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A battery of clonal assays for myeloid progenitor cells (HPP-CFC,CFU-gemm,CFU-gm,CFU-g) was utilized to evaluate the myelotoxicity of a series of alkylating agents representing the spectrum of clinical times to nadir. Bone marrow aspirates from normal volunteers were incubated with mechlorethamine,busulfan,melphalan,carmustine or lomustine for 1 h and then cultured in methylcellulose with 30% serum and cytokines. There was a concentration-dependent inhibition of colony formation and often a differential toxicity to the myeloid progenitors with the alkylators tested. On a molar basis,mechlorethamine and melphalan were the most toxic of the alkylator drugs to the myeloid precursors. The most sensitive progenitor was CFU-gemm with the lowest inhibitory concentration IC(70) concentrations for mechlorethamine,melphalan,carmustine and lomustine. Generally,there was great similarity for drug effects between CFU-g and CFU-gm with overlapping inhibition curves. HPP-CFC proved to be the least sensitive of the progenitors to the toxic actions of the drugs. While there was no correlation between the time to clinical neutropenic nadir and the most sensitive progenitor in the clonal assays,the CFU-gm assay remains a suitable method for determining the myelotoxic potential of cytotoxic agents. View Publication

Gaucher disease is caused by an inherited deficiency of glucocerebrosidase that manifests with storage of glycolipids in lysosomes,particularly in macrophages. Available cell lines modeling Gaucher disease do not demonstrate lysosomal storage of glycolipids; therefore,we set out to develop two macrophage models of Gaucher disease that exhibit appropriate substrate accumulation. We used these cellular models both to investigate altered macrophage biology in Gaucher disease and to evaluate candidate drugs for its treatment. We generated and characterized monocyte-derived macrophages from 20 patients carrying different Gaucher disease mutations. In addition,we created induced pluripotent stem cell (iPSC)-derived macrophages from five fibroblast lines taken from patients with type 1 or type 2 Gaucher disease. Macrophages derived from patient monocytes or iPSCs showed reduced glucocerebrosidase activity and increased storage of glucocerebroside and glucosylsphingosine in lysosomes. These macrophages showed efficient phagocytosis of bacteria but reduced production of intracellular reactive oxygen species and impaired chemotaxis. The disease phenotype was reversed with a noninhibitory small-molecule chaperone drug that enhanced glucocerebrosidase activity in the macrophages,reduced glycolipid storage,and normalized chemotaxis and production of reactive oxygen species. Macrophages differentiated from patient monocytes or patient-derived iPSCs provide cellular models that can be used to investigate disease pathogenesis and facilitate drug development. View Publication

Serotonergic axons from the raphe nuclei in the brainstem project to every region of the brain,where they make connections through their extensive terminal arborizations. This serotonergic innervation contributes to various normal behaviors and psychiatric disorders. The protocadherin-alpha (Pcdha) family of clustered protocadherins consists of 14 cadherin-related molecules generated from a single gene cluster. We found that the Pcdhas were strongly expressed in the serotonergic neurons. To elucidate their roles,we examined serotonergic fibers in a mouse mutant (Pcdha(Delta CR/Delta CR)) lacking the Pcdha cytoplasmic region-encoding exons,which are common to the gene cluster. In the first week after birth,the distribution pattern of serotonergic fibers in Pcdha(Delta CR/Delta CR) mice was similar to wild-type,but by 3 weeks of age,when the serotonergic axonal termini complete their arborizations,the distribution of the projections was abnormal. In some target regions,notably the globus pallidus and substantia nigra,the normally even distribution of serotonin axonal terminals was,in the mutants,dense at the periphery of each region,but sparse in the center. In the stratum lacunosum-molecular of the hippocampus,the mutants showed denser serotonergic innervation than in wild-type,and in the dentate gyrus of the hippocampus and the caudate-putamen,the innervation was sparser. Together,the abnormalities suggested that Pcdha proteins are important in the late-stage maturation of serotonergic projections. Further examination of alternatively spliced exons encoding the cytoplasmic tail showed that the A-type (but not the B-type) cytoplasmic tail was essential for the normal development of serotonergic projections. 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

OBJECTIVE: The S100A9 and S100A8 proteins are highly expressed by neutrophils and monocytes and are part of a group of damage-associated molecular pattern molecules that trigger inflammatory responses. Sera and synovial fluids of patients with rheumatoid arthritis (RA) contain high concentrations of S100A8/A9 that correlate with disease activity.backslashnbackslashnMETHODS: In this study,we investigated the importance of S100A9 in RA by using neutralizing antibodies in a murine lipopolysaccharide-synchronized collagen-induced arthritis model. We also used an in vitro model of stimulation of human immune cells to decipher the role played by S100A9 in leukocyte migration and pro-inflammatory cytokine secretion.backslashnbackslashnRESULTS: Treatment with anti-S100A9 antibodies improved the clinical score by 50%,diminished immune cell infiltration,reduced inflammatory cytokines,both in serum and in the joints,and preserved bone/collagen integrity. Stimulation of neutrophils with S100A9 protein led to the enhancement of neutrophil transendothelial migration. S100A9 protein also induced the secretion by monocytes of proinflammatory cytokines like TNFα,IL-1β and IL-6,and of chemokines like MIP-1α and MCP-1.backslashnbackslashnCONCLUSION: The effects of anti-S100A9 treatment are likely direct consequences of inhibiting the S100A9-mediated promotion of neutrophil transmigration and secretion of pro-inflammatory cytokines from monocytes. Collectively,our results show that treatment with anti-S100A9 may inhibit amplification of the immune response and help preserve tissue integrity. Therefore,S100A9 is a promising potential therapeutic target for inflammatory diseases like rheumatoid arthritis for which alternative therapeutic strategies are needed. View Publication

Induced pluripotent stem (iPS) cells are somatic cells that have been reprogrammed to a pluripotent state via the introduction of defined transcription factors. Although iPS is a potentially valuable resource for regenerative medicine and drug development,several issues regarding their pluripotency,differentiation propensity and potential for tumorigenesis remain to be elucidated. Analysis of cell surface glycans has arisen as an interesting tool for the characterization of iPS. An appropriate characterization of glycan surface molecules of human embryonic stem (hES) cells and iPS cells might generate crucial data to highlight their role in the acquisition and maintenance of pluripotency. In this study,we characterized the surface glycans of iPS generated from menstrual blood-derived mesenchymal cells (iPS-MBMC). We demonstrated that,upon spontaneous differentiation,iPS-MBMC present high amounts of terminal $\$-galactopyranoside residues,pointing to an important role of terminal-linked sialic acids in pluripotency maintenance. The removal of sialic acids by neuraminidase induces iPS-MBMC and hES cells differentiation,prompting an ectoderm commitment. Exposed $\$-galactopyranose residues might be recognized by carbohydrate-binding molecules found on the cell surface,which could modulate intercellular or intracellular interactions. Together,our results point for the first time to the involvement of the presence of terminal sialic acid in the maintenance of embryonic stem cell pluripotency and,therefore,the modulation of sialic acid biosynthesis emerges as a mechanism that may govern stem cell differentiation. View Publication

INTRODUCTION The Comprehensive in vitro Proarrhythmia Assay (CiPA) is a nonclinical Safety Pharmacology paradigm for discovering electrophysiological mechanisms that are likely to confer proarrhythmic liability to drug candidates intended for human use. TOPICS COVERED Key talks delivered at the 'CiPA on my mind' session,held during the 2015 Annual Meeting of the Safety Pharmacology Society (SPS),are summarized. Issues and potential solutions relating to crucial constituents [e.g.,biological materials (ion channels and pluripotent stem cell-derived cardiomyocytes),study platforms,drug solutions,and data analysis] of CiPA core assays are critically examined. DISCUSSION In order to advance the CiPA paradigm from the current testing and validation stages to a research and regulatory drug development strategy,systematic guidance by CiPA stakeholders is necessary to expedite solutions to pending and newly arising issues. Once a study protocol is proved to yield robust and reproducible results within and across laboratories,it can be implemented as qualified regulatory procedure. View Publication

Human monoclonal antibodies against RSV have high potential for use as prophylaxis or therapeutic molecules,and they also can be used to define the structure of protective epitopes for rational vaccine design. In the past,however,isolation of human monoclonal antibodies was difficult and inefficient. Here,we describe contemporary methods for activation and proliferation of primary human memory B cells followed by cytofusion to non-secreting myeloma cells by dielectrophoresis to generate human hybridomas secreting RSV-specific monoclonal antibodies. We also provide experimental methods for screening human B cell lines to obtain RSV-specific lines,especially lines secreting neutralizing antibodies. View Publication