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BACKGROUND: Protein 4.2 deficiency caused by mutations in the EPB42 gene results in hereditary spherocytosis with characteristic alterations of CD47,CD44 and RhAG. We decided to investigate at which stage of erythropoiesis these hallmarks of protein 4.2 deficiency arise in a novel protein 4.2 patient and whether they cause disruption to the band 3 macrocomplex. DESIGN AND METHODS: We used immunoprecipitations and detergent extractability to assess the strength of protein associations within the band 3 macrocomplex and with the cytoskeleton in erythrocytes. Patient erythroblasts were cultured from peripheral blood mononuclear cells to study the effects of protein 4.2 deficiency during erythropoiesis. RESULTS: We report a patient with two novel mutations in EPB42 resulting in complete protein 4.2 deficiency. Immunoprecipitations revealed a weakened ankyrin-1-band 3 interaction in erythrocytes resulting in increased band 3 detergent extractability. CD44 abundance and its association with the cytoskeleton were increased. Erythroblast differentiation revealed that protein 4.2 and band 3 appear simultaneously and associate early in differentiation. Protein 4.2 deficiency results in lower CD47,higher CD44 expression and increased RhAG glycosylation starting from the basophilic stage. The normal downregulation of CD44 expression was not seen during protein 4.2(-) erythroblast differentiation. Knockdown of CD47 did not increase CD44 expression,arguing against a direct reciprocal relationship. CONCLUSIONS: We have established that the characteristic changes caused by protein 4.2 deficiency occur early during erythropoiesis. We postulate that weakening of the ankyrin-1-band 3 association during protein 4.2 deficiency is compensated,in part,by increased CD44-cytoskeleton binding. View Publication

Following activation through the TCR,CD4+ T cells can differentiate into three major subsets: Th1,Th2,and Th17 cells. IL-17-secreting Th17 cells play an important role in the pathogenesis of several autoimmune diseases and in immune responses to pathogens,but little is known about the regulation of apoptosis in Th17 cells. In this study,the sensitivity of in vitro-polarized Th1,Th2,and Th17 cells to Fas-mediated apoptosis was compared directly by different methods. The order of sensitivity of T cell subsets to Fas-mediated apoptosis is: Th1 textgreater Th17 textgreater Th2. The greater sensitivity of Th17 cells to Fas-mediated apoptosis compared with Th2 cells correlated with their higher expression of FasL and comparable expression of the antiapoptotic molecule FLIP. The decreased sensitivity of Th17 compared with Th1 cells correlated with the higher expression of FLIP by Th17 cells. Transgenic overexpression of FLIP in T cells protected all three subsets from Fas-mediated apoptosis. These findings provide new knowledge for understanding how survival of different subsets of T cells is regulated. View Publication

GCS-100 is a galectin-3 antagonist with an acceptable human safety profile that has been demonstrated to have an antimyeloma effect in the context of bortezomib resistance. In the present study,the mechanisms of action of GCS-100 are elucidated in myeloma cell lines and primary tumor cells. GCS-100 induced inhibition of proliferation,accumulation of cells in sub-G(1) and G(1) phases,and apoptosis with activation of both caspase-8 and -9 pathways. Dose- and time-dependent decreases in MCL-1 and BCL-X(L) levels also occurred,accompanied by a rapid induction of NOXA protein,whereas BCL-2,BAX,BAK,BIM,BAD,BID,and PUMA remained unchanged. The cell-cycle inhibitor p21(Cip1) was up-regulated by GCS-100,whereas the procycling proteins CYCLIN E2,CYCLIN D2,and CDK6 were all reduced. Reduction in signal transduction was associated with lower levels of activated IkappaBalpha,IkappaB kinase,and AKT as well as lack of IkappaBalpha and AKT activation after appropriate cytokine stimulation (insulin-like growth factor-1,tumor necrosis factor-alpha). Primary myeloma cells showed a direct reduction in proliferation and viability. These data demonstrate that the novel therapeutic molecule,GCS-100,is a potent modifier of myeloma cell biology targeting apoptosis,cell cycle,and intracellular signaling and has potential for myeloma therapy. View Publication

We report here that butyrate,a naturally occurring fatty acid commonly used as a nutritional supplement and differentiation agent,greatly enhances the efficiency of induced pluripotent stem (iPS) cell derivation from human adult or fetal fibroblasts. After transient butyrate treatment,the iPS cell derivation efficiency is enhanced by 15- to 51-fold using either retroviral or piggyBac transposon vectors expressing 4 to 5 reprogramming genes. Butyrate stimulation is more remarkable (textgreater100- to 200-fold) on reprogramming in the absence of either KLF4 or MYC transgene. Butyrate treatment did not negatively affect properties of iPS cell lines established by either 3 or 4 retroviral vectors or a single piggyBac DNA transposon vector. These characterized iPS cell lines,including those derived from an adult patient with sickle cell disease by either the piggyBac or retroviral vectors,show normal karyotypes and pluripotency. To gain insights into the underlying mechanisms of butyrate stimulation,we conducted genome-wide gene expression and promoter DNA methylation microarrays and other epigenetic analyses on established iPS cells and cells from intermediate stages of the reprogramming process. By days 6 to 12 during reprogramming,butyrate treatment enhanced histone H3 acetylation,promoter DNA demethylation,and the expression of endogenous pluripotency-associated genes,including DPPA2,whose overexpression partially substitutes for butyrate stimulation. Thus,butyrate as a cell permeable small molecule provides a simple tool to further investigate molecular mechanisms of cellular reprogramming. Moreover,butyrate stimulation provides an efficient method for reprogramming various human adult somatic cells,including cells from patients that are more refractory to reprogramming. View Publication

HIV causes a chronic infection characterized by depletion of CD4(+) T lymphocytes and the development of opportunistic infections. Despite drugs that inhibit viral spread,HIV infection has been difficult to cure because of uncharacterized reservoirs of infected cells that are resistant to highly active antiretroviral therapy (HAART) and the immune response. Here we used CD34(+) cells from infected people as well as in vitro studies of wild-type HIV to show infection and killing of CD34(+) multipotent hematopoietic progenitor cells (HPCs). In some HPCs,we detected latent infection that stably persisted in cell culture until viral gene expression was activated by differentiation factors. A unique reporter HIV that directly detects latently infected cells in vitro confirmed the presence of distinct populations of active and latently infected HPCs. These findings have major implications for understanding HIV bone marrow pathology and the mechanisms by which HIV causes persistent infection. View Publication

UNLABELLED: Human stem cells from adult sources have been shown to contribute to the regeneration of muscle,liver,heart,and vasculature. The mechanisms by which this is accomplished are,however,still not well understood. We tested the engraftment and regenerative potential of human umbilical cord blood-derived ALDH(hi)Lin(-),and ALDH(lo)Lin(-) cells following transplantation to NOD/SCID or NOD/SCID beta2m null mice with experimentally induced acute myocardial infarction. We used combined nanoparticle labeling and whole organ fluorescent imaging to detect human cells in multiple organs 48 hours post transplantation. Engraftment and regenerative effects of cell treatment were assessed four weeks post transplantation. We found that ALDH(hi)Lin(-) stem cells specifically located to the site of injury 48 hours post transplantation and engrafted the infarcted heart at higher frequencies than ALDH(lo)Lin(-) committed progenitor cells four weeks post transplantation. We found no donor derived cardiomyocytes and few endothelial cells of donor origin. Cell treatment was not associated with any detectable functional improvement at the four week endpoint. There was,however,a significant increase in vascular density in the central infarct zone of ALDH(hi)Lin(-) cell-treated mice,as compared to PBS and ALDH(lo)Lin(-) cell-treated mice. CONCLUSIONS: Our data indicate that adult human stem cells do not become a significant part of the regenerating tissue,but rapidly home to and persist only temporarily at the site of hypoxic injury to exert trophic effects on tissue repair thereby enhancing vascular recovery. View Publication

Pilocytic astrocytoma is commonly viewed as a benign lesion. However,disease onset is most prevalent in the first two decades of life,and children are often left with residual or recurrent disease and significant morbidity. The Hedgehog (Hh) pathway regulates the growth of higher WHO grade gliomas,and in this study,we have evaluated the activation and operational status of this regulatory pathway in pilocytic astrocytomas. Expression levels of the Hh pathway transcriptional target PTCH were elevated in 45% of tumor specimens analyzed (ages 1-22 years) and correlated inversely with patient age. Evaluation of a tissue array revealed oligodendroglioma-like features,pilomyxoid features,infiltration,and necrosis more commonly in specimens from younger patients (below the median patient age of 10 years). Immunohistochemical staining for the Hh pathway components PTCH and GLI1 and the proliferation marker Ki67 demonstrated that patients diagnosed before the age of 10 had higher staining indices than those diagnosed after the age of 10. A significant correlation between Ki67 and PTCH and GLI1 staining indices was measured,and 86% of Ki67-positive cells also expressed PTCH. The operational status of the Hh pathway was confirmed in primary cell culture and could be modulated in a manner consistent with a ligand-dependent mechanism. Taken together,these findings suggest that Hh pathway activation is common in pediatric pilocytic astrocytomas and may be associated with younger age at diagnosis and tumor growth. View Publication

Lentiviruses are capable of infecting many cells irrespective of their cycling status,stably inserting DNA copies of the viral RNA genomes into host chromosomes. This property has led to the development of lentiviral vectors for high-efficiency gene transfer to a wide variety of cell types,from slowly proliferating hematopoietic stem cells to terminally differentiated neurons. Regardless of their advantage over gammaretroviral vectors,which can only introduce transgenes into target cells that are actively dividing,lentiviral vectors are still susceptible to chromosomal position effects that result in transgene silencing or variegated expression. In this chapter,various genetic regulatory elements are described that can be incorporated within lentiviral vector backbones to minimize the influences of neighboring chromatin on single-copy transgene expression. The modifications include utilization of strong internal enhancer-promoter sequences,addition of scaffold/matrix attachment regions,and flanking the transcriptional unit with chromatin domain insulators. Protocols are provided to evaluate the performance as well as the relative biosafety of lentiviral vectors containing these elements. View Publication

A subset of cells,tentatively called cancer stem cells (CSCs),in breast cancer have been associated with tumor initiation,drug resistance,and tumor persistence or aggressiveness. They are characterized by CD44 positivity,CD24 negativity,and/or ALDH1 positivity in flow cytometric studies. We hypothesized that the frequency or density of these cells may be associated with more aggressive tumor behavior. We borrowed these multiplexed,flow-based methods to develop an in situ method to define CSCs in formalin-fixed paraffin-embedded breast cancer tissue,with the goal of assessing the prognostic value of the presence of CSCs in breast cancer. Using a retrospective collection of 321 node-negative and 318 node-positive patients with a mean follow-up time of 12.6 years,we assessed TMAs using the AQUA method for quantitative immunofluorescence. Using a multiplexed assay for ALDH1,CD44,and cytokeratin to measure the coexpression of these proteins,putative CSCs appear in variable sized clusters and in 27 cases (of 490),which showed significantly worse outcome (log rank P = 0.0003). Multivariate analysis showed that this marker combination is independent of tumor size,histological grade,nodal status,ER-,PR,- and HER2-status. In this cohort,ALDH1 expression alone does not significantly predict outcome. We conclude that the multiplexed method of in situ identification of putative CSCs identifies high risk patients in breast cancer. View Publication

Complement receptor 2-negative (CR2/CD21(-)) B cells have been found enriched in patients with autoimmune diseases and in common variable immunodeficiency (CVID) patients who are prone to autoimmunity. However,the physiology of CD21(-/lo) B cells remains poorly characterized. We found that some rheumatoid arthritis (RA) patients also display an increased frequency of CD21(-/lo) B cells in their blood. A majority of CD21(-/lo) B cells from RA and CVID patients expressed germline autoreactive antibodies,which recognized nuclear and cytoplasmic structures. In addition,these B cells were unable to induce calcium flux,become activated,or proliferate in response to B-cell receptor and/or CD40 triggering,suggesting that these autoreactive B cells may be anergic. Moreover,gene array analyses of CD21(-/lo) B cells revealed molecules specifically expressed in these B cells and that are likely to induce their unresponsive stage. Thus,CD21(-/lo) B cells contain mostly autoreactive unresponsive clones,which express a specific set of molecules that may represent new biomarkers to identify anergic B cells in humans. View Publication

INTRODUCTION: Various synthetic bone-graft substitutes are used commercially as osteoconductive scaffolds in the treatment of bone defects and fractures. The role of bone-graft substitutes is changing from osteoconductive conduits for growth to an delivery system for biologic fracture treatments. Achieving optimal bone regeneration requires biologics (e.g. MSC) and using the correct scaffold incorporated into a local environment for bone regeneration. The need for an unlimited supply with high quality bone-graft substitutes continue to find alternatives for bone replacement surgery. MATERIALS AND METHODS: This in vitro study investigates cell seeding efficiency,metabolism,gene expression and growth behaviour of MSC sown on six commercially clinical available bone-graft substitutes in order to define their biological properties: synthetic silicate-substituted porous hydroxyapatite (Actifuse ABX),synthetic alpha-TCP (Biobase),synthetic beta-TCP (Vitoss),synthetic beta-TCP (Chronos),processed human cancellous allograft (Tutoplast) and processed bovines hydroxyapatite ceramic (Cerabone). 250,000 MSC derived from human bone marrow (n=4) were seeded onto the scaffolds,respectively. On days 2,6 and 10 the adherence of MSC (fluorescence microscopy) and cellular activity (MTT assay) were analysed. Osteogenic gene expression (cbfa-1) was analysed by RT-PCR and scanning electron microscopy was performed. RESULTS: The highest number of adhering cells was found on Tutoplast (e.g. day 6: 110.0+/-24.0 cells/microscopic field; ptextless0.05) followed by Chronos (47.5+/-19.5,ptextless0.05),Actifuse ABX (19.1+/-4.4),Biobase (15.7+/-9.9),Vitoss (8.8+/-8.7) and Cerabone (8.1+/-2.2). MSC seeded onto Tutoplast showed highest metabolic activity and gene expression of cbfa-1. These data are confirmed by scanning electron microscopy. The cell shapes varied from round-shaped cells to wide spread cells and cell clusters,depending on the bone-graft substitutes. Processed human cancellous allograft is a well-structured and biocompatible scaffold for ingrowing MSC in vitro. Of all other synthetical scaffolds,beta-tricalcium phosphate (Chronos) have shown the best growth behaviour for MSC. DISCUSSION: Our results indicate that various bone-graft substitutes influence cell seeding efficiency,metabolic activity and growth behaviour of MSC in different manners. We detected a high variety of cellular integration of MSC in vitro,which may be important for bony integration in the clinical setting. View Publication

Transforming growth factor (TGF)-beta superfamily proteins play a key role in the regulation of human embryonic stem cells (hESCs). Those of the TGFbeta/activin/nodal branch seem to support self-renewal and pluripotency,whereas those of the bone morphogenic protein (BMP) branch induce differentiation. In contrast to this generalization,we found that hESC remained undifferentiated after knockdown of SMAD4 with inducible short hairpin RNA interference,although the knockdown inhibited TGFbeta signaling and rendered the cells nonresponsive to BMP-induced differentiation. Moreover,the rapid differentiation of hESC after pharmacological inhibition of TGFbeta/activin/nodal receptor signaling was restricted after SMAD4 knockdown. These results suggest that TGFbeta/activin/nodal signaling supports the undifferentiated phenotype of hESC by suppressing BMP activity. During long-term culture,SMAD4 knockdown cell populations became less stable and more permissive to neural induction,a situation that was rescued by re-establishment of SMAD4 expression. These results suggest that SMAD4 is not required for maintenance of the undifferentiated state of hESC,but rather to stabilize that state. View Publication