REST suppression mediates neural conversion of adult human fibroblasts via microRNA-dependent and -independent pathways.

Direct conversion of human fibroblasts into mature and functional neurons, termed induced neurons (iNs), was achieved for the first time 6 years ago. This technology offers a promising shortcut for obtaining patient- and disease-specific neurons for disease modeling, drug screening, and other biomedical applications. However, fibroblasts from adult donors do not reprogram as easily as fetal donors, and no current reprogramming approach is sufficiently efficient to allow the use of this technology using patient-derived material for large-scale applications. Here, we investigate the difference in reprogramming requirements between fetal and adult human fibroblasts and identify REST as a major reprogramming barrier in adult fibroblasts. Via functional experiments where we overexpress and knockdown the REST-controlled neuron-specific microRNAs miR-9 and miR-124, we show that the effect of REST inhibition is only partially mediated via microRNA up-regulation. Transcriptional analysis confirmed that REST knockdown activates an overlapping subset of neuronal genes as microRNA overexpression and also a distinct set of neuronal genes that are not activated via microRNA overexpression. Based on this, we developed an optimized one-step method to efficiently reprogram dermal fibroblasts from elderly individuals using a single-vector system and demonstrate that it is possible to obtain iNs of high yield and purity from aged individuals with a range of familial and sporadic neurodegenerative disorders including Parkinson's, Huntington's, as well as Alzheimer's disease

CD8+ T cells from a novel T cell receptor transgenic mouse induce liver-stage immunity that can be boosted by blood-stage infection in rodent malaria

To follow the fate of CD8+ T cells responsive to Plasmodium berghei ANKA (PbA) infection, we generated an MHC I-restricted TCR transgenic mouse line against this pathogen. T cells from this line, termed PbT-I T cells, were able to respond to blood-stage infection by PbA and two other rodent malaria species, P. yoelii XNL and P. chabaudi AS. These PbT-I T cells were also able to respond to sporozoites and to protect mice from liver-stage infection. Examination of the requirements for priming after intravenous administration of irradiated sporozoites, an effective vaccination approach, showed that the spleen rather than the liver was the main site of priming and that responses depended on CD8α+ dendritic cells. Importantly, sequential exposure to irradiated sporozoites followed two days later by blood-stage infection led to augmented PbT-I T cell expansion. These findings indicate that PbT-I T cells are a highly versatile tool for studying multiple stages and species of rodent malaria and suggest that cross-stage reactive CD8+ T cells may be utilized in liver-stage vaccine design to enable boosting by blood-stage infections

Effects of hospital facilities on patient outcomes after cancer surgery: an international, prospective, observational study

Background Early death after cancer surgery is higher in low-income and middle-income countries (LMICs) compared with in high-income countries, yet the impact of facility characteristics on early postoperative outcomes is unknown. The aim of this study was to examine the association between hospital infrastructure, resource availability, and processes on early outcomes after cancer surgery worldwide.Methods A multimethods analysis was performed as part of the GlobalSurg 3 study-a multicentre, international, prospective cohort study of patients who had surgery for breast, colorectal, or gastric cancer. The primary outcomes were 30-day mortality and 30-day major complication rates. Potentially beneficial hospital facilities were identified by variable selection to select those associated with 30-day mortality. Adjusted outcomes were determined using generalised estimating equations to account for patient characteristics and country-income group, with population stratification by hospital.Findings Between April 1, 2018, and April 23, 2019, facility-level data were collected for 9685 patients across 238 hospitals in 66 countries (91 hospitals in 20 high-income countries; 57 hospitals in 19 upper-middle-income countries; and 90 hospitals in 27 low-income to lower-middle-income countries). The availability of five hospital facilities was inversely associated with mortality: ultrasound, CT scanner, critical care unit, opioid analgesia, and oncologist. After adjustment for case-mix and country income group, hospitals with three or fewer of these facilities (62 hospitals, 1294 patients) had higher mortality compared with those with four or five (adjusted odds ratio [OR] 3.85 [95% CI 2.58-5.75]; p<0.0001), with excess mortality predominantly explained by a limited capacity to rescue following the development of major complications (63.0% vs 82.7%; OR 0.35 [0.23-0.53]; p<0.0001). Across LMICs, improvements in hospital facilities would prevent one to three deaths for every 100 patients undergoing surgery for cancer.Interpretation Hospitals with higher levels of infrastructure and resources have better outcomes after cancer surgery, independent of country income. Without urgent strengthening of hospital infrastructure and resources, the reductions in cancer-associated mortality associated with improved access will not be realised

Investigating the role of CD8+ T cells in malaria pathogenesis

© 2011 Dr. Lei Shong LauMalaria is an infectious disease caused by infection with Plasmodium parasites transmitted by female Anopheles mosquitoes. Over 800,000 people die from malaria annually and a significant proportion is represented by young children living in malaria endemic regions. A major cause of death in this population is cerebral malaria (CM), which occurs during the blood-stage of Plasmodium falciparum infection. The mechanism underlying CM pathogenesis is not well understood, although it is thought that the sequestration of parasites to the brain is essential, and aberrant immune responses might also contribute to pathology. Murine experimental cerebral malaria (ECM) associated with Plasmodium berghei infection is thought to recapitulate many features of human CM, and thus has been used widely to examine the mechanism underlying CM pathogenesis. Studies using the ECM model have revealed that many cell types of the immune system play positive and/or negative roles in ECM pathogenesis, CD8+ T cells being a critical effector of disease. A major question relating to the role of CD8+ T cells in ECM is whether they mediate ECM in an antigen-dependent manner. My thesis aimed to address this question. The lack of knowledge of specific immunogenic antigens of blood-stage Plasmodium parasites has posted a significant challenge in addressing this issue. To overcome this barrier, my research has focused on developing approaches to investigate antigen-specific CD8+ T cell responses, generating antigen-specific reagents and identifying specific immunogenic antigens derived from the blood-stage of PbA parasite. Using several surrogate approaches to examine parasite-specific CD8+ T cell responses, my studies revealed an extensive induction of parasite-specific CD8+ T cells with a range of specificities to this pathogen, and we successfully identified several epitopes using an MHC I prediction algorithm (chapters 3 and 6). We have generated CD8+ T cell hybridomas from parasite-specific CD8+ T cells, which enabled us to investigate the role of dendritic cells in the cross-presentation of authentic parasite antigens, and to generate a parasite-specific CD8+ T cell receptor transgenic mouse model, termed PbT-I (chapter 5). Studies using PbT-I transgenic cells demonstrated that antigen-specific CD8+ T cell responses induced during the blood-stage of PbA infection contributed to ECM pathogenesis. PbT-I cells were cross-reactive with PbA sporozoites and Plasmodium chabaudi, suggesting that the antigen of interest is expressed by other stages and species of Plasmodium. Collectively, our studies provided compelling evidence that CD8+ T cells specific for a broad range of authentic blood-stage PbA antigens are induced during the blood-stage of PbA infection. The new reagents generated within this study will help further clarify the role of antigen-specific CD8+ T cells in malaria immunity and immuno-pathology associated with infection with different species of Plasmodium

Direct Neural Conversion from Human Fibroblasts Using Self-Regulating and Nonintegrating Viral Vectors

Summary: Recent findings show that human fibroblasts can be directly programmed into functional neurons without passing via a proliferative stem cell intermediate. These findings open up the possibility of generating subtype-specific neurons of human origin for therapeutic use from fetal cell, from patients themselves, or from matched donors. In this study, we present an improved system for direct neural conversion of human fibroblasts. The neural reprogramming genes are regulated by the neuron-specific microRNA, miR-124, such that each cell turns off expression of the reprogramming genes once the cell has reached a stable neuronal fate. The regulated system can be combined with integrase-deficient vectors, providing a nonintegrative and self-regulated conversion system that rids problems associated with the integration of viral transgenes into the host genome. These modifications make the system suitable for clinical use and therefore represent a major step forward in the development of induced neurons for cell therapy. : Lau et al. now use miRNA targeting to build a self-regulating neural conversion system. Combined with nonintegrating vectors, this system can efficiently drive conversion of human fibroblasts into functional induced neurons (iNs) suitable for clinical applications

In Vivo Reprogramming of Striatal NG2 Glia into Functional Neurons that Integrate into Local Host Circuitry

The possibility of directly converting non-neuronal cells into neurons in situ in the brain would open therapeutic avenues aimed at repairing the brain after injury or degenerative disease. We have developed an adeno-associated virus (AAV)-based reporter system that allows selective GFP labeling of reprogrammed neurons. In this system, GFP is turned on only in reprogrammed neurons where it is stable and maintained for long time periods, allowing for histological and functional characterization of mature neurons. When combined with a modified rabies virus-based trans-synaptic tracing methodology, the system allows mapping of 3D circuitry integration into local and distal brain regions and shows that the newly reprogrammed neurons are integrated into host brain