Lymphocyte reconstitution following autologous stem cell transplantation for progressive MS

BACKGROUND: Autologous stem cell transplantation (ASCT) for progressive multiple sclerosis (MS) may reset the immune repertoire. OBJECTIVE: The objective of this paper is to analyse lymphocyte recovery in patients with progressive MS treated with ASCT. METHODS: Patients with progressive MS not responding to conventional treatment underwent ASCT following conditioning with high-dose cyclophosphamide and antithymocyte globulin. Lymphocyte subset analysis was performed before ASCT and for two years following ASCT. Neurological function was assessed by the EDSS before ASCT and for three years post-ASCT. RESULTS: CD4+ T-cells fell significantly post-transplant and did not return to baseline levels. Recent thymic emigrants and naïve T-cells fell sharply post-transplant but returned to baseline by nine months and twelve months, respectively. T-regulatory cells declined post-transplant and did not return to baseline levels. Th1 and Th2 cells did not change significantly while Th17 cells fell post-transplant but recovered to baseline by six months. Neurological function remained stable in the majority of patients. Progression-free survival was 69% at three years. CONCLUSION: This study demonstrates major changes in the composition of lymphocyte subsets following ASCT for progressive MS. In particular, ablation and subsequent recovery of thymic output is consistent with the concept that ASCT can reset the immune repertoire in MS patients

Deoxyribonucleic acid methylation profiling of single human blastocysts by methylated CpG-island amplification coupled with CpG-island microarray

Objective To study whether methylated CpG-island (CGI) amplification coupled with microarray (MCAM) can be used to generate DNA (deoxyribonucleic acid) methylation profiles from single human blastocysts. Design A pilot microarray study with methylated CpG-island amplification applied to human blastocyst genomic DNA and hybridized on CpG-island microarrays. Setting University research laboratory. Patient(s) Five cryopreserved sibling 2-pronuclear zygotes that were surplus to requirements for clinical treatment by in vitro fertilization were donated with informed consent from a patient attending Bourn Hall Clinic, Cambridge, United Kingdom. Intervention(s) None. Main Outcome Measure(s) Successful generation of genome-wide DNA methylation profiles at CpG islands from individual human blastocysts, with common genomic regions of DNA methylation identified between embryos. Result(s) Between 472 and 734 CpG islands were methylated in each blastocyst, with 121 CpG islands being commonly methylated in all 5 blastocysts. A further 159 CGIs were commonly methylated in 4 of the 5 tested blastocysts. Methylation was observed at a number of CGIs within imprinted-gene, differentially methylated regions (DMRs), including placental and preimplantation-specific DMRs. Conclusion(s) The MCAM method is capable of providing comprehensive DNA methylation data in individual human blastocysts

In vitro models of medulloblastoma: choosing the right tool for the job

The recently-defined four molecular subgroups of medulloblastoma have required updating of our understanding of in vitro models to include molecular classification and risk stratification features from clinical practice. This review seeks to build a more comprehensive picture of the in vitro systems available for modelling medulloblastoma. The subtype classification and molecular characterisation for over 40 medulloblastoma cell-lines has been compiled, making it possible to identify the strengths and weaknesses in current model systems. Less than half (18/44) of established medulloblastoma cell-lines have been subgrouped. The majority of the subgrouped cell-lines (11/18) are Group 3 with MYC-amplification. SHH cell-lines are the next most common (4/18), half of which exhibit TP53 mutation. WNT and Group 4 subgroups, accounting for 50% of patients, remain underrepresented with 1 and 2 cell-lines respectively. In vitro modelling relies not only on incorporating appropriate tumour cells, but also on using systems with the relevant tissue architecture and phenotype as well as normal tissues. Novel ways of improving the clinical relevance of in vitro models are reviewed, focusing on 3D cell culture, extracellular matrix, co-cultures with normal cells and organotypic slices. This paper champions the establishment of a collaborative online-database and linked cell-bank to catalyse preclinical medulloblastoma research

The role of MDM2-p53 axis in pathogenesis of embryonal brain tumors

During nervous system development, neural stem cells (NSCs) and their derivative progenitor cells undergo a proliferative expansion prior to differentiation and migration to their final destination. Aberrations in this tightly controlled process that shift the balance from differentiation toward proliferation is implicated in the pathogenesis of embryonal brain tumors including medulloblastoma (MB), the most common malignant brain tumor in children. Despite advances in current therapeutic strategies, young patients with embryonal brain tumors retain life-long post-treatment complications due to lack of treatment specificity and toxicity to developing nervous system. A better tumor-targeted therapy for embryonal brain tumors is critically needed. Activation of the p53 tumor suppressor in embryonal brain tumor might have therapeutic potentials as these tumors rarely carry p53 mutation. Our laboratory has previously demonstrated that MDM2, a key negative regulator of p53, is required for MB tumorigensis. Accordingly, reduction of the MDM2 level induces apoptosis in a subset of cerebellar granular neuronal progenitors (GNPs) in a p53-dependent manner. Our previous findings further suggested that GNPs lacking MDM2 are also prone to premature differentiation. In current study, we elucidated the inhibitory role of MDM2 on p53 in regulation of apoptosis and differentiation of premature neuronal cells. Given that MB tumors contain undifferentiated neuronal cells, first, the biological outcome of MDM2 inhibition by small molecule, nutlin-3 was studied in a panel of human MB cells. Nutlin-3 appears to release p53 inhibition from MDM2 in human MB cells harboring wild-type TP53 and thereby, induces p53 target genes leading to apoptosis and/or cell cycle arrest. Nutlin-3 potentiates anti-proliferative effects of doxorubicin inTP53 wild-type MB cells through enhancement of p53 function. In contrast, TP53 mutant MB cell lines are highly resistant to nutlin-3 treatment. However, at high concentrations, nutlin-3 could induce apoptosis in TP53 mutant MB cell suggesting that nutlin-3 may also be effective in regression of TP53 mutant MB tumors through an alternative mechanism. Next, to gain a better insight in mechanism(s) by which MDM2 determines cellular fate toward differentiation or apoptosis, regulatory role of MDM2 in neuronal differentiation was elucidated in anin vitro model employing the human embryonal teratocarcinoma NT2/D1 cell line. NT2/D1 cells can differentiate to mature neurons in response to retinoic acid (RA). Induction of neuronal differentiation in these cells resulted in activation of p53 concomitant with a decrease in the level of MDM2. Accordingly, inhibition of MDM2 by nutlin-3 promoted differentiation of NT2/D1 to mature neurons as shown by up-regulation of neuronal markers, revealing that release of p53 inhibition from MDM2 promotes neuronal differentiation. The reduction of MDM2 in differentiating NT2/D1 cells was at the protein level and mediated by proteasomal degradation. In keeping with the importance of ubiquitination as an obligatory step for proteasomal degradation of proteins, a change in the levels of several enzymes that regulate MDM2 stability via ubiquitin-mediated mechanisms was observed in differentiating NT2/D1 cells. In these cells, MDM2 deubiquitinating enzymes, USP2a and HAUSP were down-regulated while the ubiquitinating enzyme, PCAF was up-regulated. Together, my findings suggest that proteasomal degradation of MDM2 and the subsequent activation of p53 may play a role in promoting the neuronal differentiation. This further provides the new insights regarding the potential molecular mechanism that may provoke prodifferentiation function of MDM2-p53 axis. Overall, my findings identify MDM2 as a potential target to suppress the growth of TP53 wild-type embryonic brain tumors through the induction of either differentiation or cell death

Streptococcal mAb10F5 interacts with synaptic vesicles due to antiphospholipod activity

Hypermetabolism, observed in Sydenham's chorea (SC); a complication of acute rheumatoid fever (ARF) involving binding of streptococcal M protein antibodies in the brain, may result from an increase in glutamate release. The interaction of mAb 1 OF5, a specific M protein antibody subtype, with brain proteins (e.g. Rabphilin-3A), synaptic vesicles (SVs) and synaptosomal fraction (SF) was examined. Rat brain slices immunostained with mAb l OF5 revealed an interaction with choroid plexus and elements appearing to be neuropils. Dot blotting demonstrated an interaction of mAb I OF5 with both SVs and SFs. Western blotting revealed a smear from mAb 10F5 against the SF fraction. However, both modified SVs and pure liposomes examined by fluorescent and confocal microscopy bound mAbl0F5 suggesting a direct interaction with phospholipids. ELISA demonstrated binding of mAb1OF5 with negatively charged phospholipids involved in antiphospholipid syndrome (APS). Hypermetabolism and binding at the choroid plexus is observed in SC and APS supporting the connection between these disorders.Thesis (M.S.)Department of Physiology and Health Scienc

Junctional complexes in the early mammalian embryo

Preimplantation embryos generate intercellular junctions during differentiation of the trophectoderm epithelium and the formation of the blastocyst. These membrane complexes comprise gap junctions, adherens junctions, tight junctions, and desmosomes, each performing fundamental roles in cellular communication, adhesion, and differentiation. The mouse embryo has been used as a model for the biogenesis of cell junctions. Their construction is achieved by temporally regulated gene expression programs. Mechanisms of junction membrane assembly include the timing of transcription, translation, and posttranslational modifications of specific junctional proteins. Human embryos exhibit similar expression programs, and defects in these programs may contribute to reduced embryo viability

In vitro characterization of the a-thalassemia point mutation HBA2:c.95+1G>A [IVS-I-1(G>A) (a2)]

The a-thalassemias are a group of disorders occurring as a result of decreased synthesis of a-globin chains, most commonly due to deletions of a-globin genes. Detection of a-thalassemia (a-thal) caused by point mutations has increased during the past few years and more than 70 different point mutations have been reported for the a1- and a2-globin genes. The mutation at the splice donor site of the first intervening sequence [IVS-I-1 (G>A)] of the a2-globin gene, HBA2:c.95+1G>A, is thought to cause a thalassemic phenotype by interfering with and preventing the normal splicing of pre-mRNA. We developed an in vitro expression system to study a-globin gene point mutations at the molecular and cellular levels. The expression vector carrying the HBA2:c.95+1G>A mutation (a2G IVS-I-1G>A) was created using site-directed mutagenesis of a wild type (WT) construct of the a2-globin gene (a2G 2034WT). Gene expression experiments in human bladder carcinoma 5637 cells were carried out using sequence verified WT and mutated clones. Complementary DNA synthesis and polymerase chain reaction (PCR) analysis showed normal a2-globin transcripts from cells transfected with the WT vector, but aberrant transcripts from cells transfected with the mutated vector carrying the splice donor site mutation. In the presence of the G>A mutation, normal splicing does not occur, and a cryptic splice site 49 bp upstream of the normal site is used. The translation of this product produces a premature termination codon, thus resulting in a thalassemic phenotype. © 2012 Informa Healthcare USA, Inc

Advancing towards a tissue-engineered tympanic membrane: Silk fibroin as a substratum for growing human eardrum keratinocytes

Human tympanic membrane cells (hTMCs), harvested from tympanic membrane (TM) explants, were grown in culture and then seeded on membranes prepared from silkworm (Bombyx mori) silk fibroin (BMSF) and on tissue-culture plastic membranes (PET). Fibroin was isolated from silk cast into membranes with a thickness of 10-15 μm. The hTMCs were cultured on both materials for 15 days in a serum-containing culture medium. The cells grown on both substrata were subjected to nuclear staining (DAPI) and counted. Further, the cultures were immunostained for a number of protein markers related to the epithelial/keratinocyte phenotype and cell adhesion complexes. The BMSF membranes supported levels of hTMC growth higher than that observed on the PET membranes. The immunofluorochemical analysis indicated unequivocally that BMSF is a more suitable substratum than PET with respect to the growth patterns, proliferation, and cell-cell contact and adhesion. BMSF appear as a promising substratum in the tissue-engineered constructs for the replacement of TM in case of nonhealing perforations