Transplantation of gene-modified haematopoietic stem cells: Application and clinical considerations.

Autologous and allogeneic haematopoietic stem cell (HSC) transplantation has been performed in patients with various malignant and non-malignant haematological disorders for more than 50 years. Ex vivo gene modification of HSCs for autologous transplantation opens up new therapeutic avenues for genetic and infectious diseases. Major advances have been made over the last three decades with respect to gene modification of HSCs and transplantation strategies, ultimately culminating in the approval of two such therapies in Europe (Strimvelis for a rare primary immune deficiency, and LentiGlobin for beta-thalassaemia). Newer gene-modifying technologies and treatment regimens have also recently come to the fore, which hold great promise for the development of safer and more effective treatments. We provide an overview of the current state of gene-modified HSC therapies, highlighting success stories, limitations and important considerations for achieving successful translation of these therapies to the clinic.

Transplantation of gene-modified haematopoietic stem cells: Application and clinical considerations

Autologous and allogeneic haematopoietic stem cell (HSC) transplantation has been performed in patients with various malignant and non-malignant haematological disorders for more than 50 years. Ex vivo gene modification of HSCs for autologous transplantation opens up new therapeutic avenues for genetic and infectious diseases. Major advances have been made over the last three decades with respect to gene modification of HSCs and transplantation strategies, ultimately culminating in the approval of two such therapies in Europe (Strimvelis for a rare primary immune deficiency, and LentiGlobin for beta-thalassaemia). Newer gene-modifying technologies and treatment regimens have also recently come to the fore, which hold great promise for the development of safer and more effective treatments. We provide an overview of the current state of gene-modified HSC therapies, highlighting success stories, limitations and important considerations for achieving successful translation of these therapies to the clinic

GROW: a model for mentorship to advance women's leadership in global health.

In this essay, we discuss the under-representation of women in leadership positions in global health (GH) and the importance of mentorship to advance women's standing in the field. We then describe the mentorship model of GROW, Global Research for Women. We describe the theoretical origins of the model and an adapted theory of change explaining how the GROW model for mentorship advances women's careers in GH. We present testimonials from a range of mentees who participated in a pilot of the GROW model since 2015. These mentees describe the capability-enhancing benefits of their mentorship experience with GROW. Thus, preliminary findings suggest that the GROW mentorship model is a promising strategy to build women's leadership in GH. We discuss supplemental strategies under consideration and next steps to assess the impact of GROW, providing the evidence to inform best practices for curricula elsewhere to build women's leadership in GH.

Decreased NOX2 expression in the brain of patients with bipolar disorder: association with valproic acid prescription and substance abuse.

Neuroinflammation and increased oxidative stress are believed to contribute to the development of psychiatric diseases. Animal studies have implicated NADPH oxidases (NOX) as relevant sources of reactive oxygen species in the brain. We have analyzed the expression of NOX isoforms in post-mortem brain samples from patients with psychiatric disorders (schizophrenia, bipolar disorder) and non-psychiatric subjects. Two collections from the Stanley Medical Research Institute were studied: the Array Collection (RNA, 35 individuals per group), and a neuropathology consortium collection (paraffin-embedded sections, 15 individuals per group). Quantitative PCR analysis revealed expression of NOX2 and NOX4 in prefrontal cortex. No impact of psychiatric disease on NOX4 levels was detected. Remarkably, the expression of NOX2 was specifically decreased in prefrontal and cingulate cortices of bipolar patients, as compared with controls and schizophrenic patients. NOX2 expression was not statistically associated with demographic parameters and post-mortem interval, but correlated with brain pH. Immunostaining demonstrated that NOX2 was predominantly expressed in microglia, which was corroborated by a decrease in the microglial markers CD68 and CD11b in the cingulate cortex of bipolar disorder patients. The analysis of potentially confounding parameters showed association of valproic acid prescription and heavy substance abuse with lower levels of NOX2. Taken together, we did not observe changes of NOX2 in schizophrenic patients, but a marked decrease of microglial markers and NOX2 in the brain of bipolar patients. This might be an underlying feature of bipolar disorder and/or a consequence of valproic acid treatment and substance abuse.

[Cell transplantation: current treatments and future prospects]. / Transplantation cellulaire: traitements actuels et perspectives d'avenir.

Regenerative medicine aims to replace a body function or specific cell loss. It includes therapies at the forefront of modem medicine, issuing from translational biomedical research. Transplantation of organs and cells has revolutionized the management of patients for whom medical treatment is a failure. Unfortunately, organ shortage is limiting treatment possibility. As an example, among the 15,000 patients with type I diabetes in Switzerland, only approximately 30 can receive a pancreas or an islet transplant per year. Second example, 500 patients die each year in Switzerland from alcoholic cirrhosis because no treatment is available. Transplantation of islet cells, hepatocytes, mesenchymal stem cells or dopaminergic neurons represents hope fora therapy available for large populations of patients.

Profiling of drugs and environmental chemicals for functional impairment of neural crest migration in a novel stem cell-based test battery.

Developmental toxicity in vitro assays have hitherto been established as stand-alone systems, based on a limited number of toxicants. Within the embryonic stem cell-based novel alternative tests project, we developed a test battery framework that allows inclusion of any developmental toxicity assay and that explores the responses of such test systems to a wide range of drug-like compounds. We selected 28 compounds, including several biologics (e.g., erythropoietin), classical pharmaceuticals (e.g., roflumilast) and also six environmental toxicants. The chemical, toxicological and clinical data of this screen library were compiled. In order to determine a non-cytotoxic concentration range, cytotoxicity data were obtained for all compounds from HEK293 cells and from murine embryonic stem cells. Moreover, an estimate of relevant exposures was provided by literature data mining. To evaluate feasibility of the suggested test framework, we selected a well-characterized assay that evaluates 'migration inhibition of neural crest cells.' Screening at the highest non-cytotoxic concentration resulted in 11 hits (e.g., geldanamycin, abiraterone, gefitinib, chlorpromazine, cyproconazole, arsenite). These were confirmed in concentration-response studies. Subsequent pharmacokinetic modeling indicated that triadimefon exerted its effects at concentrations relevant to the in vivo situation, and also interferon-ß and polybrominated diphenyl ether showed effects within the same order of magnitude of concentrations that may be reached in humans. In conclusion, the test battery framework can identify compounds that disturb processes relevant for human development and therefore may represent developmental toxicants. The open structure of the strategy allows rich information to be generated on both the underlying library, and on any contributing assay.

Reactive oxygen species: from health to disease.

Upon reaction with electrons, oxygen is transformed into reactive oxygen species (ROS). It has long been known that ROS can destroy bacteria and destroy human cells, but research in recent decades has highlighted new roles for ROS in health and disease. Indeed, while prolonged exposure to high ROS concentrations may lead to non-specific damage to proteins, lipids, and nucleic acids, low to intermediate ROS concentrations exert their effects rather through regulation of cell signalling cascades. Biological specificity is achieved through the amount, duration, and localisation of ROS production. ROS have crucial roles in normal physiological processes, such as through redox regulation of protein phosphorylation, ion channels, and transcription factors. ROS are also required for biosynthetic processes, including thyroid hormone production and crosslinking of extracellular matrix. There are multiple sources of ROS, including NADPH oxidase enzymes; similarly, there are a large number of ROS-degrading systems. ROS-related disease can be either due to a lack of ROS (e.g., chronic granulomatous disease, certain autoimmune disorders) or a surplus of ROS (e.g., cardiovascular and neurodegenerative diseases). For diseases caused by a surplus of ROS, antioxidant supplementation has proven largely ineffective in clinical studies, most probably because their action is too late, too little, and too non-specific. Specific inhibition of ROS-producing enzymes is an approach more promising of clinical efficacy.

NADPH oxidase elevations in pyramidal neurons drive psychosocial stress-induced neuropathology.

Oxidative stress is thought to be involved in the development of behavioral and histopathological alterations in animal models of psychosis. Here we investigate the causal contribution of reactive oxygen species generation by the phagocyte NADPH oxidase NOX2 to neuropathological alterations in a rat model of chronic psychosocial stress. In rats exposed to social isolation, the earliest neuropathological alterations were signs of oxidative stress and appearance of NOX2. Alterations in behavior, increase in glutamate levels and loss of parvalbumin were detectable after 4 weeks of social isolation. The expression of the NOX2 subunit p47(phox) was markedly increased in pyramidal neurons of isolated rats, but below detection threshold in GABAergic neurons, astrocytes and microglia. Rats with a loss of function mutation in the NOX2 subunit p47(phox) were protected from behavioral and neuropathological alterations induced by social isolation. To test reversibility, we applied the antioxidant/NOX inhibitor apocynin after initiation of social isolation for a time period of 3 weeks. Apocynin reversed behavioral alterations fully when applied after 4 weeks of social isolation, but only partially after 7 weeks. Our results demonstrate that social isolation induces rapid elevations of the NOX2 complex in the brain. Expression of the enzyme complex was strongest in pyramidal neurons and a loss of function mutation prevented neuropathology induced by social isolation. Finally, at least at early stages, pharmacological targeting of NOX2 activity might reverse behavioral alterations.

Deficiency in the NADPH oxidase 4 predisposes towards diet-induced obesity.

OBJECTIVE: NADPH oxidase 4 (NOX4) is a reactive oxygen species (ROS) producing NADPH oxidase that regulates redox homeostasis in diverse insulin-sensitive cell types. In particular, NOX4-derived ROS is a key modulator of adipocyte differentiation and mediates insulin receptor signaling in mature adipocytes in vitro. Our study was aimed at investigating the role of NOX4 in adipose tissue differentiation, whole body metabolic homeostasis and insulin sensitivity in vivo. DESIGN: Mice with genetic ablation of NOX4 (NOX4-deficient mice) were subjected to chow or high-fat-containing diet for 12 weeks. Body weight gain, adiposity, insulin sensitivity, and adipose tissue and liver gene and protein expression were analyzed and compared with similarly treated wild-type mice. RESULTS: Here, we report that NOX4-deficient mice display latent adipose tissue accumulation and are susceptible to diet-induced obesity and early onset insulin resistance. Obesity results from accelerated adipocyte differentiation and hypertrophy, and an increase in whole body energy efficiency. Insulin resistance is associated with increased adipose tissue hypoxia, inflammation and adipocyte apoptosis. In the liver, more severe diet-induced steatosis was observed due to the lack of proper upregulation of mitochondrial fatty acid ß-oxidation. CONCLUSION: These findings identify NOX4 as a regulator of metabolic homeostasis. Moreover, they indicate an anti-adipogenic role for NOX4 in vivo and reveal its function as a protector against the development of diet-induced obesity, insulin resistance and hepatosteatosis.

Generation and applications of human pluripotent stem cells induced into neural lineages and neural tissues.

Human pluripotent stem cells (hPSCs) represent a new and exciting field in modern medicine, now the focus of many researchers and media outlets. The hype is well-earned because of the potential of stem cells to contribute to disease modeling, drug screening, and even therapeutic approaches. In this review, we focus first on neural differentiation of these cells. In a second part we compare the various cell types available and their advantages for in vitro modeling. Then we provide a "state-of-the-art" report about two major biomedical applications: (1) the drug and toxicity screening and (2) the neural tissue replacement. Finally, we made an overview about current biomedical research using differentiated hPSCs.

Telomere length, comorbidity, functional, nutritional and cognitive status as predictors of 5 years post hospital discharge survival in the oldest old.

BACKGROUND: Telomere length has been considered in many cross-sectional studies as a biomarker of aging. However the association between shorter telomeres with lower survival at advanced ages remains a controversial issue. This association could reflect the impact of other health conditions than a direct biological effect. OBJECTIVE: To test whether leukocyte telomere length is associated with 5-year survival beyond the impact of other risk factors of mortality like comorbidity, functional, nutritional and cognitive status. DESIGN: Prospective study. SETTING AND PARTICIPANTS: A population representative sample of 444 patients (mean age 85 years; 74% female) discharged from the acute geriatric hospital of Geneva University Hospitals (January-December 2004), since then 263 (59.2%) had died (December 2009). MEASUREMENTS: Telomere length in leukocytes by flow cytometry. RESULTS: In univariate model, telomere length at baseline and cognitive status were not significantly associated with mortality even when adjusting for age (R²=9.5%) and gender (R²=1.9%). The best prognostic predictor was the geriatric index of comorbidity (GIC) (R²=8.8%; HR=3.85) followed by more dependence in instrumental (R²=5.9%; HR=3.85) and based (R²=2.3%; HR=0.84) activities of daily living and lower albumin levels (R²=1.5%; HR=0.97). Obesity (BMI>30: R²=1.6%; HR=0.55) was significantly associated with a two-fold decrease in the risk of mortality compared to BMI between 20-25. When all independent variables were entered in a full multiple Cox regression model (R²=21.4%), the GIC was the strongest risk predictor followed by the nutritional and functional variables. CONCLUSION: Neither telomeres length nor the presence of dementia are predictors of survival whereas the weight of multiple comorbidity conditions, nutritional and functional impairment are significantly associated with 5-year mortality in the oldest old.

Diabetes, comorbidities and increased long-term mortality in older patients admitted for geriatric inpatient care.

AIMS: To study the specific impact of diabetes on long-term mortality in very old subjects with multiple comorbidities and functional disabilities. METHODS: The prevalence of vascular disorders, global comorbidity load (cumulative illness rating scale [CIRS]) and functional disabilities (activities of daily living [ADL] and Lawton's instrumental ADL [IADL] scores) were determined according to diabetes status in a cohort of 444 patients (mean age 85.3±6.7 years; 74.0% women) admitted to our geriatric service. Also, the specific impact of diabetes on 4-year mortality was analyzed using Cox proportional-hazards models. RESULTS: Diabetic patients had higher BMI scores (27.1±4.9 vs. 23.4±4.7 kg/m(2) in controls; P<0.001), and higher prevalences of hypertension (81.9% vs. 65.1%, respectively; P=0.003) and ischaemic heart disease (33.7% vs. 22.2%, respectively; P=0.033), but not of stroke and renal insufficiency. They also had more comorbidities (CIRS score excluding diabetes: 15.1±4.5 vs. 13.8±4.8, respectively; P=0.016) and functional disabilities. Diabetes was associated with mortality (HR: 1.42, 95% CI: 1.02-1.99; P=0.041) after adjusting for age, gender and BMI, and this persisted after adjusting for individual vascular comorbidities, but disappeared after adjusting for CIRS, ADL or IADL scores. CONCLUSION: Diabetes was associated with 4-year mortality after adjusting for the inverse relationship between mortality and BMI. This association was better accounted for by the global comorbidity load and functional disabilities than by the individual vascular comorbidities. These findings suggest that the active management of all--rather than selected--comorbidities is the key to improving the prognosis for older diabetic patients.

The miR 302-367 cluster drastically affects self-renewal and infiltration properties of glioma-initiating cells through CXCR4 repression and consequent disruption of the SHH-GLI-NANOG network.

Glioblastoma multiforme (GBM) is the most common form of primary brain tumor in adults, often characterized by poor survival. Glioma-initiating cells (GiCs) are defined by their extensive self-renewal, differentiation, and tumor initiation properties. GiCs are known to be involved in tumor growth and recurrence, and in resistance to conventional treatments. One strategy to efficiently target GiCs in GBM consists in suppressing their stemness and consequently their tumorigenic properties. In this study, we show that the miR-302-367 cluster is strongly induced during serum-mediated stemness suppression. Stable miR-302-367 cluster expression is sufficient to suppress the stemness signature, self-renewal, and cell infiltration within a host brain tissue, through inhibition of the CXCR4 pathway. Furthermore, inhibition of CXCR4 leads to the disruption of the sonic hedgehog (SHH)-GLI-NANOG network, which is involved in self-renewal and expression of the embryonic stem cell-like signature. In conclusion, we demonstrated that the miR-302-367 cluster is able to efficiently trigger a cascade of inhibitory events leading to the disruption of GiCs stem-like and tumorigenic properties.

Primate-specific RFPL1 gene controls cell-cycle progression through cyclin B1/Cdc2 degradation.

Ret finger protein-like 1 (RFPL1) is a primate-specific target gene of Pax6, a key transcription factor for pancreas, eye and neocortex development. However, its cellular activity remains elusive. In this article, we report that Pax6-elicited expression of the human (h)RFPL1 gene in HeLa cells can be enhanced by in vivo p53 binding to its promoter and therefore investigated the hypothesis that hRFPL1 regulates cell-cycle progression. Upon expression in these cells, hRFPL1 decreased cell number through a kinase-dependent mechanism as PKC activates and Cdc2 inhibits hRFPL1 activity. hRFPL1 antiproliferative activity led to an increased cell population in G(2)/M phase and specific cyclin B1 and Cdc2 downregulations, which were precluded by a proteasome inhibitor. Specifically, cytoplasm-localized hRFPL1 prevented cyclin B1 and Cdc2 accumulation during interphase. Consequently, cells showed a delayed entry into mitosis and cell-cycle lengthening resulting from a threefold increase in G(2) phase duration. Given previous reports that RFPL1 is expressed during cell differentiation, its impact on cell-cycle lengthening therefore provides novel insights into primate-specific development.

Increased brain damage after ischaemic stroke in mice lacking the chemokine receptor CCR5.

BACKGROUND AND PURPOSE: The chemokine receptor CCR5 is well known for its function in immune cells; however, it is also expressed in the brain, where its specific role remains to be elucidated. Because genetic factors may influence the risk of developing cerebral ischaemia or affect its clinical outcome, we have analysed the role of CCR5 in experimental stroke. EXPERIMENTAL APPROACH: Permanent cerebral ischaemia was performed by occlusion of the middle cerebral artery in wild-type and CCR5-deficient mice. Locomotor behaviour, infarct size and histochemical alterations were analysed at different time points after occlusion. KEY RESULTS: The cerebral vasculature was comparable in wild-type and CCR5-deficient mice. However, the size of the infarct and the motor deficits after occlusion were markedly increased in CCR5-deficient mice as compared with wild type. No differences between wild-type and CCR5-deficient mice were elicited by occlusion with respect to the morphology and abundance of astrocytes and microglia. Seven days after occlusion the majority of CCR5-deficient mice displayed neutrophil invasion in the infarct region, which was not observed in wild type. As compared with wild type, the infarct regions of CCR5-deficient mice were characterized by increased neuronal death. CONCLUSIONS AND IMPLICATIONS: Lack of CCR5 increased the severity of brain injury following occlusion of the middle cerebral artery. This is of particular interest with respect to the relatively frequent occurrence of CCR5 deficiency in the human population (1-2% of the Caucasian population) and the advent of CCR5 inhibitors as novel drugs.

Neural commitment of embryonic stem cells: molecules, pathways and potential for cell therapy.

The study of neuronal differentiation of embryonic stem cells has raised major interest over recent years. It allows a better understanding of fundamental aspects of neurogenesis and, at the same time, the generation of neurons as tools for various applications ranging from drug testing to cell therapy and regenerative medicine. Since the first report of human embryonic stem (ES) cells derivation, many studies have shown the possibility of directing their differentiation towards neurons. However, there are still many challenges ahead, including gaining a better understanding of the mechanisms involved and developing techniques to allow the generation of homogeneous neuronal and glial subtypes. We review the current state of knowledge of embryonic neurogenesis which has been acquired from animal models and discuss its translation into in vitro strategies of neuronal differentiation of ES cells. We also highlight several aspects of current protocols which need to be optimized to generate high-quality embryonic stem cell-derived neuronal precursors suitable for clinical applications. Finally, we discuss the potential of embryonic stem cell-derived neurons for cell replacement therapy in several central nervous system diseases.

Branched fungal beta-glucan causes hyperinflammation and necrosis in phagocyte NADPH oxidase-deficient mice.

Chronic granulomatous disease (CGD), a genetic disorder characterized by the absence of a functional phagocyte NADPH oxidase, is a severe immune deficiency. However, non-infectious hyperinflammation is a second hallmark of the disease. In CGD mouse models, sterile hyperinflammation can be induced by A. fumigatus cell wall preparations. In this study, we used subcutaneous injection of microbial cell walls and cell wall components to identify causes of CGD hyperinflammation and to characterize its histological features. Sterile cell wall preparations from fungi (A. fumigatus, C. albicans, S. cerevisiae), but not from bacteria (S. aureus, P. aeruginosa, E. coli), caused prolonged and severe skin inflammation in CGD mice. To identify fungal cell wall elements responsible for this process, we investigated microbial cell wall-derived monosubstances. Injection of beta(1-3)(1-6)-glucan induced severe hyperinflammation in CGD mice, while other fungal cell components [mannan, (1-3) beta-glucan] or bacterial cell wall components (lipopolysaccharide, lipoteichoic acid) caused no or only moderate inflammation. beta-glucan-induced hyperinflammation was predominantly due to a defect in termination of inflammation, as in the initial stage (2 days), the severity of inflammation and the extent of cell death were comparable in wild-type and CGD mice. At later stages (7 days), beta(1-3)(1-6)-glucan-induced inflammation had subsided in wild-type mice. In contrast, CGD mice showed persistent severe inflammation with central necrosis, containing abundant apoptotic and necrotic cells. In summary, branched fungal beta-glucan induces a severe inflammatory reaction in the absence of phagocyte NADPH oxidase. As opposed to the commonly perceived notion that reactive oxygen species are the cause of cell death, our results demonstrate that tissue necrosis can be caused by the absence of a superoxide-producing enzyme.

Scapuloperoneal syndrome type Kaeser and a wide phenotypic spectrum of adult-onset, dominant myopathies are associated with the desmin mutation R350P.

In 1965, an adult-onset, autosomal dominant disorder with a peculiar scapuloperoneal distribution of weakness and atrophy was described in a large, multi-generation kindred and named 'scapuloperoneal syndrome type Kaeser' (OMIM #181400). By genetic analysis of the original kindred, we discovered a heterozygous missense mutation of the desmin gene (R350P) cosegregating with the disorder. Moreover, we detected DES R350P in four unrelated German families allowing for genotype-phenotype correlations in a total of 15 patients carrying the same mutation. Large clinical variability was recognized, even within the same family, ranging from scapuloperoneal (n = 2, 12%), limb girdle (n = 10, 60%) and distal phenotypes (n = 3, 18%) with variable cardiac (n = 7, 41%) or respiratory involvement (n = 7, 41%). Facial weakness, dysphagia and gynaecomastia were frequent additional symptoms. Overall and within each family, affected men seemingly bear a higher risk of sudden, cardiac death as compared to affected women. Moreover, histological and immunohistochemical examination of muscle biopsy specimens revealed a wide spectrum of findings ranging from near normal or unspecific pathology to typical, myofibrillar changes with accumulation of desmin. This study reveals that the clinical and pathological variability generally observed in desminopathies may not be attributed to the nature of the DES mutation alone, but may be influenced by additional genetic and epigenetic factors such as gender. In addition, mutations of the desmin gene should be considered early in the diagnostic work-up of any adult-onset, dominant myopathy, even if specific myofibrillar pathology is absent.

Microcebus murinus: a useful primate model for human cerebral aging and Alzheimer's disease?

Age-associated dementia, in particular Alzheimer's disease (AD), will be a major concern of the 21st century. Research into normal brain aging and AD will therefore become increasingly important. As for other areas of medicine, the availability of good animal models will be a limiting factor for progress. Given the complexity of the human brain, the identification of appropriate primate models will be essential to further knowledge of the disease. In this review, we describe the features of brain aging and age-associated neurodegeneration in a small lemurian primate, the Microcebus murinus, also referred to as the mouse lemur. The mouse lemur has a relatively short life expectancy, and animals over 5 years of age are considered to be elderly. Among elderly mouse lemurs, the majority show normal brain aging, whereas approximately 20% develop neurodegeneration. This Microcebus age-associated neurodegeneration is characterized by a massive brain atrophy, abundant amyloid plaques, a cytoskeletal Tau pathology and a loss of cholinergic neurons. While elderly mouse lemurs with normal brain aging maintain memory function and social interaction, animals with age-associated neurodegeneration lose their cognitive and social capacities and demonstrate certain similarities with age-associated human AD. We conclude that M. murinus is an interesting primate model for the study of normal brain aging and the biochemical dysfunctions occurring in age-associated neurodegeneration. Mouse lemurs might also become an increasingly important model for the development of novel treatments in this domain.