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Fluoride is a ubiquitous natural substance that is often used in dental products to prevent dental caries. The biphasic actions of fluoride imply that excessive systemic exposure to fluoride can cause harmful effects on embryonic development in both animal models and humans. However,insufficient information is available on the effects of fluoride on human embryonic stem cells (hESCs),which is a novel in vitro humanized model for analyzing the embryotoxicities of chemical compounds. Therefore,we investigated the effects of sodium fluoride (NaF) on the proliferation,differentiation and viability of H9 hESCs. For the first time,we showed that 1 mM NaF did not significantly affect the proliferation of hESCs but did disturb the gene expression patterns of hESCs during embryoid body (EB) differentiation. Higher doses of NaF (2 mM and above) markedly decreased the viability and proliferation of hESCs. The mode and underlying mechanism of high-dose NaF-induced cell death were further investigated by assessing the sub-cellular morphology,mitochondrial membrane potential (MMP),caspase activities,cellular reactive oxygen species (ROS) levels and activation of mitogen-activated protein kinases (MAPKs). High-dose NaF caused the death of hESCs via apoptosis in a caspase-mediated but ROS-independent pathway,coupled with an increase in the phospho-c-Jun N-terminal kinase (p-JNK) levels. Pretreatment with a pJNK-specific inhibitor (SP600125) could effectively protect hESCs from NaF-induced cell death in a concentration- and time-dependent manner. These findings suggest that NaF might interfere with early human embryogenesis by disturbing the specification of the three germ layers as well as osteogenic lineage commitment and that high-dose NaF could cause apoptosis through a JNK-dependent pathway in hESCs. View Publication

Ectopic retroviral expression of homeobox B4 (HOXB4) causes an accelerated and enhanced regeneration of murine hematopoietic stem cells (HSCs) and is not known to compromise any program of lineage differentiation. However,HOXB4 expression levels for expansion of human stem cells have still to be established. To test the proposed hypothesis that HOXB4 could become a prime tool for in vivo expansion of genetically modified human HSCs,we retrovirally overexpressed HOXB4 in purified cord blood (CB) CD34+ cells together with green fluorescent protein (GFP) as a reporter protein,and evaluated the impact of ectopic HOXB4 expression on proliferation and differentiation in vitro and in vivo. When injected separately into nonobese diabetic-severe combined immunodeficient (NOD/SCID) mice or in competition with control vector-transduced cells,HOXB4-overexpressing cord blood CD34+ cells had a selective growth advantage in vivo,which resulted in a marked enhancement of the primitive CD34+ subpopulation (P =.01). However,high HOXB4 expression substantially impaired the myeloerythroid differentiation program,and this was reflected in a severe reduction of erythroid and myeloid progenitors in vitro (P textless.03) and in vivo (P =.01). Furthermore,HOXB4 overexpression also significantly reduced B-cell output (P textless.01). These results show for the first time unwanted side effects of ectopic HOXB4 expression and therefore underscore the need to carefully determine the therapeutic window of HOXB4 expression levels before initializing clinical trials. View Publication

The extended substrate specificity of granzyme B (GrB) was used to identify substrates among the chaperone superfamily. This approach identified Hsp90 and Bag1-L as novel GrB substrates,and an additional GrB cleavage site was identified in the Hsc70/Hsp70-Interacting Protein,Hip. Hsp90,Bag1L,and Hip were validated as GrB substrates in vitro,and mutational analysis confirmed the additional cleavage site in Hip. Because the role of Hip in apoptosis is unknown,its proteolysis by GrB was used as a basis to test whether it has anti-apoptotic activity. Previous work on Hip was limited to in vitro characterization; therefore,it was important to demonstrate Hip cleavage in a physiological context and to show its relevance to natural killer (NK) cell-mediated death. Hip is cleaved at both GrB cleavage sites during NK-mediated cell death in a caspase-independent manner,and its cleavage is due solely to GrB and not other granule components. Furthermore,Hip is not cleaved upon stimulation of the Fas receptor in the Jurkat T-cell line,suggesting that Hip is a substrate unique to GrB. RNA interference-mediated reduction of Hip within the K562 cell line rendered the cells more susceptible to NK cell-mediated lysis,indicating that proteolysis by GrB of Hip contributes to death induction. The small effect of RNA interference-mediated Hip deficiency on cytotoxicity is in agreement with the inherent redundancy of NK cell-mediated cell death. The identification of additional members of the chaperone superfamily as GrB substrates and the validation of Hip as an anti-apoptotic protein contribute to understanding the interplay between stress response and apoptosis. View Publication

Imatinib mesylate has shown remarkable efficacy in the treatment of patients in the chronic phase of chronic myeloid leukemia. However,despite an overall significant hematological and cytogenetic response,imatinib therapy may favor the emergence of drug-resistant clones,ultimately leading to relapse. Some imatinib resistance mechanisms had not been fully elucidated yet. In this study we used sensitive and resistant sublines from a Bcr-Abl positive cell line to investigate the putative involvement of telomerase in the promotion of imatinib resistance. We showed that sensitivity to imatinib can be partly restored in imatinib-resistant cells by targeting telomerase expression,either by the introduction of a dominant-negative form of the catalytic protein subunit of the telomerase (hTERT) or by the treatment with all-trans-retinoic acid,a clinically used drug. Furthermore,we showed that hTERT overexpression favors the development of imatinib resistance through both its antiapoptotic and telomere maintenance functions. Therefore,combining antitelomerase strategies to imatinib treatment at the beginning of the treatment should be promoted to reduce the risk of imatinib resistance development and increase the probability of eradicating the disease. View Publication

In February 2013,zoonotic transmission of a novel influenza A virus of the H7N9 subtype was reported in China. Although at present no sustained human-to-human transmission has been reported,a pandemic outbreak of this H7N9 virus is feared. Since neutralizing antibodies to the hemagglutinin (HA) globular head domain of the virus are virtually absent in the human population,there is interest in identifying other correlates of protection,such as cross-reactive CD8(+) T cells (cytotoxic T lymphocytes [CTLs]) elicited during seasonal influenza A virus infections. These virus-specific CD8(+) T cells are known to recognize conserved internal proteins of influenza A viruses predominantly,but it is unknown to what extent they cross-react with the newly emerging H7N9 virus. Here,we assessed the cross-reactivity of seasonal H3N2 and H1N1 and pandemic H1N1 influenza A virus-specific polyclonal CD8(+) T cells,obtained from HLA-typed study subjects,with the novel H7N9 virus. The cross-reactivity of CD8(+) T cells to H7N9 variants of known influenza A virus epitopes and H7N9 virus-infected cells was determined by their gamma interferon (IFN-γ) response and lytic activity. It was concluded that,apart from recognition of individual H7N9 variant epitopes,CD8(+) T cells to seasonal influenza viruses display considerable cross-reactivity with the novel H7N9 virus. The presence of these cross-reactive CD8(+) T cells may afford some protection against infection with the new virus. View Publication

Several progenitor cell populations have been reported to exist in hearts that play a role in cardiac turnover and/or repair. Despite the presence of cardiac stem and progenitor cells within the myocardium,functional repair of the heart after injury is inadequate. Identification of the signaling pathways involved in the expansion and differentiation of cardiac progenitor cells (CPCs) will broaden insight into the fundamental mechanisms playing a role in cardiac homeostasis and disease and might provide strategies for in vivo regenerative therapies. To understand and exploit cardiac ontogeny for drug discovery efforts,we developed an in vitro human induced pluripotent stem cell-derived CPC model system using a highly enriched population of KDR(pos)/CKIT(neg)/NKX2.5(pos) CPCs. Using this model system,these CPCs were capable of generating highly enriched cultures of cardiomyocytes under directed differentiation conditions. In order to facilitate the identification of pathways and targets involved in proliferation and differentiation of resident CPCs,we developed phenotypic screening assays. Screening paradigms for therapeutic applications require a robust,scalable,and consistent methodology. In the present study,we have demonstrated the suitability of these cells for medium to high-throughput screens to assess both proliferation and multilineage differentiation. Using this CPC model system and a small directed compound set,we identified activin-like kinase 5 (transforming growth factor-β type 1 receptor kinase) inhibitors as novel and potent inducers of human CPC differentiation to cardiomyocytes. Significance: Cardiac disease is a leading cause of morbidity and mortality,with no treatment available that can result in functional repair. This study demonstrates how differentiation of induced pluripotent stem cells can be used to identify and isolate cell populations of interest that can translate to the adult human heart. Two separate examples of phenotypic screens are discussed,demonstrating the value of this biologically relevant and reproducible technology. In addition,this assay system was able to identify novel and potent inducers of differentiation and proliferation of induced pluripotent stem cell-derived cardiac progenitor cells. View Publication

Systemic mastocytosis (SM) is a myeloid neoplasm involving mast cells (MCs) and their progenitors. In most cases,neoplastic cells display the D816V-mutated variant of KIT. KIT D816V exhibits constitutive tyrosine kinase (TK) activity and has been implicated in increased survival and growth of neoplastic MCs. Recent data suggest that the proapoptotic BH3-only death regulator Bim plays a role as a tumor suppressor in various myeloid neoplasms. We found that KIT D816V suppresses expression of Bim in Ba/F3 cells. The KIT D816-induced down-regulation of Bim was rescued by the KIT-targeting drug PKC412/midostaurin. Both PKC412 and the proteasome-inhibitor bortezomib were found to decrease growth and promote expression of Bim in MC leukemia cell lines HMC-1.1 (D816V negative) and HMC-1.2 (D816V positive). Both drugs were also found to counteract growth of primary neoplastic MCs. Furthermore,midostaurin was found to cooperate with bortezomib and with the BH3-mimetic obatoclax in producing growth inhibition in both HMC-1 subclones. Finally,a Bim-specific siRNA was found to rescue HMC-1 cells from PKC412-induced cell death. Our data show that KIT D816V suppresses expression of proapoptotic Bim in neoplastic MCs. Targeting of Bcl-2 family members by drugs promoting Bim (re)-expression,or by BH3-mimetics such as obatoclax,may be an attractive therapy concept in SM. View Publication

Bat immunity has received increasing attention because some bat species are being decimated by the fungal disease,White Nose Syndrome,while other species are potential reservoirs of zoonotic viruses. Identifying specific immune processes requires new specific tools and reagents. In this study,we describe a new mouse monoclonal antibody (mAb) reactive with Eptesicus fuscus immunoglobulins. The epitope recognized by mAb BT1-4F10 was localized to immunoglobulin light (lambda) chains; hence,the mAb recognized serum immunoglobulins and B lymphocytes. The BT1-4F10 epitope appeared to be restricted to Microchiropteran immunoglobulins and absent from Megachiropteran immunoglobulins. Analyses of sera and other E. fuscus fluids showed that most,if not all,secreted immunoglobulins utilized lambda light chains. Finally,mAb BT1-4F10 permitted the identification of B cell follicles in splenic white pulp. This Microchiropteran-specific mAb has potential utility in seroassays; hence,this reagent may have both basic and practical applications for studying immune process. View Publication

IL-17A is a T cell-derived proinflammatory cytokine required for microbial host defense. In vivo expression profoundly stimulates granulopoiesis. At baseline,the hemopoietic system of IL-17R knockout mice (IL-17Ra(-/-)) is,with the exception of increased splenic progenitor numbers,indistinguishable from normal control mice. However,when challenged with gamma irradiation,hemopoietic toxicity is significantly more pronounced in IL-17Ra(-/-) animals,with the gamma irradiation-associated LD(50) being reduced by 150 rad. In spleen-derived T cells,gamma irradiation induces significant murine IL-17A expression in vivo but not in vitro. After sublethal radiation injury (500 rad),the infusion of purified CD4(+) T cells enhances hemopoietic recovery. This recovery is significantly impaired in IL-17Ra(-/-) animals or after in vivo blockade of IL-17Ra in normal mice,resulting in a reduction of hemopoietic precursors by 50% and of neutrophils by 43%. Following sublethal radiation-induced myelosuppression,in vivo overexpression of murine IL-17A in normal mice substantially enhanced granulopoietic restoration in mice with a 4-fold increase in neutrophils and splenic precursors on day 8 (CFU-granulocyte-macrophage/granulocyte-erythrocyte-megakaryocyte-monocyte,CFU-high proliferative potential),as well as 2- and 3-fold increases of bone marrow precursors,respectively. This establishes IL-17A as a hemopoietic response cytokine to radiation injury in mice and an inducible mechanism that is required for recovery of granulopoiesis after radiation injury. View Publication

Since protein kinases are frequently mutated or otherwise deregulated in human malignancies,they serve as a target for differentiating between tumor cells and normal tissues. Imatinib mesylat (IM),an inhibitor of the BCR-ABL tyrosine kinase was introduced in 2001 and has revolutionized the treatment of patients with chronic myeloid leukemia (CML). Since 2005 a second generation of tyrosine kinase inhibitors is to follow in Imatinib's footsteps: The development of these new small molecules was promoted by the identification of potential target kinases within the cellular signaling apparatus. Modern biochemical tools provide relevant amounts of these target kinases necessary for high throughput screening (HTS) campaigns and for elucidation of their 3-D structure by crystallography. Supported by computational chemistry the resulting data have enabled rational drug design. In this review low molecular weight inhibitors used for the CML treatment are summarized,pointing out their chemical similarities and differences. View Publication

Xylosylated and core alpha1,3-fucosylated N-glycans from plants are immunogenic,and they play a still obscure role in allergy and in the field of plant-made protein pharmaceuticals. We immunized mice to generate monoclonal antibodies (mAbs) binding plant N-glycans specifically via the epitope containing either the xylose or the core alpha1,3-fucose residue. Splenocytes expressing N-glycan-specific antibodies derived from C57BL/6 mice previously immunized with plant glycoproteins were preselected by cell sorting to generate hybridoma lines producing specific antibodies. However,we obtained only mAbs unable to distinguish fucosylated from xylosylated N-glycans and reactive even with the pentasaccharide core Man3GlcNAc2. In contrast,immunization of rabbits yielded polyclonal sera selectively reactive with either fucosylated or xylosylated N-glycans. Purification of these sera using glyco-modified neoglycoproteins coupled to a chromatography matrix provided polyclonal sera suitable for affinity determination. Surface plasmon resonance measurements using sensor chips with immobilized glyco-modified transferrins revealed dissociation constants of around 10(-9) M. This unexpectedly high affinity of IgG antibodies toward carbohydrate epitopes has repercussions on our conception of the binding strength and significance of antiglycan IgE antibodies in allergy. View Publication

Immunomodulatory derivative (IMiD) CC-4047,a new analog of thalidomide,directly inhibits growth of B-cell malignancies in vivo and in vitro and exhibits stronger antiangiogenic activity than thalidomide. However,there is little information on whether CC-4047 affects normal hematopoiesis. Here we investigated the effect of CC-4047 on lineage commitment and differentiation of hematopoietic stem cells. We found that CC-4047 effectively inhibits erythroid cell colony formation from CD34+ cells and increases the frequency of myeloid colonies. We also demonstrate that development of both erythropoietin-independent and erythropoietin-dependent red cell progenitors was strongly inhibited by CC-4047,while terminal red cell differentiation was unaffected. DNA microarray analysis revealed that red cell transcription factors,including GATA-1,GATA-2,erythroid Kruppel-like factor (EKLF),and growth factor independence-1B (Gfi-1b),were down-regulated in CC-4047-treated CD34+ cells,while myeloid transcription factors such as CCAAT/enhancer binding protein-alpha (C/EBPalpha),C/EBPdelta,and C/EBPepsilon were induced. Analysis of cytokine secretion indicated that CC-4047 induced secretion of cytokines that enhance myelopoiesis and inhibit erythropoiesis. In conclusion,these data indicate that CC-4047 might directly influence lineage commitment of hematopoietic cells by increasing the propensity of stem and/or progenitor cells to undergo myeloid cell development and concomitantly inhibiting red cell development. Therefore,CC-4047 provides a valuable tool to study the mechanisms underlying lineage commitment. View Publication