populations contained a high proportion of PDX1-expressing cells (~85%-90%) but were distinguished by their relatively high (~80%) or low (~25%) expression of NKX6.1. NKX6.1-high and NKX6.1-low progenitor populations were transplanted subcutaneously within macroencapsulation devices into diabetic mice. Mice transplanted with NKX6.1-low cells remained hyperglycemic throughout the 5-month post-transplant period whereas diabetes was reversed in NKX6.1-high recipients within 3 months. Fasting human C-peptide levels were similar between groups throughout the study, but only NKX6.1-high grafts displayed robust meal-, glucose- and arginine-responsive insulin secretion as early as 3 months post-transplant. NKX6.1-low recipients displayed elevated fasting glucagon levels. Theracyte devices from both groups contained almost exclusively pancreatic endocrine tissue, but NKX6.1-high grafts contained a greater proportion of insulin-positive and somatostatin-positive cells, whereas NKX6.1-low grafts contained mainly glucagon-expressing cells. Insulin-positive cells in NKX6.1-high, but not NKX6.1-low grafts expressed nuclear MAFA. Collectively, this study demonstrates that a pancreatic endoderm-enriched population can mature into highly functional ß-cells with only a minor contribution from the endocrine subpopulation.

Maturation and function of human embryonic stem cell-derived pancreatic progenitors in macroencapsulation devices following transplant into mice.

AIMS/HYPOTHESIS: Islet transplantation is a promising cell therapy for patients with diabetes, but it is currently limited by the reliance upon cadaveric donor tissue. We previously demonstrated that human embryonic stem cell (hESC)-derived pancreatic progenitor cells matured under the kidney capsule in a mouse model of diabetes into glucose-responsive insulin-secreting cells capable of reversing diabetes. However, the formation of cells resembling bone and cartilage was a major limitation of that study. Therefore, we developed an improved differentiation protocol that aimed to prevent the formation of off-target mesoderm tissue following transplantation. We also examined how variation within the complex host environment influenced the development of pancreatic progenitors in vivo. METHODS: The hESCs were differentiated for 14 days into pancreatic progenitor cells and transplanted either under the kidney capsule or within Theracyte (TheraCyte, Laguna Hills, CA, USA) devices into diabetic mice. RESULTS: Our revised differentiation protocol successfully eliminated the formation of non-endodermal cell populations in 99% of transplanted mice and generated grafts containing >80% endocrine cells. Progenitor cells developed efficiently into pancreatic endocrine tissue within macroencapsulation devices, despite lacking direct contact with the host environment, and reversed diabetes within 3 months. The preparation of cell aggregates pre-transplant was critical for the formation of insulin-producing cells in vivo and endocrine cell development was accelerated within a diabetic host environment compared with healthy mice. Neither insulin nor exendin-4 therapy post-transplant affected the maturation of macroencapsulated cells. CONCLUSIONS/INTERPRETATION: Efficient differentiation of hESC-derived pancreatic endocrine cells can occur in a macroencapsulation device, yielding glucose-responsive insulin-producing cells capable of reversing diabetes.

Maturation of human embryonic stem cell-derived pancreatic progenitors into functional islets capable of treating pre-existing diabetes in mice.

Diabetes is a chronic debilitating disease that results from insufficient production of insulin from pancreatic ß-cells. Islet cell replacement can effectively treat diabetes but is currently severely limited by the reliance upon cadaveric donor tissue. We have developed a protocol to efficiently differentiate commercially available human embryonic stem cells (hESCs) in vitro into a highly enriched PDX1+ pancreatic progenitor cell population that further develops in vivo to mature pancreatic endocrine cells. Immature pancreatic precursor cells were transplanted into immunodeficient mice with streptozotocin-induced diabetes, and glycemia was initially controlled with exogenous insulin. As graft-derived insulin levels increased over time, diabetic mice were weaned from exogenous insulin and human C-peptide secretion was eventually regulated by meal and glucose challenges. Similar differentiation of pancreatic precursor cells was observed after transplant in immunodeficient rats. Throughout the in vivo maturation period hESC-derived endocrine cells exhibited gene and protein expression profiles that were remarkably similar to the developing human fetal pancreas. Our findings support the feasibility of using differentiated hESCs as an alternative to cadaveric islets for treating patients with diabetes.

Characterization of platelet-derived growth factor-C (PDGF-C): expression in normal and tumor cells, biological activity and chromosomal localization.

The predicted platelet-derived growth factor-C (PDGF-C) polypeptide contains an N-terminal CUB-like domain and a C-terminal domain with homology to members of the PDGF/vascular endothelial growth factor (VEGF) family. PDGF-C mRNA is widely expressed in normal tissues and does not appear to be up-regulated in the tumor cell lines tested. The PDGF-C gene was mapped to human chromosome 4q31-32. PDGF-C protein and the CUB domain of PDGF-C expressed in Escherichia coli, were able to stimulate proliferation of human artery smooth muscle cells, but were inactive on umbilical vein endothelial cells, osteoblasts, fibroblasts, skeletal muscle cells (SkMC), bovine chondrocytes, and rat myocardium cells. Although the mitogenic activity of PDGF-C and the CUB domain was only observed at concentrations ranging from 1 to 10 microg/ml, substitution of Cys(124) by Ser or deletion of Cys(124) significantly reduced the mitogenic activity. Our data suggest a possible role of the CUB domain of PDGF-C in addition to its role in maintaining latency of the PDGF domain.