Gene therapy for Wiskott-Aldrich syndrome in a severely affected adult

Until recently, hematopoietic stem cell transplantation was the only curative option for Wiskott-Aldrich syndrome (WAS). The first attempts at gene therapy for WAS using a \u3d2-retroviral vector improved immunological parameters substantially but were complicated by acute leukemia as a result of insertional mutagenesis in a high proportion of patients. More recently, treatment of children with a state-of-the-art self-inactivating lentiviral vector (LV-w1.6 WASp) has resulted in significant clinical benefit without inducing selection of clones harboring integrations near oncogenes. Here, we describe a case of a presplenectomized 30-year-old patient with severe WAS manifesting as cutaneous vasculitis, inflammatory arthropathy, intermittent polyclonal lymphoproliferation, and significant chronic kidney disease and requiring long-term immunosuppressive treatment. Following reduced-intensity conditioning, there was rapid engraftment and expansion of a polyclonal pool of transgene-positive functional T cells and sustained gene marking in myeloid and B-cell lineages up to 20 months of observation. The patient was able to discontinue immunosuppression and exogenous immunoglobulin support, with improvement in vasculitic disease and proin-flammatory markers. Autologous gene therapy using a lentiviral vector is a viable strategy for adult WAS patients with severe chronic disease complications and for whom an allogeneic procedure could present an unacceptable risk. This trial was registered at www.clinicaltrials.gov as #NCT01347242

PPAR<i>γ</i> agonist-loaded PLGA -PEG nanocarriers as a potential treatment for Alzheimer’s disease: in vitro and in vivo studies

Objective: The first aim of this study was to develop a nanocarrier that could transport the peroxisome proliferator-activated receptor agonist, pioglitazone (PGZ) across brain endothelium and examine the mechanism of nanoparticle transcytosis. The second aim was to determine whether these nanocarriers could successfully treat a mouse model of Alzheimer’s disease (AD). Methods: PGZ-loaded nanoparticles (PGZ-NPs) were synthesized by the solvent displacement technique, following a factorial design using poly (lactic-co-glycolic acid) polyethylene glycol (PLGA-PEG). The transport of the carriers was assessed in vitro, using a human brain endothelial cell line, cytotoxicity assays, fluorescence-tagged nanocarriers, fluorescence-activated cell sorting, confocal and transmission electron microscopy. The effectiveness of the treatment was assessed in APP/PS1 mice in a behavioral assay and by measuring the cortical deposition of β-amyloid. Results: Incorporation of PGZ into the carriers promoted a 50x greater uptake into brain endothelium compared with the free drug and the carriers showed a delayed release profile of PGZ in vitro. In the doses used, the nanocarriers were not toxic for the endothelial cells, nor did they alter the permeability of the blood–brain barrier model. Electron microscopy indicated that the nanocarriers were transported from the apical to the basal surface of the endothelium by vesicular transcytosis. An efficacy test carried out in APP/PS1 transgenic mice showed a reduction of memory deficit in mice chronically treated with PGZ-NPs. Deposition of β-amyloid in the cerebral cortex, measured by immunohistochemistry and image analysis, was correspondingly reduced. Conclusion: PLGA-PEG nanocarriers cross brain endothelium by transcytosis and can be loaded with a pharmaceutical agent to effectively treat a mouse model of AD

Endothelial-Derived Extracellular Vesicles Induce Cerebrovascular Dysfunction in Inflammation

Blood–brain barrier (BBB) dysfunction is a key hallmark in the pathology of many neuroinflammatory disorders. Extracellular vesicles (EVs) are lipid membrane-enclosed carriers of molecular cargo that are involved in cell-to-cell communication. Circulating endothelial EVs are increased in the plasma of patients with neurological disorders, and immune cell-derived EVs are known to modulate cerebrovascular functions. However, little is known about whether brain endothelial cell (BEC)-derived EVs themselves contribute to BBB dysfunction. Human cerebral microvascular cells (hCMEC/D3) were treated with TNFα and IFNy, and the EVs were isolated and characterised. The effect of EVs on BBB transendothelial resistance (TEER) and leukocyte adhesion in hCMEC/D3 cells was measured by electric substrate cell-substrate impedance sensing and the flow-based T-cell adhesion assay. EV-induced molecular changes in recipient hCMEC/D3 cells were analysed by RT-qPCR and Western blotting. A stimulation of naïve hCMEC/D3 cells with small EVs (sEVs) reduced the TEER and increased the shear-resistant T-cell adhesion. The levels of microRNA-155, VCAM1 and ICAM1 were increased in sEV-treated hCMEC/D3 cells. Blocking the expression of VCAM1, but not of ICAM1, prevented sEV-mediated T-cell adhesion to brain endothelia. These results suggest that sEVs derived from inflamed BECs promote cerebrovascular dysfunction. These findings may provide new insights into the mechanisms involving neuroinflammatory disorders

The rs10993994 risk allele for prostate cancer results in clinically relevant changes in microseminoprotein-beta expression in tissue and urine

Microseminoprotein-beta (MSMB) regulates apoptosis and using genome-wide association studies the rs10993994 single nucleotide polymorphism in the MSMB promoter has been linked to an increased risk of developing prostate cancer. The promoter location of the risk allele, and its ability to reduce promoter activity, suggested that the rs10993994 risk allele could result in lowered MSMB in benign tissue leading to increased prostate cancer risk

Bi-allelic Variants in the GPI Transamidase Subunit PIGK Cause a Neurodevelopmental Syndrome with Hypotonia, Cerebellar Atrophy, and Epilepsy

Glycosylphosphatidylinositol (GPI)-anchored proteins are critical for embryogenesis, neurogenesis, and cell signaling. Variants in several genes participating in GPI biosynthesis and processing lead to decreased cell surface presence of GPI-anchored proteins (GPI-APs) and cause inherited GPI deficiency disorders (IGDs). In this report, we describe 12 individuals from nine unrelated families with 10 different bi-allelic PIGK variants. PIGK encodes a component of the GPI transamidase complex, which attaches the GPI anchor to proteins. Clinical features found in most individuals include global developmental delay and/or intellectual disability, hypotonia, cerebellar ataxia, cerebellar atrophy, and facial dysmorphisms. The majority of the individuals have epilepsy. Two individuals have slightly decreased levels of serum alkaline phosphatase, while eight do not. Flow cytometric analysis of blood and fibroblasts from affected individuals showed decreased cell surface presence of GPI-APs. The overexpression of wild-type (WT) PIGK in fibroblasts rescued the levels of cell surface GPI-APs. In a knockout cell line, transfection with WT PIGK also rescued the GPI-AP levels, but transfection with the two tested mutant variants did not. Our study not only expands the clinical and known genetic spectrum of IGDs, but it also expands the genetic differential diagnosis for cerebellar atrophy. Given the fact that cerebellar atrophy is seen in other IGDs, flow cytometry for GPI-APs should be considered in the work-ups of individuals presenting this feature

Lewis Base Mediated β-Elimination and Lewis Acid Mediated Insertion Reactions of Disilazido Zirconium Compounds

The reactivity of a series of disilazido zirconocene complexes is dominated by the migration of anionic groups (hydrogen, alkyl, halide, OTf) between the zirconium and silicon centers. The direction of these migrations is controlled by the addition of two-electron donors (Lewis bases) or two-electron acceptors (Lewis acids). The cationic nonclassical [Cp2ZrN(SiHMe2)2]+ ([2]+) is prepared from Cp2Zr{N(SiHMe2)2}H (1) and B(C6F5)3 or [Ph3C][B(C6F5)4], while reactions of B(C6F5)3 and Cp2Zr{N(SiHMe2)2}R (R = Me (3), Et (5), n-C3H7 (7), CH═CHSiMe3 (9)) provide a mixture of [2]+ and [Cp2ZrN(SiHMe2)(SiRMe2)]+. The latter products are formed through B(C6F5)3 abstraction of a β-H and R group migration from Zr to the β-Si center. Related β-hydrogen abstraction and X group migration reactions are observed for Cp2Zr{N(SiHMe2)2}X (X = OTf (11), Cl (13), OMe (15), O-i-C3H7 (16)). Alternatively, addition of DMAP (DMAP = 4-(dimethylamino)pyridine) to [2]+ results in coordination to a Si center and hydrogen migration to zirconium, giving the cationic complex [Cp2Zr{N(SiHMe2)(SiMe2DMAP)}H]+ ([19]+). Related hydrogen migration occurs from [Cp2ZrN(SiHMe2)(SiMe2OCHMe2)]+ ([18]+) to give [Cp2Zr{N(SiMe2DMAP)(SiMe2OCHMe2)}H]+ ([22]+), whereas X group migration is observed upon addition of DMAP to [Cp2ZrN(SiHMe2)(SiMe2X)]+ (X = OTf ([12]+), Cl ([14]+)) to give [Cp2Zr{N(SiHMe2)(SiMe2DMAP)}X]+ (X = OTf ([26]+), Cl ([20]+)). The species involved in these transformations are described by resonance structures that suggest β-elimination. Notably, such pathways are previously unknown in early metal amide chemistry. Finally, these migrations facilitate direct Si–H addition to carbonyls, which is proposed to occur through a pathway that previously had been reserved for later transition metal compounds

Molecular definition of group 1 innate lymphoid cells in the mouse uterus

Determining the function of uterine lymphocytes is challenging because of the rapidly changing nature of the organ in response to sex hormones and, during pregnancy, to the invading fetal trophoblast cells. Here we provide the first genome-wide transcriptome atlas of mouse uterine group 1 innate lymphoid cells (g1 ILCs) at mid-gestation. The composition of g1 ILCs fluctuates throughout reproductive life, with Eomes-veCD49a+ ILC1s dominating before puberty and specifically expanding in second pregnancies, when the expression of CXCR6, a marker of memory cells, is upregulated. Tissue-resident Eomes+CD49a+ NK cells (trNK), which resemble human uterine NK cells, are most abundant during early pregnancy, and showcase gene signatures of responsiveness to TGF-β, connections with trophoblast, epithelial, endothelial and smooth muscle cells, leucocytes, as well as extracellular matrix. Unexpectedly, trNK cells express genes involved in anaerobic glycolysis, lipid metabolism, iron transport, protein ubiquitination, and recognition of microbial molecular patterns. Conventional NK cells expand late in gestation and may engage in crosstalk with trNK cells involving IL-18 and IFN-γ. These results identify trNK cells as the cellular hub of uterine g1 ILCs at mid-gestation and mark CXCR6+ ILC1s as potential memory cells of pregnancy.This work was funded by a Wellcome Trust Investigator Award 200841/Z/16/Z, the Centre for Trophoblast Research (CTR), and the Cambridge NIHR BRC Cell Phenotyping Hub to FC, the Associazione Italiana Ricerca per la Ricerca sul Cancro (AIRC) - Special Project 5x1000 no. 9962, AIRC IG 2017 Id.19920 and AIRC 2014 Id. 15283 to LM, and Ministero della Salute RF-2013, GR-2013-02356568 to PV. IF was funded by a CTR PhD fellowship

Transcriptional Profiling of Human Brain Endothelial Cells Reveals Key Properties Crucial for Predictive In Vitro Blood-Brain Barrier Models

Brain microvascular endothelial cells (BEC) constitute the blood-brain barrier (BBB) which forms a dynamic interface between the blood and the central nervous system (CNS). This highly specialized interface restricts paracellular diffusion of fluids and solutes including chemicals, toxins and drugs from entering the brain. In this study we compared the transcriptome profiles of the human immortalized brain endothelial cell line hCMEC/D3 and human primary BEC. We identified transcriptional differences in immune response genes which are directly related to the immortalization procedure of the hCMEC/D3 cells. Interestingly, astrocytic co-culturing reduced cell adhesion and migration molecules in both BECs, which possibly could be related to regulation of immune surveillance of the CNS controlled by astrocytic cells within the neurovascular unit. By matching the transcriptome data from these two cell lines with published transcriptional data from freshly isolated mouse BECs, we discovered striking differences that could explain some of the limitations of using cultured BECs to study BBB properties. Key protein classes such as tight junction proteins, transporters and cell surface receptors show differing expression profiles. For example, the claudin-5, occludin and JAM2 expression is dramatically reduced in the two human BEC lines, which likely explains their low transcellular electric resistance and paracellular leakiness. In addition, the human BEC lines express low levels of unique brain endothelial transporters such as Glut1 and Pgp. Cell surface receptors such as LRP1, RAGE and the insulin receptor that are involved in receptor-mediated transport are also expressed at very low levels. Taken together, these data illustrate that BECs lose their unique protein expression pattern outside of their native environment and display a more generic endothelial cell phenotype. A collection of key genes that seems to be highly regulated by the local surroundings of BEC within the neurovascular unit are presented and discussed

Influence of firm size on the competencies required to management engineers in the Jordanian telecommunications sector

This is an Accepted Manuscript of an article published by Taylor & Francis in European Journal of Engineering Education on [13 jun 2016], available online:http://www.tandfonline.com/ doi/abs/10.1080/03043797.2016.1197890.[EN] The objective of this study is to identify the competencies required to achieve success in the transition from higher education to the labour market based on the perceptions of employers. This paper analyses the assessments made by a group of engineering company employers. An item-battery of 20 competencies was grouped into 3 dimensions by using factor analysis. Subsequently, respondents scores were also clustered into three groups and characterised through contingency tables. The competencies demanded by employers were grouped into business and finance, problem-solving and strategic planning. 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