Factors predictive of leg-ulcer healing in sickle cell disease: a multicentre, prospective cohort study.

BACKGROUND: Leg ulcers (LUs) are a chronic and severe complication of sickle cell disease (SCD). A prospective study in patients with SCD to identify factors associated with complete healing and recurrence of LUs is lacking. OBJECTIVES: To determine clinical and biological factors associated with SCD-LU complete healing and recurrence. METHODS: This prospective, observational cohort study was conducted at two adult SCD referral-centre sites (2009-2015) and included 98 consecutive patients with at least one LU lasting ≥ 2 weeks. The primary end points compared patients with healed vs. nonhealed LUs at week 24, and patients with vs. without recurrence during follow-up. RESULTS: The median (interquartile range) LU area, duration and follow-up were, respectively, 6·2 cm2 (3-12·8), 9 weeks (4-26) and 65·8 weeks (23·8-122·1). At week 24, LUs were healed in 47% of patients, while 49% of LUs recurred. Univariate analyses identified inclusion LU area < 8 cm2 (82% vs. 35%; P < 0·001), inclusion LU duration < 9 weeks (65% vs. 35%; P = 0·0013) and high median fetal haemoglobin level (P = 0·008) as being significantly associated with complete healing at week 24, and low lactate dehydrogenase level (P = 0·038) as being associated with recurrence. Multivariate analyses retained LU area < 8 cm2 (odds ratio 6·73, 95% confidence interval 2·35-19. 31; P < 0·001) and < 9 weeks' duration (OR 3·19, 95% confidence interval 1·16-8·76; P = 0·024) as being independently associated with healing at week 24. Factors independently associated with recurrence could not be identified. CONCLUSIONS: SCD-LU complete healing is independently associated with the clinical characteristics of LUs rather than the clinical or biological characteristics of SCD.

Drug screening on Hutchinson Gilford progeria pluripotent stem cells reveals aminopyrimidines as new modulators of farnesylation.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder characterized by a dramatic appearance of premature aging. HGPS is due to a single-base substitution in exon 11 of the LMNA gene (c.1824C>T) leading to the production of a toxic form of the prelamin A protein called progerin. Because farnesylation process had been shown to control progerin toxicity, in this study we have developed a screening method permitting to identify new pharmacological inhibitors of farnesylation. For this, we have used the unique potential of pluripotent stem cells to have access to an unlimited and relevant biological resource and test 21,608 small molecules. This study identified several compounds, called monoaminopyrimidines, which target two key enzymes of the farnesylation process, farnesyl pyrophosphate synthase and farnesyl transferase, and rescue in vitro phenotypes associated with HGPS. Our results opens up new therapeutic possibilities for the treatment of HGPS by identifying a new family of protein farnesylation inhibitors, and which may also be applicable to cancers and diseases associated with mutations that involve farnesylated proteins.

Differentiation from human pluripotent stem cells of cortical neurons of the superficial layers amenable to psychiatric disease modeling and high-throughput drug screening.

Cortical neurons of the superficial layers (II-IV) represent a pivotal neuronal population involved in the higher cognitive functions of the human and are particularly affected by psychiatric diseases with developmental manifestations such as schizophrenia and autism. Differentiation protocols of human pluripotent stem cells (PSC) into cortical neurons have been achieved, opening the way to in vitro modeling of neuropsychiatric diseases. However, these protocols commonly result in the asynchronous production of neurons typical for the different layers of the cortex within an extended period of culture, thus precluding the analysis of specific subtypes of neurons in a standardized manner. Addressing this issue, we have successfully captured a stable population of self-renewing late cortical progenitors (LCPs) that synchronously and massively differentiate into glutamatergic cortical neurons of the upper layers. The short time course of differentiation into neurons of these progenitors has made them amenable to high-throughput assays. This has allowed us to analyze the capability of LCPs at differentiating into post mitotic neurons as well as extending and branching neurites in response to a collection of selected bioactive molecules. LCPs and cortical neurons of the upper layers were successfully produced from patient-derived-induced PSC, indicating that this system enables functional studies of individual-specific cortical neurons ex vivo for disease modeling and therapeutic purposes.

Direct myocardial implantation of human embryonic stem cells in a dog model of Duchenne cardiomyopathy reveals poor cell survival in dystrophic tissue.

Duchenne muscular dystrophy is characterized by progressive muscle weakness and early death resulting from dystrophin deficiency. Spontaneous canine muscular disorders are interesting settings to evaluate the relevance of innovative therapies in human using pre-clinical trials.

Progress and prospects: gene therapy clinical trials (part 2).

This is the second part of a review summarizing progress and prospects in gene therapy clinical research. Twenty key diseases/strategies are succinctly described and commented on by leaders in the field. This part includes clinical trials for skin diseases, neurological disorders, HIV/AIDS, ornithine transcarbamylase deficiency, alpha(1)-antitrypsin deficiency, haemophilia and cancer.

Pig xenografts to the immunocompetent rat brain: Survival rates using distinct neurotoxic lesions in the nigrostriatal pathway and two rat strains.

Porcine foetal neurons for xenotransplantation in Parkinson's disease (PD) is an alternative source to human fetuses. One of the obstacles facing brain xenotransplantation is the existence of an immune response, which prevents long-term graft survival. Experimental results concerning the survival time of porcine foetal neurons implanted into the brain of immunocompetent rats have been quite different from one study to another, suggesting an effect on graft survival of uncontrolled experimental parameters. To identify such parameters, we have first analyzed the survival of porcine foetal nigral neurons at 5 and 10 weeks after implantation into the striatum of immunocompetent rats having different types of brain lesion affecting cells (quinolinic acid) or projections to the striatum (MPP+, 6-OHDA). In a second experiment, graft survival was analyzed in two strains of recipient rats (female Sprague-Dawley and male Lewis rats) in conditions of ipsilateral dopaminergic denervation using 6-OHDA. The characteristics of surviving grafts were assessed by measuring the graft volume, the number of TH+ neurons, the size of TH+ neurons soma, and CD5+ cell infiltration. Long-term survival (> or = 10 weeks) of porcine neurons could be observed in all experimental models. However, there was no significant difference in graft survival rates and characteristics of the surviving grafts between the lesioned groups, or between Sprague-Dawley and Lewis rats. Altogether, results were highly variable within groups of grafts exposed to similar experimental procedures at both 5 and 10 weeks post-grafting. We conclude that the distinct neurotoxins and host rat strains used in our experimental design are not major factors influencing the rejection time-course of primary neural xenografts.

Neuroprotective gene therapy for Huntington's disease, using polymer-encapsulated cells engineered to secrete human ciliary neurotrophic factor: results of a phase I study.

Huntington's disease (HD) is a monogenic neurodegenerative disease that affects the efferent neurons of the striatum. The protracted evolution of the pathology over 15 to 20 years, after clinical onset in adulthood, underscores the potential of therapeutic tools that would aim at protecting striatal neurons. Proteins with neuroprotective effects in the adult brain have been identified, among them ciliary neurotrophic factor (CNTF), which protected striatal neurons in animal models of HD. Accordingly, we have carried out a phase I study evaluating the safety of intracerebral administration of this protein in subjects with HD, using a device formed by a semipermeable membrane encapsulating a BHK cell line engineered to synthesize CNTF. Six subjects with stage 1 or 2 HD had one capsule implanted into the right lateral ventricle; the capsule was retrieved and exchanged for a new one every 6 months, over a total period of 2 years. No sign of CNTF-induced toxicity was observed; however, depression occurred in three subjects after removal of the last capsule, which may have correlated with the lack of any future therapeutic option. All retrieved capsules were intact but contained variable numbers of surviving cells, and CNTF release was low in 13 of 24 cases. Improvements in electrophysiological results were observed, and were correlated with capsules releasing the largest amount of CNTF. This phase I study shows the safety, feasibility, and tolerability of this gene therapy procedure. Heterogeneous cell survival, however, stresses the need for improving the technique.

Efficient early and sustained transduction of human fetal mesencephalon using adeno-associated virus type 2 vectors.

The success of transplantation of human fetal mesencephalic tissue into the putamen of patients with Parkinson's disease (PD) is still limited by the poor survival of the graft. In animal models of fetal transplantation for PD, antiapoptotic agents, such as growth factors or caspase inhibitors, or agents counteracting oxidative stress enhance the survival and reinnervation potential of the graft. Genetic modification of the transplant could allow a local and continuous delivery of these factors at physiologically relevant doses. The major challenge remains the development of strategies to achieve both early and sustained gene delivery in the absence of vector-mediated toxicity. We recently reported that E14 rat fetal mesencephalon could be efficiently tranduced by adeno-associated virus type 2 (AAV2) vectors and that gene expression was maintained until at least 3 months after transplantation in the adult rat striatum. Here we report that an AAV2 vector can mediate the expression of the EGFP reporter gene under the control of a CMV promoter in organotypic cultures of freshly explanted solid fragments of human fetal mesencephalic tissue as early as 3 days to at least 6 weeks postinfection. These results suggest that AAV2 vectors could be used to genetically modify the human fetal tissue prior to transplantation to Parkinson's patients to promote graft survival and integration.

Correlation of in vitro infiltration with glioma histological type in organotypic brain slices.

Diffuse invasion of the brain, an intrinsic property of gliomas, renders these tumours incurable, and is a principal determinant of their spatial and temporal growth. Knowledge of the invasive potential of gliomas is highly desired in order to understand their behaviour in vivo. Comprehensive ex vivo invasion studies including tumours of different histological types and grades are however lacking, mostly because reliable physiological invasion assays have been difficult to establish. Using an organotypic rodent brain slice assay, we evaluated the invasiveness of 42 grade II-IV glioma biopsy specimens, and correlated it with the histological phenotype, the absence or presence of deletions on chromosomes 1p and 19q assessed by fluorescent in situ hybridisation, and proliferation and apoptosis indices assessed by immunocytochemistry. Oligodendroglial tumours with 1p/19q loss were less invasive than astrocytic tumours of similar tumour grade. Correlation analysis of invasiveness cell proliferation and apoptosis further suggested that grade II-III oligodendroglial tumours with 1p/19q loss grow in situ as relatively circumscribed compact masses in contrast to the more infiltrative and more diffuse astrocytomas. Lower invasiveness may be an important characteristic of oligodendroglial tumours, adding to our understanding of their more indolent clinical evolution and responsiveness to therapy.

AAV2 vectors mediate efficient and sustained transduction of rat embryonic ventral mesencephalon.

The success of transplantation of human embryonic mesencephalic tissue to treat parkinsonian patients is limited by the poor survival of the transplant. We show that an AAV2 vector mediates efficient expression of the egfp reporter gene in organotypic cultures of freshly explanted solid fragments of rat embryonic ventral mesencephalon (VM). We observed early and sustained transgene expression (4 days to > or = 6 weeks). Furthermore, rAAV-infected rat embryonic VM transplanted in the adult striatum continued to express EGFP for > or = 3 months. More than 95% of the transduced cells were neurons. Dopaminergic neurons were transduced at low frequency at earlier time points. This method of gene delivery could prove useful to achieve local, continuous secretion of neurotrophic factors at physiologically relevant doses to treat Parkinson's disease.

[10 years of substitution therapy for neurodegenerative diseases using fetal neuron grafts: a positive outcome but with questions for the future]. / Dix ans de traitement substitutif de maladies neurodégénératives par greffe de neurones foetaux: un bilan positif mais des interrogations sur l'avenir.

Fetal neural allografts have already proven their therapeutic value in several hundreds of patients with Parkinson's disease, and have very recently provided promising results for patients with Huntington's disease in our center. Fetal neurons integrate readily into the neural parenchyma of the adult hosts, differentiate into mature neurons and substitute, anatomically and functionally for lost host neurons. Notable clinical improvements have been obtained using this procedure. Nevertheless, a major obstacle hampers the development of the technique, that provoked by the logistic difficulty in retrieving and preparing the tissue. Indeed, this requires, for each surgical session, the organization of a chain of expertise which cannot be taken up by an external provider (e.g. a biotech company). This is difficult to organize outside of specialized research centers. The future of the technique relies, therefore, upon the design of alternative sources of tissue. Two different ways are currently explored very actively, namely xenografting of neurons of porcine origin and human stem cells, in particular derived from ES cells. In both cases, but in different ways, the goal of both techniques is to allow the organisation of cell banking systems, relieving the constraints of obtaining the collaboration of specialized obstetricians and biologists. Obstacles foreseen for these two alternative ways of fetal neurons to be are identified and research laboratories are actively exploring ways to overcome them.

Protection against experimental autoimmune encephalomyelitis by a proteasome modulator.

The capacity of interferon beta to alter the course of multiple sclerosis has promoted a new therapeutic concept, based upon the modulation of the immune response rather than its suppression. As the proteasome plays a crucial role in the control of the inflammatory process and immune cell survival, targeting the proteasome appears as a novel approach for the prevention and treatment of inflammatory autoimmune diseases. We have previously shown that ritonavir, an HIV-1 protease inhibitor used in AIDS therapy, can modulate the proteasome function by inhibiting the chymotrypsin-like activity and enhancing the trypsin-like activity. We have, therefore, explored its therapeutic potential on experimental autoimmune encephalomyelitis (EAE), an experimental model of multiple sclerosis, in Lewis rats and SJL mice. Daily administration of ritonavir during autoimmune antigen stimulation prevented clinical symptoms of EAE in a dose- and time-dependent manner. This protection was accompanied by an inhibition of the mononuclear cell infiltration into the central nervous system usually observed in EAE. Despite a complete absence of clinical symptoms during first EAE induction, ritonavir-treated animals became resistant to further induction of EAE, suggesting an immune mechanism of protection. These results suggest that proteasome modulation using ritonavir or analogues may be of interest for patients with multiple sclerosis.

Migration pathways of human glioblastoma cells xenografted into the immunosuppressed rat brain.

Diffuse invasion of the brain by tumor cells is a hallmark of human glioblastomas and a major cause for the poor prognosis of these tumors. This phenomenon is only partially reproduced by rodent models of gliomas that display a very high rate of proliferation and limited cell migration. We have analyzed the development of human glioblastoma cells (GL15) xenografted into the brain of immunosuppressed rats, in order to define the characteristics of tumor cell invasion. As identified by the specific immunolabeling of the tumor cells for the human HLA-ABC antigen, GL15 tumors reproduced the three types of intraparenchymal invasion observed in patients. First, a majority of multipolar tumor cells intermingled rapidly and profusely with host neural cells in the margin of the injection site. This progressively enlarging area was principally responsible for the tumor growth over time. Second, in the gray matter, columns of thin bipolar tumor cells aligned along capillary walls. Third, in the white matter, elongated bipolar isolated tumor cells were observed scattered between axonal fibers. The maximum migration distances along white matter fibers remained significantly higher than the maximum migration distances along blood vessels, up to two months after injection. Development of the tumor was associated with a significant increase of vascularization in the area of tumor spread. Xenografting of human GL15 glioblastoma cells into the immunosuppressed rat brain allowed to differentiate between the three classical types of invasion identified in the clinic, to quantify precisely the distances of migration, and to evaluate cell morphology for each of these routes. The present results support the existence of host/tumor cells interactions with specific characteristics for each type of invasion.

Shc signaling in differentiating neural progenitor cells.

Previously we found that the availability of ShcA adapter is maximal in neural stem cells but that it is absent in mature neurons. Here we report that ShcC, unlike ShcA, is not present in neural stem/progenitor cells, but is expressed after cessation of their division and becomes selectively enriched in mature neurons. Analyses of its activity in differentiating neural stem/progenitor cells revealed that ShcC positively affects their viability and neuronal maturation via recruitment of the PI3K-Akt-Bad pathway and persistent activation of the MAPK pathway. We suggest that the switch from ShcA to ShcC modifies the responsiveness of neural stem/progenitor cells to extracellular stimuli, generating proliferation (with ShcA) or survival/differentiation (with ShcC).

Retest effects and cognitive decline in longitudinal follow-up of patients with early HD.

OBJECTIVE: To assess the natural progression of cognitive impairment in Huntington's disease (HD) and to reveal factors that may mask this progression. BACKGROUND: Although numerous cross-sectional studies reported cognitive deterioration at different stages of the disease, progressive cognitive deterioration has been, up to now, difficult to demonstrate in neuropsychological longitudinal studies. METHODS: The authors assessed 22 patients in early stages of HD at yearly intervals for 2 to 4 years (average, 31.2 +/- 10 months), using a comprehensive neuropsychological battery based on the Core Assessment Program for Intracerebral Transplantation in Huntington's Disease (CAPIT-HD). RESULTS: The authors observed a significant decline in different cognitive functions over time: these involved primarily attention and executive functions but also involved language comprehension, and visuospatial immediate memory. Episodic memory impairment that was already present at the time of enrollment did not show significant decline. The authors found a significant retest effect at the second assessment in many tasks. CONCLUSION: Many attention and executive tasks adequately assess the progression of the disease at an early stage. For other functions, the overlapping of retest effects and disease progression may confuse the results. High interindividual and intraindividual variability seem to be hallmarks of the disease.

Differential microglial response to progressive neurodegeneration in the murine mutant Wobbler.

Activated microglia is associated with neurodegenerative processes, but the precise role of this cell population is difficult to identify. Most experimental models employed to examine microglial responses involve acute alterations of neuronal integrity, in contrast to the progressive nature of neurodegenerative diseases. In order to approach the clinical situation better, the microglial response was analyzed in the murine mutant Wobbler, which exhibits a well-characterized neurodegenerative pathology, manifested by motoneuronal death following a period of cellular dysfunction with characteristic morphological features. Microglial cells were identified using anti-Mac1 or anti-IgG antibodies. Examination of the changes in density, localization, and phenotype of microglia differentiated two types of responses in Wobblers. A first type of response was observed as early as in the third week after birth, when the only apparent neuronal defect was the morphological alteration of a subset of motoneurons in the cervical spinal cord, which was maintained later on. The activated microglia extended long processes that selectively ensheathed vacuolated motoneurons. At later stages, when motoneuron death became prominent, an additional type of response was characterized by an increased density of reactive microglia that was seen extending throughout the cervical enlargement. This secondary microglial response occurred in parallel to the infiltration of T-lymphocytes. Thus, these results point to a differential response of microglial cells to a progressive neurodegenerative process.