Search

Yap1 and its paralogue Taz largely control epithelial tissue growth. We have identified that hematopoietic stem cell (HSC) fitness response to stress depends on Yap1 and Taz. Deletion of Yap1 and Taz induces a loss of HSC quiescence, symmetric self-renewal ability, and renders HSC more vulnerable to serial myeloablative 5-fluorouracil treatment. This effect depends on the predominant cytosolic polarization of Yap1 through a PDZ domain-mediated interaction with the scaffold Scribble. Scribble and Yap1 coordinate to control cytoplasmic Cdc42 activity and HSC fate determination in vivo. Deletion of Scribble disrupts Yap1 copolarization with Cdc42 and decreases Cdc42 activity, resulting in increased self-renewing HSC with competitive reconstitution advantages. These data suggest that Scribble/Yap1 copolarization is indispensable for Cdc42-dependent activity on HSC asymmetric division and fate. The combined loss of Scribble, Yap1, and Taz results in transcriptional upregulation of Rac-specific guanine nucleotide exchange factors, Rac activation, and HSC fitness restoration. Scribble links Cdc42 and the cytosolic functions of the Hippo signaling cascade in HSC fate determination.

Hematopoietic stem cell (HSC) activity is tightly controlled to ensure the integrity of the hematopoietic system during the organism’s lifetime. How the HSC compartment maintains its long-term fitness in conditions of chronic stresses associated with systemic metabolic disorders is poorly understood. In this study, we show that obesity functionally affects the long-term function of the most immature engrafting HSC subpopulation. We link this altered regenerative activity to the oxidative stress and the aberrant constitutive activation of the AKT signaling pathway that characterized the obese environment. In contrast, we found minor disruptions of the HSC function in obese mice at steady state, suggesting that active mechanisms could protect the HSC compartment from its disturbed environment. Consistent with this idea, we found that FOXO proteins in HSCs isolated from obese mice become insensitive to their normal upstream regulators such as AKT, even during intense oxidative stress. We established that hyperglycemia, a key condition associated with obesity, is directly responsible for the alteration of the AKT-FOXO axis in HSCs and their abnormal oxidative stress response. As a consequence, we observed that HSCs isolated from a hyperglycemic environment display enhanced resistance to oxidative stress and DNA damage. Altogether, these results indicate that chronic metabolic stresses associated with obesity and/or hyperglycemia affect the wiring of the HSCs and modify their oxidative stress response. These data suggest that the uncoupling of FOXO from its environmental regulators could be a key adaptive strategy that promotes the survival of the HSC compartment in obesity.

Severe congenital neutropenia (SCN) is frequently associated with mutations in the ELANE gene encoding neutrophil elastase (NE), an azurophilic granule protein which represents a major fraction of all protein synthesized by neutrophilic promyelocytes and myelocytes. The reduction of mutant ELANE translation inversely correlates with neutrophil differentiation. Chronic administration of granulocyte colony-stimulating-factor (G-CSF) has been successfully used to treat some of these patients, especially those ones with mutations in C-terminus translating exons-4 and -5. Patients with proximal mutations of ELANE tend to be resistant to G-CSF therapy. The SCN pathobiology is poorly understood due to the lack of efficient disease animal models, and difficulty in obtaining relevant primary cell populations from patients. We have demonstrated that ELANE exon-3 mutations, sensitive to higher doses of G-CSF administration, cause maturation arrest and apoptosis of promyelocyte and myelocyte stages due to mutant NE inducing unfolded protein response (UPR) and endoplasmic reticulum (ER) stress. We have also shown that SCN patients with mutations in ELANE at its translation initiation codon (ELANE-ATG mut) result in synthesis of alternate truncated neutrophil elastase peptides from downstream in-frame ATG due to the presence of internal ribosomal entry sites. However, the mechanisms of SCN pathogenesis associated with mutations at the translation initiation codon of ELANE gene associated with G-CSF resistance remain unclear. We employed a disease-in-a-dish approach by directed hematopoietic and granulopoietic differentiation of gene edited (CRISPR/Cas9) isogenic patient-derived iPSC lines. We used four iPSC lines (healthy donor 28L and its start codon knock-in isogenic 28L-GTG; and patient-derived ATG mut SCN110 and its isogenic corrected line SCN110C) to evaluate if ELANE start codon mutations are both necessary and sufficient to cause disease pathogenesis, and to unravel the underlying molecular mechanisms. We found that myeloid precursors derived from 28-GTG and SCN110 iPSC lines generate low molecular weight truncated NE that is detected by a C-terminal region (C225-H238) specific anti-NE antibody (Ab). The survival, expansion and differentiation of ELANE start codon mutant hematopoietic progenitors were significantly reduced in cytokine supplemented in vitro myelopoiesis cultures, and the correction of the mutation (SCN110C) restored the granulocytic precursor expansion and differentiation. The reduced expansion in 28L-GTG and SCN110 lines was associated with both increased apoptosis and expression of proapoptotic BH3-only proteins in comparison to their isogenic 28L and SCN110C lines. However, this apoptosis was not associated with UPR/ER-stress in ELANE-ATG mut myeloid precursors. Granulopoietic differentiation of the 28L-GTG and SCN110 iPSC derived hematopoietic progenitors was significantly reduced, while it was rescued in corrected isogenic lines. Correlating with their clinical behavior, high-dose G-CSF in vitro did not restore the granulopoietic differentiation of ELANE ATG mutant hematopoietic progenitors. Mechanistically, 28L-GTG and SCN110 myeloid precursors contained high-molecular weight, sodium dodecyl sulfate (SDS) resistant NE bands that were detected by anti-NE Ab specific for the C-terminal (C225-238), but not the N-terminal region (C19-38), suggesting that truncated mutant NE generates insoluble NE aggregates. The association of truncated NE with classical autophagy marker LC3B strongly suggested the activation of an aggrephagy process in these cells. We found that SERF1 (small EDRK-rich factor), an RNA-chaperoning protein, known to localize in misfolded protein aggregates of neurodegenerative diseases, was highly upregulated in GTG knock-in and SCN patient myeloid precursors. SERF1 was distributed in both nucleus and cytoplasm, co-localized and physically interacted with mutant truncated NE in the cytoplasm of ELANE-ATG mut cells as shown by proximity ligation assays. Silencing of SERF1 made the survival and differentiation of 28L-GTG and SCN110 derived myeloid precursors sensitive to G-CSF. ELANE translation initiation mutant was associated with truncated NE aggregates and induced apoptosis mediated by SERF1. This data opens a path for therapeutic intervention of G-CSF resistant ELANEmut SCN. Disclosures Kalfa: Agios Pharmaceuticals, Inc.: Other: Steering Committee, Research Funding; FORMA Therapeutics, Inc: Research Funding. Lutzko: Aruvant Sciences: Patents & Royalties: preclinical vector development. Cancelas: Hemerus LLC: Research Funding; Vascular Solutions Inc.: Research Funding; Westat Inc: Research Funding; Fresenius-Kabi LLC: Research Funding; Cytosorbents Inc: Research Funding; Hemanext: Membership on an entity's Board of Directors or advisory committees, Research Funding; TerumoBCT: Research Funding; Cerus Co: Research Funding; University of South Florida/MEQU Inc: Research Funding.

Epigenetically regulated transcriptional plasticity has been proposed as a mechanism of differentiation arrest and resistance to therapy. BCR-ABL leukemias result from leukemic stem cell/progenitor transformation and represent an opportunity to identify epigenetic progress contributing to lineage leukemogenesis. Primary human and murine BCR-ABL+ leukemic progenitors have increased activation of Cdc42 and the downstream atypical protein kinase C (aPKC). While the isoform aPKCζ behaves as a leukemic suppressor, aPKCλ/ι is critically required for oncogenic progenitor proliferation, survival, and B-cell differentiation arrest, but not for normal B-cell lineage differentiation. In vitro and in vivo B-cell transformation by BCR-ABL requires the downregulation of key genes in the B-cell differentiation program through an aPKC λ/ι-Erk dependent Etv5/Satb2 chromatin repressive signaling complex. Genetic or pharmacological targeting of aPKC impairs human oncogenic addicted leukemias. Therefore, the aPKCλ/ι-SATB2 signaling cascade is required for leukemic BCR-ABL+ B-cell progenitor transformation and is amenable to non-tyrosine kinase inhibition., The upstream pathways regulating leukemic transcriptional plasticity for differentiation arrest and resistance to therapy are unclear. Here the authors show that aPKC λ/ι-controls leukemic B-cell precursor differentiation arrest trough RAC/MEK/ERK/SATB2 epigenetic repression

Severe congenital neutropenia (SCN) is often associated with inherited heterozygous point mutations in ELANE, which encodes neutrophil elastase (NE). However, a lack of appropriate models to recapitulate SCN has substantially hampered the understanding of the genetic etiology and pathobiology of this disease. To this end, we generated both normal and SCN patient-derived induced pluripotent stem cells (iPSCs), and performed genome editing and differentiation protocols that recapitulate the major features of granulopoiesis. Pathogenesis of ELANE point mutations was the result of promyelocyte death and differentiation arrest, and was associated with NE mislocalization and activation of the unfolded protein response/ER stress (UPR/ER stress). Similarly, high-dose G-CSF (or downstream signaling through AKT/BCL2) rescues the dysgranulopoietic defect in SCN patient-derived iPSCs through C/EBPβ-dependent emergency granulopoiesis. In contrast, sivelestat, an NE-specific small-molecule inhibitor, corrected dysgranulopoiesis by restoring normal intracellular NE localization in primary granules; ameliorating UPR/ER stress; increasing expression of CEBPA, but not CEBPB; and promoting promyelocyte survival and differentiation. Together, these data suggest that SCN disease pathogenesis includes NE mislocalization, which in turn triggers dysfunctional survival signaling and UPR/ER stress. This paradigm has the potential to be clinically exploited to achieve therapeutic responses using lower doses of G-CSF combined with targeting to correct NE mislocalization.

Diagnosis and monitoring of diseases by measurement of biochemical markers has most commonly been performed on samples of peripheral blood. However, no such markers are available for clinical use in the major diseases of the central nervous system (CNS). In Parkinson's disease circulating biomarkers would find clinical utility in early diagnosis and also monitoring of disease progression. Of particular interest is early diagnosis as this would create .a window of opportunity for treatment with neuroprotective drugs. We have developed a novel strategy for monitoring disease activity in the CNS based on the recognition that tissue injuries incite inflammation and recruitment of phagocytes that engulf debris. We postulated that some of these debris laden phagocytes may return to the peripheral blood and their cargo of CNS proteins could be measured. If CNS antigens can be measured in PBMCs it may be an indicator of active neurodegeneration as the debris engulfed by phagocytes is completely degraded within days. To make this approach more specific to Parkinson's disease we probed PBMC lysates for neuromelanin as a marker of degeneration within the substancia nigra. We performed a proof of principle study in ten subjects with early PD and ten age and sex matched controls. The biomarkers neuromelanin, Tau protein, UCH-L1 and HPCAL-1 were measured in PBMC lysates from these two groups. Neuromelanin and Tau protein mean levels were elevated in PD compared with controls and was extremely statistically significant in both cases. UCH-L1 and HPCAL-1 mean levels were elevated in PD over controls and were not quite significant in both cases. These results suggest that this is a promising new approach for diagnosis and monitoring of PD and potentially other CNS diseases.

Human bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) are manufactured using many different methods, but little is known about the spectrum of manufacturing methods used and their effects on BMSC characteristics and function. Seven centers using, and one developing, Good Manufacturing Practices (GMP) processes were surveyed as to their production methods. Among the seven centers, all used marrow aspirates as the starting material, but no two centers used the same manufacturing methods. Two to four BMSC lots from each center were compared using global gene expression. Among the twenty-four BMSC lots from the eight centers intra-center transcriptome variability was low and similar among centers. Principal component analysis and unsupervised hierarchical clustering analysis separated all the lots from five centers into five distinct clusters. BMSCs from six of the eight centers were tested for their ability to form bone and support hematopoiesis by in vivo transplantation (defining features of BMSCs). Those from all six centers tested formed bone, but the quantity formed was highly variable and BMSCs from only three centers supported hematopoiesis. These results show that differences in manufacturing resulted in variable BMSC characteristics including their ability to form bone and support hematopoiesis.

SummaryIn the bone marrow (BM), hematopoietic progenitors (HPs) reside in specific anatomical niches near osteoblasts (Obs), macrophages (MΦs), and other cells forming the BM microenvironment. A connection between immunosurveillance and traffic of HP has been demonstrated, but the regulatory signals that instruct the immune regulation of HP circulation are unknown. We discovered that the BM microenvironment deficiency of p62, an autophagy regulator and signal organizer, results in loss of autophagic repression of macrophage contact-dependent activation of Ob NF-κB signaling. Consequently, Ob p62-deficient mice lose bone, Ob Ccl4 expression, and HP chemotaxis toward Cxcl12, resulting in egress of short-term hematopoietic stem cells and myeloid progenitors. Finally, Ccl4 expression and myeloid progenitor egress are reversed by deficiency of the p62 PB1-binding partner Nbr1. A functional “MΦ-Ob niche” is required for myeloid progenitor/short-term stem cell retention, in which Ob p62 is required to maintain NF-κB signaling repression, osteogenesis, and BM progenitor retention.

Lifelong maintenance of the blood system requires the preservation of a healthy hematopoietic stem cell (HSC) pool. Integrity of the HSC compartment is disrupted by severe homeostatic perturbations following acute infection or irradiation, which result in HSC loss and lead to dramatic hematological dysfunctions. HSC fitness is also affected by organismal patho-physiological conditions, which are associated with chronic low-grade stress. These chronic conditions present, over time, a unique challenge to the maintenance of the long-lived HSCs. However, the mechanisms by which the HSC pool adapts to chronic, low-grade stress conditions remain largely unknown. We focus on obesity as a model of chronic stress that potentially affects the health of the HSC compartment. Obesity is a chronic pathological state, which is established and persists over long period of time and therefore, is a prime candidate to alter long-lived HSCs. Obesity becomes progressively associated with an array of systemic and local alterations that include metabolic dysregulations, unresolved low-grade inflammation andalteration of BM microenvironment, all conditions that have been linked to HSC defects (Nagareddy et al, Cell Metabol., 2013; Naveiras et al, Nature, 2009; Luo et al, Cell Metabol., 2015; Ambrosi et al, Cell Stem Cell, 2017).Consistent with these studies, we recently showed that obesity alters the composition of the HSC-SLAM (Lin- ckit+ Sca1+ CD48+ CD150-) compartment and leads to a progressive loss of HSC fitness upon serial competitive transplantation assays (Lee et al, J. Exp. Med.,2018). Mechanistically, we established that the oxidative stress induced by obesity dysregulates the expression of the transcription factor Gfi1 and contributes to the long-term alteration of the HSC functions. Following this work, we confirmed that obesity mainly affects the short and long-term stress response of the most primitive HSC compartment (defined as HSC-SLAM CD34- CD49b-) but not of the downstream short-term HSC subsets, therefore indicating that obesitydisrupts intrinsic mechanisms regulating self-renewal activity. Genome-wide gene expression analyses indicated that HSCs isolated from obese mice dysregulate multiples genes involved in the phosphatidylinositol signaling pathway (e.g. Pik3c2a, Pik3c2b, Pi3kap1, Pi3kip1), upstream of the Akt signaling molecule. Phospho-flow cytometry analyses showed that HSCs freshly isolated from obese mice display constitutive Akt activation. In vivopharmacological Akt inhibition did not significantly change the size or phenotype of the HSC-SLAM compartment in obesity but led to the normalization of their functions in condition of transplantation-induced regenerative stress. These results indicate that the aberrant Akt activation in obesity-primed HSCs directly contributes to their hyperactivity upon transplantation, which ultimately results in the erosion of their self-renewal potential. We next explored the mechanisms that allow HSCs to remain quiescent in obesity despite the constitutive activation of Akt and the continued presence of oxidative stress condition. We focused on FoxO3, which is a key transcriptional regulator of HSC quiescence and direct Akt target. We found that FoxO3 in obesity-primed HSCs become insensitive to its normal upstream regulators such as Akt and p38, leading to the maintenance of its nuclear location in oxidative stress condition. As consequence, we observed that obesity-primed HSCs were more resistant to oxidative stress than their normal counterparts, suggesting an increase protection against ROS-mediated senescence and apoptosis. Altogether, these results suggest that chronic metabolic stresses associated with obesity progressively affect the wiring of the HSCs and modify their oxidative stress response. The uncoupling of FoxO3 to its environmental regulators in obesity could be viewed as a key adaptive strategy to ensure the survival and function of the HSC compartment in condition of chronic metabolic stress. More broadly, these results highlight how patho-physiological conditions associated with chronic low-grade stress (such as obesity) could shape the characteristic of the HSC compartment at steady state. Progressive alteration of the normal HSC stress response in such conditions could be an unrecognized contributing factor for the development of hematological diseases. Disclosures Cancelas: Cerus Co.: Research Funding; TerumoBCT: Consultancy, Research Funding; Macopharma Inc: Research Funding; Cytosorbents: Research Funding; Cellphire: Research Funding; Velico: Consultancy, Research Funding; Fresenius-Kabi: Research Funding; Hemanext: Consultancy, Research Funding.

The Rho family of guanosine triphosphatases (GTPases) is composed of members of the Ras superfamily of proteins. They are GTP-bound molecules with a modest intrinsic GTPase activity that can be accelerated upon activation/localization of specialized guanine nucleotide exchange factors. Members of this family act as molecular switches and are required for coordinated cytoskeletal rearrangements that are crucial in a set of specialized functions of mammalian stem cells. These functions include self-renewal, adhesion, and migration. Mouse gene-targeting studies have provided convincing evidence of the indispensable and dispensable roles of individual members of the Rho GTPase family and the putative upstream and downstream mediators in stem cell-specific functions. The role of Rho GTPases and related signaling pathways previously seen in other cell types and organisms have been confirmed in mammalian hematopoietic stem cells (HSCs), and new signaling pathways and unexpected functions unique to HSCs have been identified and dissected. This review summarizes our current understanding of the role of Rho family of GTPases on HSC and progenitor activity through cytoskeleton-mediated signaling pathways, providing insight about relevant signaling pathways that regulate mammalian stem cell self-renewal, adhesion, and migration.

Hematopoiesis is regulated by components of the microenvironment, so-called niche. Here, we show that p190-B GTPase-activating protein (p190-B) deletion in mice causes hematopoietic failure during ontogeny, in p190-B(-/-) fetal liver and bones, and in p190-B(+/-) adult bones and spleen. These defects are non-cell autonomous, as we previously showed that transplantation of p190-B(-/-) hematopoietic cells into wild-type (WT) hosts leads to normal hematopoiesis. Coculture of mesenchymal stem (MSC)/progenitor cells and wild-type bone marrow (BM) cells reveals that p190-B(-/-) MSCs are dysfunctional in supporting hematopoiesis owing to impaired Wnt signaling. Furthermore, p190-B loss causes alteration in BM niche composition, including abnormal colony-forming unit (CFU)-fibroblast, CFU-adipocyte and CFU-osteoblast numbers. This is due to altered MSC lineage fate specification to osteoblast and adipocyte lineages. Thus, p190-B organizes a functional mesenchymal/microenvironment for normal hematopoiesis during development.

Macrophages are a major component of the innate immune response, comprising the first line of defense against various intracellular pathogens, including Mycobacterium tuberculosis. In this report, we studied the factors that regulate growth of M. tuberculosis H37Rv in subpopulations of human monocyte-derived macrophages (MDMs). In healthy donors, M. tuberculosis H37Rv grew 5.6-fold more rapidly in CD14hi MDMs compared with that in CD14loCD16+ MDMs. Compared with CD14loCD16+ cells, M. tuberculosis H37Rv-stimulated CD14hi monocytes produced more IL-10 and had increased mRNA expression for c-Maf, a transcription factor that upregulates IL-10 gene expression. c-Maf small interfering RNA (siRNA) inhibited IL-10 production and growth of M. tuberculosis in CD14hi cells. Compared with CD14loCD16+ monocytes, M. tuberculosis H37Rv-stimulated CD14hi cells had increased expression of 22 genes whose promoters contained a c-Maf binding site, including hyaluronan synthase 1 (HAS1). c-Maf siRNA inhibited HAS1 expression in M. tuberculosis-stimulated CD14hi monocytes, and HAS1 siRNA inhibited growth of M. tuberculosis in CD14hi MDMs. M. tuberculosis H37Rv upregulated expression of HAS1 protein and its product, hyaluronan, in CD14hi MDMs. We conclude that M. tuberculosis grows more rapidly in CD14hi than in CD14loCD16+ MDMs because CD14hi cells have increased expression of c-Maf, which increases production of two key factors (hyaluronan and IL-10) that promote growth of M. tuberculosis.

Despite the introduction of tyrosine kinase inhibitor and CAR-T cell therapies, the prognosis for Ph+ and Ph-like acute lymphoblastic leukemia remains poor. In the present study, we show the role of the Scribble protein in both lymphoid and myeloid leukemogenesis. The polarity protein Scribble is a member of the basal polarity complex, which is down-regulated in many cancers, suggesting a possible tumor suppressor role, especially in so-called cancer initiating cells. Its effect and mechanisms of activity in leukemic cell fate along with its potential activity on leukemic initiating cells have only been recently started to elucidated. Using interferon-responsive inducible (Mx1-Cre) Scribble-deficient mice, we have characterized the role of Scribble in both retroviral transduction, transplantation animal models and binary, inducible stem cell initiated (Scl-tTA/TRE-BCR-ABL) serial propagation models of BCR-ABL induced leukemia. We found that Scribble expression is upregulated at both transcriptional and translational levels in p210- or p190-BCR-ABL induced leukemic progenitors. In vitro, leukemic colony formation was impaired in Scribble deficient leukemic progenitors (~48% reduction; p≤ 0.05, compared to Wt leukemic progenitors) demonstrating that Scribble is important for leukemogenesis. In vivo, the deletion of Scribble abrogates the development of myeloproliferative disease induced by p210-BCR-ABL (median survival: 70 vs 47 days in Scribble deficient and Wt chimeric mice, respectively; p≤0.05); and significantly impairs B-cell lymphoid leukemogenesis induced by p190-BCR-ABL (median survival: 80 vs 60 days for Scribble deficient and Wt chimeric animals, respectively). Mechanistically, BCR-ABL activates the apical polarity regulator Cdc42 in leukemic progenitors and this activation is inhibited by the deficiency of Scribble. The deficiency of Cdc42 does not impair leukemogenesis but the combined deficiency of Cdc42 and Scribble restores the in vivo survival (median survival: 47 days, p≤0.01 compared to Scribble deficient mice) in chimeric p190-BCR-ABL+ leukemic mice to levels similar to wild-type leukemic cells. These data indicate that Scribble-deficient leukemogenesis is dependent on oncogene induced Cdc42 activity in lymphoid progenitors. Furthermore, Scribble deficiency in leukemic progenitors increases the activation of the AMPK/mTORC1 signaling pathway and the protein expression and transcriptional activity of its downstream effector hypoxia-inducing factor-1α (HIF-1α). HIF-1α silencing by constitutive shRNA expression or inducible deletion in Scribble deleted B-lymphoid leukemic cells restored leukemic progenitor clonogenic efficiency (CFU average: 52 vs 110 per 1,000 B220+/EGFP+ BM cells, in Scribble and double Scribble/HIF-1α deficient, respectively; p≤0.01) and B-lymphoid leukemogenesis in vivo (median survival of 62 days; p≤0.05 compared with Scribble deficient chimeric animals). In addition, double deficiency of Scribble and HIF-1α restored AMPK/mTORC1 signaling to Wt leukemic levels. This data indicates that Scribble is a negative regulator of HIF-1α expression and activity, and the restoration of HIF-1α expression and activity to normal leukemic levels is necessary to restore leukemogenesis. Altogether, our data indicates that Scribble is a positive regulator of oncogenesis in leukemic progenitors, in vitro and in vivo, through Cdc42 and HIF-1α activities. Disclosures No relevant conflicts of interest to declare.

Background: Recent clinical studies suggest that the prophylactic use of recombinant factor VIIa (rFVIIa) markedly reduces the number of bleeding episodes in hemophilic patients with inhibitors. Given the short biological half-life of rFVIIa, it is unclear how rFVIIa could be effective in prophylactic treatment. Objectives: To examine the extravascular distribution of pharmacologically administered rFVIIa to obtain clues on how rFVIIa could work in prophylaxis. Methods: Recombinant mouse FVIIa tagged with AF488 fluorophore (AF488-FVIIa) was administered into mice via the tail vein. At different time intervals following the administration, mice were exsanguinated and various tissues were collected. The tissue sections were processed for immunohistochemistry to evaluate distribution of rFVIIa. Results: rFVIIa, immediately following the administration, associated with the endothelium lining of large blood vessels. Within 1 h, rFVIIa bound to endothelial cells was transferred to the perivascular tissue surrounding the blood vessels and thereafter diffused throughout the tissue. In the liver, rFVIIa was localized to sinusoidal capillaries and accumulated in hepatocytes. In bone, rFVIIa was accumulated in the zone of calcified cartilage and some of it was retained there for a week. The common finding of the present study is that rFVIIa in extravascular spaces was mostly localized to regions that contain TF expressing cells. Conclusions: The present study demonstrates that pharmacologically administered rFVIIa readily associates with the vascular endothelium and subsequently enters into extravascular spaces where it is likely to bind to TF and is retained for extended time periods. This may explain the prolonged pharmacological effect of rFVIIa. (Less)

Although the binding of endothelial cell protein C receptor (EPCR) to its ligands is well characterized at the biochemical level, it remains unclear how EPCR interaction with its ligands at the cell surface impacts its cellular trafficking. We characterized the cellular localization and trafficking of EPCR in endothelial cells and a heterologous expression system. Immunofluorescence confocal microscopy studies revealed that a majority of EPCR is localized on the cell surface in membrane microdomains that are positive for caveolin-1. A small fraction of EPCR is also localized intracellularly in the recycling compartment. Factor VIIa (FVIIa) or activated protein C binding to EPCR promoted the internalization of EPCR. EPCR and EPCR-bound ligands were endocytosed rapidly via a dynamin- and caveolar-dependent pathway. The endocytosed receptor-ligand complexes were accumulated in a recycling compartment before being targeted back to the cell surface. EPCR-mediated FVIIa endocytosis/recycling also resulted in transport of FVIIa from the apical to the basal side. In vivo studies in mice showed that blockade of EPCR with EPCR-blocking antibodies impaired the early phase of FVIIa clearance. Overall, our results show that FVIIa or activated protein C binding to EPCR promotes EPCR endocytosis, and EPCR-mediated endocytosis may facilitate the transcytosis of FVIIa and its clearance from the circulation.

We demonstrate for the first time the expression of 14.3.3sigma, an epithelial cell differentiation marker, in human corneal epithelium. 14.3.3sigma appeared at 30 kDa, pI 4-5, in 2D gels of corneal extracts. We found no significant differences in 14.3.3sigma levels between healthy corneas and corneas from keratoconus, corneal dystrophy, and corneal edema patients. 14.3.3sigma immunofluorescence was observed in the cytoplasm and nucleus of epithelial cells and colocalized with cyclin-B1. 14.3.3sigma was secreted by HCE-2 cells; HCE-2-conditioned medium induced matrix metalloproteinase-1 in cultured keratocytes. In summary, our work presents evidence of 14.3.3sigma expression in corneal epithelium and elaborates over its possible implications in corneal pathologic conditions.

Diabetic retinopathy is a sight-threatening complication of diabetes, and loss of pericytes represents early signs of its development. We tested the hypothesis that high glucose levels may induce signs of oxidative stress in cultured bovine retinal pericytes. Pericytes were exposed to either normal (5.5 mM) or high (22 mM) glucose levels for 1, 3, and 5 days. Signs of oxidative stress were measured by expression of copper/zinc superoxide dismutase, manganese superoxide dismutase, catalase, and glutathione peroxidase using real-time RTPCR. To elucidate the role of oxidative stress, we also measured glutathione (GSH) concentration in the cells and investigated the impact of thiol-reactive metal ions and hydrogen peroxide (H(2)O(2)) on intracellular GSH. Despite the stimulation with high glucose, thiol-reactive metal ions, or H(2)O(2), there was no clear increased expression of antioxidant enzymes or influence of GSH levels. Lipid peroxidation (malondialdehyde level) was increased in bovine aortic smooth muscle cells, but not in bovine retinal pericytes. The data indicate that pericytes do not develop oxidative stress in response to hyperglycemia. However, it is not definitively excluded that oxidative stress may occur after longer time periods of glucose stimulation.

Aims: To determine whether albuminuria, hypertension, or HbA(1c) are independently associated with antipericyte autoantibodies (APAAs) in type 2 diabetes mellitus. Methods: Two hundred ninety-nine subjects with different degrees of retinopathy according to the Early Treatment Diabetic Retinopathy Study Scale participated in this study. Albuminuria was defined as an albumin/creatinine ratio above the normal cutoff limit, that is, 2.0 g/mol for men and 2.8 g/mol for women. Hypertension was defined as a diastolic blood pressure more than 90 mm Hg, a systolic blood pressure more than 140 mm Hg, or pharmacological antihypertensive treatment. Serum APAAs were detected by immunofluorescence on tissue-cultured bovine retinal pericytes. Association analysis was performed using univariate and multivariate statistical tools. Results: In type 2 diabetes, APAAs were independently associated with albuminuria (OR = 0.56; P < .04), hypertension (OR = 2.21; P < .01), as well as with proliferative retinopathy (OR = 0.39; P < .01). Conclusions: The increased prevalence of APAA in patients with hypertension may suggest that these antibodies are related to tissue damage and repair and that the decline in frequency with albuminuria may serve as a marker for more advanced angiopathy. Future longitudinal studies are needed to determine whether the frequency of APAA is associated with the progression of angiopathy, and to determine the biological activity and antigens recognized by the antibody. (Less)

Life-long hematopoietic demands are met by a pool of hematopoietic stem cells (HSC) with self-renewal and multipotential differentiation ability. Humoral and paracrine signals from the bone marrow (BM) hematopoietic microenvironment control HSC activity. Cell-to-cell communication through connexin (Cx) containing gap junctions (GJs) allows pluricellular coordination and synchronization through transfer of small molecules with messenger activity. Hematopoietic and surrounding nonhematopoietic cells communicate each other through GJs, which regulate fetal and postnatal HSC content and function in hematopoietic tissues. Traffic of HSC between peripheral blood and BM is also dependent on Cx proteins. Cx mutations are associated with human disease and hematopoietic dysfunction and Cx signaling may represent a target for therapeutic intervention. In this review, we illustrate and highlight the importance of Cxs in the regulation of hematopoietic homeostasis under normal and pathological conditions.

The Rho family of GTPases is composed of members of the Ras superfamily of proteins. They are GTP-bound molecules with a modest intrinsic GTPase activity that can be accelerated upon activation/localization of specialized guanine nucleotide exchange factors. Members of this family act as molecular switches, and are required for coordinated cytoskeletal rearrangements which are crucial in a set of specialized functions of mammalian stem cells. These functions include self-renewal, adhesion, and migration. Mouse gene-targeting studies have provided convincing evidence of the indispensable and dispensable roles of individual members of the Rho GTPase family, and the putative upstream and downstream mediators in stem cell-specific functions. The role of Rho GTPases and related signaling pathways previously seen in other cell types and organisms have been confirmed in mammalian hematopoietic stem cells (HSC), and new signaling pathways and unexpected functions unique to HSC have been identified and dissected. This review intends to summarize our current understanding of the role of the Rho family of GTPases on HSC and progenitor activity through cytoskeleton-mediated signaling pathways, and to provide insight on relevant signaling pathways that regulate mammalian stem cell self-renewal, adhesion and migration in health and HSC disease.

Search of molecular targets to expand functional hematopoietic stem cells (HSC) for transplantation and hematological cancer therapy remains paramount, particularly when manipulation of cellular quiescence is required. HSC are highly quiescent cells with the ability to rapidly enter the cell cycle in response to microenvironment cues and divide through changes in their polarity. Despite cellular polarity being one of the most basic properties of all living cells, the role that polarity regulators have on polarization and function of HSC remains controversial. Scribble, of the Scribble Polarity Complex, controls the spatial organization of intracellular proteins and negatively regulates the cell cycle. By using a combination of constitutive and inducible hematopoietic-specific Scribble-deficient animal models, hematopoietic reconstitution assays, structure-function mutants of Scribble and intracellular protein trafficking analysis, we identified the functional relevance of Scribble in HSC activity. We observed that Scribble is polarized in HSC and deletion of Scribble leads to a ~ 50% reduction in the bone marrow (BM) HSC population (p30 days median survival vs 16 days for KO mice, p Disclosures No relevant conflicts of interest to declare.

Background: Pericytes cover the abluminal surface of microvessels and play an important role in capillary regulation and pathology. Studies on pericytes have been hindered by the lack of specific markers with which to facilitate microscopic identification of this cell type. Expression of the cell surface 3G5 ganglioside antigen has been reported in cultured retinal and cardiac pericytes. The objective of this study was to determine the usefulness of monoclonal antibody 3G5 as a pericyte marker in human skin. Methods: Cryosections of 21 skin biopsies were examined by direct fluorescence technique with anti-3G5, anti-von Willebrand factor, anti-α-smooth muscle actin or DNA fluorochrome. Results: In human dermis, 3G5 expression is limited to pericytes discriminating this cell type from all other cells including smooth muscle cells, myofibroblasts and myoepithelial cells. We found a pericyte: endothelial cell ratio of 1:12.4 (±7.1), and a difference of α-smooth muscle actin expression between the subpapillary plexus and the microvessels of the Stratum reticulare. Conclusions: 3G5 mAB is an excellent and so far the only reported tool for identification of dermal pericytes by fluorescent light microscopy. Moreover, this is the first report of the application of 3G5 technique to the microvasculature in tissue sections at the light microscopic level.

Glycolipid antigens are emerging as important markers of differentiated cells in vitro and in vivo. The study of the expression of these antigens in whole tissues by immunoelectron microscopy, using standard techniques, does not give acceptable results. We have established conditions for the specific demonstration of antibody binding to tissue glycolipid antigens by immunoelectron microscopy. Dehydration of tissues with alcohol is to be avoided as it extracts the glycolipid antigen out of the tissue. Dehydration in acetone provides good results. Embedding of the tissue in Araldite 512 results in high non-specific binding of the primary antibody and a decreased effective titre of the primary antibody. Embedding of tissues in Lowicryl HM20 resin resulted in low non-specific binding. We also describe a method of curing the Lowicryl resin that does not require a purpose built curing chamber. Quantitative analysis of immunogold binding reveals that acetone dehydration of tissues and embedding in Lowicryl gives greatly superior results in comparison with dehydration in alcohol and embedding in Araldite.

Using the extended Thomas-Fermi plus Strutinsky integral approximation to the Skyrme-Hartree-Fock method we show that provided the nuclear mass data are well fitted extrapolations to neutron-rich and super-heavy nuclei are quite insensitive to generalizations intended to simulate the main effects of relativistic mean-field theory. In particular, as far as energy-related quantities are concerned, it is sufficient to use the conventional spin-orbit force.

Hereditary neutropenia is usually caused by heterozygous germline mutations in the ELANE gene encoding neutrophil elastase (NE). How mutations cause disease remains uncertain, but two hypotheses have been proposed. In one, ELANE mutations lead to mislocalization of NE. In the other, ELANE mutations disturb protein folding, inducing an unfolded protein response in the endoplasmic reticulum (ER). In this study, we describe new types of mutations that disrupt the translational start site. At first glance, they should block translation and are incompatible with either the mislocalization or misfolding hypotheses, which require mutant protein for pathogenicity. We find that start-site mutations, instead, force translation from downstream in-frame initiation codons, yielding amino-terminally truncated isoforms lacking ER-localizing (pre) and zymogen-maintaining (pro) sequences, yet retain essential catalytic residues. Patient-derived induced pluripotent stem cells recapitulate hematopoietic and molecular phenotypes. Expression of the amino-terminally deleted isoforms in vitro reduces myeloid cell clonogenic capacity. We define an internal ribosome entry site (IRES) within ELANE and demonstrate that adjacent mutations modulate IRES activity, independently of protein-coding sequence alterations. Some ELANE mutations, therefore, appear to cause neutropenia via the production of amino-terminally deleted NE isoforms rather than by altering the coding sequence of the full-length protein.

Krüppel-like factor 5 regulates pluripotent stem cell self-renewal, but its role in somatic stem cells is unknown. Here we show that Krüppel-like factor 5-deficient haematopoietic stem cells and progenitors fail to engraft after transplantation. This haematopoietic stem cell and progenitor defect is associated with impaired bone marrow homing and lodging and decreased retention in bone marrow, and with decreased adhesion to fibronectin and expression of membrane-bound β1/β2-integrins. In vivo-inducible gain-of-function of Krüppel-like factor 5 in haematopoietic stem cells increases haematopoietic stem cell and progenitor adhesion. The expression of Rab5 family members, mediators of β1/β2-integrin recycling in the early endosome, is decreased in Klf5(Δ/Δ) haematopoietic stem cells and progenitors. Krüppel-like factor 5 binds directly to the promoter of Rab5a/b, and overexpression of Rab5b rescues the expression of activated β1/β2-integrins, adhesion and bone marrow homing of Klf5(Δ/Δ) haematopoietic stem cells and progenitors. Altogether, these data indicate that Krüppel-like factor 5 is indispensable for adhesion, homing, lodging and retention of haematopoietic stem cells and progenitors in the bone marrow through Rab5-dependent post-translational regulation of β1/β2 integrins.

Multiple sclerosis (MS) is assumed to be an autoimmune disease initiated by autoreactive T cells that recognize central nervous system antigens. Although adaptive immunity is clearly involved in MS pathogenesis, innate immunity increasingly appears to be implicated in the disease. We and others have presented evidence that natural killer (NK) cells may be involved in immunoregulation in MS, leading to the question of whether a particular NK cell subtype will account for this effect. Changes of NK cell functionality in MS were associated with MS activity, and depletion of NK cells exacerbated the course of disease in a murine model of MS, experimental autoimmune encephalomyelitis. Several studies described a deficiency and transient "valleys" in NK cell killing activity in human MS, which may coincide with symptomatic relapse. However, the molecular basis of the defect in killing activity has not been determined. We discuss results on the expression of perforin in CD16(+) NK cells and the existence of an inverse relationship between myelin loaded phagocytes and the proportion of CD16(+) NK cells expressing perforin in the circulation. This inverse relationship is consistent with a role for NK cell killing activity in dampening autoimmunity. On the other hand, it has been broadly reported that first line MS therapies, such as interferon-beta, glatiramer acetate as well as escalation therapies such as fingolimod, daclizumab, or mitoxantrone seem to affect NK cell functionality and phenotype in vivo. Therefore, in this review we consider evidence for the immunoregulatory role of NK cells in MS and its animal models. Furthermore, we discuss the effect of MS treatments on NK cell activity.

Whereas actin-binding proteins (ABPs) regulate network formation during the cell cycle, it is not known whether ABPs also function to sequester or target isoactins to specific subcellular compartments. Recently, we have shown that ezrin indirectly associates with beta, but not alpha actin filaments in a calcium- and cytochalasin-sensitive manner [Shuster and Herman, 1995: J. Cell Biol. 128:837-848]. To identify the beta actin-specific binding protein that fosters ezrin-beta actin interactions, we developed an isoactin affinity fractionation and F-isoactin overlay/Western blotting technique. Results reveal that a 73 kd polypeptide that co-precipitates with ezrin and beta actin [Shuster and Herman, 1995: J. Cell Biol. 128:837-848] can also binds directly to filaments of beta, but not alpha actin by isoactin overlay. In an effort to establish whether p73 plays a role in regulating beta actin dynamics in cells, we produced monoclonal antibodies by immunizing BALB/c mice with p73-containing lamellar lysates or high salt elutions from beta actin affinity columns. Two monoclonal antibodies were cloned that react with p73 present in fractions released from beta actin Sepharose-4B or purified to homogeneity by DEAE chromatography. Anti-p73 Western blots reveal that there is a 16-fold difference in p73 binding to beta actin vs. alpha actin affinity columns when experiments are performed in physiological salts. To characterize p73-beta actin binding in vitro and establish whether p73 binds along the lengths or at the barbed end of the beta actin filament, we asked whether cytochalasin D (CD) could displace p73 pre-bound to beta actin-Sepharose 4B. Anti-p73 Western blotting reveals that nanomolar concentrations of CD are capable of selectively eluting p73 and ezrin from beta actin Sepharose 4B, indicating that p73 binds beta actin via the barbed end. Simultaneous double antibody localization studies using anti-beta actin IgG and anti-p73 IgM reveal that p73 and beta actin are co-localized in the forward aspects of motile cytoplasmic domains, in close proximity to the plasma membrane. Because of its isoform-specific interactions with the barbed end of beta actin filaments, we have named this molecule beta cap73. These results indicate that isoform-specific actin-binding proteins can be identified from cortical cytoplasm, and suggest that beta cap73 may not only act to spatially regulate the intracellular distribution of isoactins, but may also facilitate forward protrusion formation through the regulated release of free filament ends during cell motility.

Neutrophils are a key component in the innate immune system and are crucial in the protection against bacterial and fungal infections. Patients with neutropenias are at high risk to develop serious life threatening bacterial and fungal infections. Transfusion of donor neutrophils into neutropenic patients may help alleviate disease burden, but difficulty in collecting and transfusing sufficient quantities of viable donor neutrophils has limited the clinical adaptation of neutrophil replacement therapies. Induced pluripotent stem cells (iPSC) are an attractive alternative source to donor derived neutrophils due to their ability to self-renew and differentiate into cells of the three embryonic germ layers, including neutrophils. Successful implementation of iPSC for neutrophil replacement therapies will require a) derivation of iPSC, b) large scale culture of iPSC, and c) efficient differentiation of these iPSC into functional neutrophils in xeno-fee, GMP-compliant conditions. First, normal human fibroblasts were reprogrammed in xeno-free, GMP compliant conditions though nucleofection of a non-integrating 3 plasmid system carrying OCT4, SOX2, KLF-4, l-MYC, Lin28 and shp53. The reprogrammed fibroblasts were cultured in defined conditions on Vitronectin XF in TeSR-E7 media until colony formation when they were transitioned to E8 medium. We generated 3 lines under these conditions and all iPSC derived and cultured under these conditions retain a normal karyotype, express pluripotency markers SSEA-4, Tra-1-60, Tra-1-81, and CD9 at >85% as determined by FACs analysis, and differentiate into all three germ layers in vitro. Once iPSC lines were established, cultures were adapted from adherent cultures to non-adherent, aggregate suspension cultures in spinner flask bioreactor systems in mTeSR or E8 medium. Briefly, adherent iPSC are dissociated into single cells and seeded into a spinner flask at a concentration of 2×105 cells/ml and spun at 60 RPM. We evaluated 2 lines in 3 independent experiments and cells readily formed clusters and were able to achieve 4-log expansion (range: 2-4 log) within approximately 40 days (10 passages). All lines retained a normal karyotype, expressed pluripotency markers SSEA-4, Tra-1-60, Tra-1-81, and CD9 (>85%), and displayed a normal morphology when re-plated in adherent culture. We next adapted a neutrophil differentiation system for differentiation from iPSC from the defined media/spinnerflask system. Clusters of cells from spinner flask cultures were induced to form embryoid bodies (EBs) by transitioning the cultures to EB specification medium for 5 days, followed by a neutrophil specification medium containing IL-3 and G-CSF in adherent conditions. After 7-14 days in neutrophil specification conditions, EBs attached to the plate and CD45+CD11b+CD16+ neutrophils were shed into the medium and were harvested twice weekly for 30-60 days. In 10 experiments, we collected an average of 6.5×10^5 CD45+ cells per well of differentiation representing a 6.5 fold expansion (range: 3-10 fold). In conclusion, iPSC have been derived in GMP compliant conditions and can be cultured at a large scale using spinner flask bioreactors, and the iPSC from spinner flask bioreactors can then successfully be differentiated into CD45+ cells. Thus, iPSC represent an attractive, self-renewing resource of neutrophils for neutrophil replacement therapies.

To the editor: Endothelial cell protein C receptor (EPCR) is the cellular receptor for protein C (PC) and activated protein C (APC). Studies from our laboratory[1][1] and others[2][2],[3][3] have shown that FVII/FVIIa also binds to EPCR in a true-ligand fashion. Recently, it has been suggested that

Expression of a monoclonal antibody (3G5) defined ganglioside antigen in the renal cortex. The monoclonal antibody (mAb) 3G5 was found, by indirect immunofluorescence, to bind to renal cortical structures in frozen sections of human, rat and calf kidneys. Double indirect immunofluorescence studies on frozen sections of rat kidneys showed that 3G5 stained only the glomerulus and the distribution of the 3G5 antigen on the glomerulus was more extensive than the staining observed with antibodies to Factor VIII antigen. 3G5 stained the proximal convoluted tubules and collecting tubules in bovine renal sections but glomeruli did not stain with 3G5. The 3G5 mAb did not stain tissue cultured bovine glomerular endothelial cells or mesangial cells, but did stain bovine glomerular epithelial cell cultures. 3G5 did not stain MDCK cell cultures. The binding of mAb 3G5 to glomeruli was investigated by immunoelectron microscopy of rat renal tissue. In contrast to the podocyte specificity on bovine glomerular cells in vitro, it was found that the specificity of 3G5 expression on rat glomerular cells in vivo was broader. No binding of mAb 3G5 was found outside the glomerulus in the rat renal cortex. Podocytes, endothelial cells and capsular epithelial cells expressed the 3G5 antigen most strongly. A lesser amount of binding was found in the glomerular basement membrane. The mesangium showed a little binding of mAb 3G5 and no binding at all was found to other cortical structures. The 3G5 antigen in rat renal tissue was found to be a glycolipid that migrated between the ganglioside markers GM 2 and GM 1 by immunostaining of thin layer chromatograms. The 3G5 mAb has previously been shown to react with microvascular pericytes, resting T-lymphocytes and cells of the brain, thyroid gland and adrenal gland as well as various malignancies. The significance of the tissue specific patterns of expression remains unclear and will be understood only when the function of this ganglioside is elucidated.

We determined whether human NK cells could contribute to immune defenses against Mycobacterium tuberculosis through production of IL-22. CD3−CD56+ NK cells produced IL-22 when exposed to autologous monocytes and γ-irradiated M. tuberculosis, and this depended on the presence of IL-15 and IL-23, but not IL-12 or IL-18. IL-15-stimulated NK cells expressed 10.6 times more DAP10 mRNA compared with control NK cells, and DAP10 siRNA inhibited IL-15-mediated IL-22 production by NK cells. Soluble factors produced by IL-15-activated NK cells inhibited growth of M. tuberculosis in macrophages, and this effect was reversed by anti-IL-22. Addition of rIL-22 to infected macrophages enhanced phagolysosomal fusion and reduced growth of M. tuberculosis. We conclude that NK cells can contribute to immune defenses against M. tuberculosis through production of IL-22, which inhibits intracellular mycobacterial growth by enhancing phagolysosomal fusion. IL-15 and DAP-10 elicit IL-22 production by NK cells in response to M. tuberculosis.

In general, coronins play an important role in actin-based processes, and are expressed in a variety of eukaryotic cells, including Leishmania. Here, we show that Leishmania coronin preferentially distributes to the distal tip during cytokinesis, and interacts with microtubules through a microtubule-based motor, kinesin K39. We further show that reduction in coronin levels by 40-50% in heterozygous coronin mutants results in generation of bipolar cells (25-30%), specifically in the log phase, owing to unregulated growth of the corset microtubules. Further analysis of bipolar cells revealed that the main cause of generation of bipolar cell morphology is the intrusion of the persistently growing corset microtubules into the other daughter cell corset from the opposite direction. This defect in cytokinesis, however, disappears upon episomal gene complementation. Additionally, our attempts to prepare homozygous mutants were unsuccessful, as only the aneuploid cells survive the selection process. These results indicate that coronin regulates microtubule remodeling during Leishmania cytokinesis and is essentially required for survival of these parasites in culture.

The presence of actin in Leishmania has recently been demonstrated, but the functional form of this protein (filamentous actin) has not yet been identified. We report here that the putative coronin homologue identified in the Leishmania genome is invariably associated with the filament-like structures of actin in Leishmania promastigotes. The occurrence of filamentous structures is significantly increased upon overexpression of Leishmania coronin as its GFP fusion product in Leishmania cells. However, expression of Leishmania actin or coronin alone in mammalian cells does not result in formation of any filament-like structures of Leishmania actin or association of Leishmania coronin with mammalian filamentous actin, but coexpression of both the proteins in these cells leads to formation of filamentous structures containing Leishmania actin and coronin. The high specificity of Leishmania coronin for Leishmania actin could be attributed to its unique structure as it differs from other coronins not only in the unique region but also in the actin-binding site and leucine zipper motif. These results taken together indicate that Leishmania contains a novel form of coronin which colocalizes with actin in filament-like structures in these cells.

The 3G5-reactive ganglioside antigen (3G5 antigen) is expressed on the surface of various cell types including pericytes, pancreatic islet cells, thyroid follicular cells, and cells of the pituitary and the adrenal medulla. Expression on melanocytes has not yet been reported. We examined 148 5-microm cryosections of 12 normal skin samples and 45 skin tumors (21 melanocytic nevi, 8 malignant melanoma primaries, 4 metastases of malignant melanoma, 3 basal cell carcinomas, and 9 pigmented seborrheic keratoses) by triple fluorescence technique with the monoclonal antibody 3G5, DNA fluorochrome, and the anti-melanocytic antibody A103 (Anti-Melan-A). In normal skin, 3G5 reactivity was detected in epidermal melanocytes of 4 of 12 cases with 14.8 +/- 24.1% positive melanocytes; 20 of 21 nevi (72.2 +/- 29.1% positive nevus cells, mean +/- SD), 8 of 8 primary melanomas (83.9 +/- 12.3% positive melanoma cells), and 4 of 4 melanoma metastases (82.5 +/- 6.5% positive melanoma cells) expressed the 3G5 antigen. All tumor cells of investigated basal cell carcinoma or seborrheic keratosis were 3G5 negative. This is the first report of 3G5 antigen expression in melanocytes. The data demonstrate high expression of this ganglioside in the aggregated melanocytes of malignant or benign tumors but low or absent expression in singular melanocytes (normal epidermis, seborrheic keratoses) reflecting a different biologic state.

PURPOSE To evaluate the anti-ganglioside monoclonal antibody 3G5 as a marker of corneal keratocytes. METHODS 3G5 expression on keratocytes was investigated by immunofluorescence microscopy. Studies were performed on frozen sections of normal human, bovine, porcine, rabbit, rat, and mouse corneas and on repairing rabbit cornea. In vitro studies were performed on tissue-cultured human, bovine, porcine, mouse, and rabbit keratocytes. RESULTS 3G5 stained frozen sections of human, bovine, porcine, rat, and rabbit cornea but not mouse cornea and the staining pattern followed the distribution of stromal keratocytes but did not stain epithelium or endothelium. Subconfluent human and bovine keratocyte cultures were 3G5 negative. Almost 100% of the human and bovine cells that were maintained at confluence without replacement of serum-containing culture medium for 2 weeks became 3G5 positive. The 3G5 antigen was constitutively expressed on cultured rabbit and porcine keratocytes under all conditions examined. Mouse keratocyte cultures did not express 3G5. The 3G5 antigen was not present on myofibroblastic cells in the repairing area of a full-thickness wound in rabbit cornea that had been healing for 20 days. The area surrounding the healing wound expressed 3G5 antigen in an altered distribution, whereas 3G5 antigen was distributed in the expected pattern in areas that were distant from the wound. When rabbit keratocytes were induced to express the myofibroblast marker alpha-smooth muscle actin by treatment with TGFbeta1 in vitro, the pattern of 3G5 staining was altered. CONCLUSIONS The 3G5 antigen is a useful marker for the identification of corneal keratocytes and for documenting their response to environmental stimuli associated with wound repair.

The growth of new capillaries from existing vessels (angiogenesis) is of fundamental importance in wound healing and in pathological situations such as proliferative diabetic retinopathy (1), rheumatoid arthritis (2), and tumor growth. Consequently, considerable interest in vascular cell biology has arisen in apparently disparate clinical and experimental fields. Held in common, however, is the hope that an understanding of the cellular and molecular mechanisms that regulate angiogenesis will lead to novel therapeutic agents and targets.

Aims/Hypothesis. This study aims to determine the prevalence of anti-pericyte autoantibodies in Type 2 diabetes and to characterize these autoantibodies as new markers of disease activity in diabetic retinopathy. Methods. A total of 299 patients with Type 2 diabetes participated in this study. Retinopathy was assessed by 7-field stereo fundus photography and was graded according to the ETDRS scale. Serum anti-pericyte autoantibodies were detected by immunofluorescence on tissue cultured bovine retinal pericytes. Results. The prevalence of anti-pericyte autoantibodies in Type 2 diabetic patients was 54% and was approximately equal in men and women. The prevalence was approximately 55% with retinopathy at grades from 10 to 53. At grades above 53 the prevalence declined to 23% (p

In the bone marrow (BM), hematopoietic stem cells and progenitors (HSC/P) reside in specific anatomical niches. Among these niches, a functional osteoblast (Ob)-macrophage (MΦ) niche has been described where Ob and MΦ (so called "osteomacs") are in direct relationship. A connection between innate immunity surveillance and traffic of hematopoietic stem cells/progenitors (HSC/P) has been demonstrated but the regulatory signals that instruct immune regulation from MΦ and Ob on HSC/P circulation are unknown. The adaptor protein sequestosome 1 (Sqstm1), contains a Phox bemp1 (PB1) domain which regulates signal specificities through PB1-PB1 scaffolding and processes of autophagy. Using microenvironment and osteoblast-specific mice deficient in Sqstm1, we discovered that the deficiency of Sqstm1 results in macrophage contact-dependent activation of Ob IKK/NF-κB, in vitro and in vivo repression of Ccl4 (a CCR5 binding chemokine that has been shown to modulate microenvironment Cxcl12-mediated responses of HSC/P), HSC/P egress and deficient BM homing of wild-type HSC/P. Interestingly, while Ccl4 expression is practically undetectable in wild-type or Sqstm1-/- Ob, primary Ob co-cultured with wild-type BM-derived MΦ strongly upregulate Ccl4 expression, which returns to normal levels upon genetic deletion of Ob Sqstm1. We discovered that MΦ can activate an inflammatory pathway in wild-type Ob which include upregulation of activated focal adhesion kinase (p-FAK), IκB kinase (IKK), nuclear factor (NF)-κB and Ccl4 expression through direct cell-to-cell interaction. Sqstm1-/- Ob cocultured with MΦ strongly upregulated p-IKBα and NF-κB activity, downregulated Ccl4 expression and secretion and repressed osteogenesis. Forced expression of Sqstm1, but not of an oligomerization-deficient mutant, in Sqstm1-/- Ob restored normal levels of p-IKBα, NF-κB activity, Ccl4 expression and osteogenic differentiation, indicating that Sqstm1 dependent Ccl4 expression depends on localization to the autophagosome formation site. Finally, Ob Sqstm1 deficiency results in upregulation of Nbr1, a protein containing a PB1 interacting domain. Combined deficiency of Sqstm1 and Nbr1 rescues all in vivo and in vitro phenotypes of Sqstm1 deficiency related to osteogenesis and HSC/P egression in vivo. Together, this data indicated that Sqstm1 oligomerization and functional repression of its PB1 binding partner Nbr1 are required for Ob dependent Ccl4 production and HSC/P retention, resulting in a functional signaling network affecting at least three cell types. A functional ‘MΦ-Ob niche’ is required for HSC/P retention where Ob Sqstm1 is a negative regulator of MΦ dependent Ob NF-κB activation, Ob differentiation and BM HSC/P traffic to circulation. Disclosures Starczynowski: Celgene: Research Funding. Cancelas:Cerus Co: Research Funding; P2D Inc: Employment; Terumo BCT: Research Funding; Haemonetics Inc: Research Funding; MacoPharma LLC: Research Funding; Therapure Inc.: Consultancy, Research Funding; Biomedical Excellence for Safer Transfusion: Research Funding; New Health Sciences Inc: Consultancy.

Pericytes cover the abluminal surface of capillaries and venules and are thought to play an important role in microvascular regulation and pathology. The purpose of this study was to isolate and characterize human dermal microvascular pericytes (HDMPC), a minor cell type in the skin but a relatively easily obtainable human source of tissue. We developed and compared two procedures that differed in the preselection method. Isolation of dermal microvessel fragments from neonatal foreskins by trypsin digestion was followed by mechanical release of subepidermal tissue, collagenase treatment, and sieving through 100- and 30-microm meshes. After subcultivation, pericytes were preselected either by isolation of outgrowing capillary fragments or by 3G5-coupled magnetic beads. Pericytes were selected finally by cultivation of single cells in endothelial cell-conditioned media. Cultured HDMPC were seen to be large and well spread with irregular edges and prominent stress fibers. They lack contact inhibition, are positive for 3G5 antigen, alpha-smooth muscle actin, and vimentin, and are negative for the endothelial cell marker CD31, diI-acetylated low-density lipoprotein uptake, cytokeratin 5, 6, and 18, and S100 protein. Using both preselection methods, we could establish purified cell cultures of HDMPC. The results of these studies represent the first report of HDMPC isolation.

Two different types of blood glucose self-monitoring device, designed for use by diabetics (Accu-Chek Advantage* and Accu-Chek III*), were evaluated for the purpose of monitoring glucose concentration in tissue culture media. The Accu-Chek Advantage* meter was found to systematically overestimate the glucose concentration in a variety of commonly used tissue culture media by 50-90%, in comparison with their formulated glucose concentration. The Accu-Chek III* meter reliably estimated glucose concentrations from 100 to 300 mg/dl and overestimated glucose concentrations above 300 mg/dl. The systematic overestimation of glucose concentration in tissue culture fluids by the Accu-Chek Advantage* meter was further investigated. A standard curve was constructed and the meter reading was found to be linearly related to the actual glucose concentration and the best linear fit was given by the formula y = -43.504 + 1.9246x, where y is the meter reading and x is the actual glucose concentration. Rearranging the equation to make x the subject gave the following algorithm x = (y + 43.504) divided by 1.9246 which could be used to correct the 'raw' meter reading. The mean corrected glucose concentrations deviated from the formulated glucose concentration by less than 3.5% in five media tested, indicating that this meter is more than adequate for monitoring glucose consumption by cells growing in hollow fiber cartridge bioreactors, when used in conjunction with this correction factor.

Severe congenital neutropenia (SCN) is a hereditary neutropenia characterized by granulocytic precursor differentiation arrest in which the pathogenic mechanism is poorly understood. Over half of SCN patients associate with autosomal dominant point mutations in the gene encoding neutrophil elastase (ELANE). High-dose G-CSF therapy succeeds in increasing neutrophil counts in many SCN patients, but the molecular explanation for high dose G-CSF rescue is unknown. Mutations in the G-CSF receptor (CSF3R) associated with a dramatic risk for transformation provides a cautionary note for high dose G-CSF treatment. The pathogenic mechanism of ELANE mutations is incompletely understood due to the lack of disease recapitulation in murine models of ELANE-mutant hematopoiesis and the difficulty to obtain primary, relevant myeloid cell populations from patients. Using induced pluripotent stem cell (iPSC) derived myelopoietic cultures from peripheral blood mononuclear cells of patients with ELANE exon 3 point mutations (Q97P and I118N), we designed experiments to recapitulate G-CSF signaling in SCN myelopoiesis. The patient-derived iPSC lines were characteristically similar to human embryonic stem cell lines and normal blood-derived iPSC lines with expression of SSEA-4, Tra-1-60, Tra-1-81, and CD9 pluripotency markers >85% in all lines as determined by FACS analysis at passage 10-15. All iPSC retained a normal karyotype and ELANE locus mutations of the original specimens. Both SCN iPSC lines and control iPSC lines (normal and immune-mediated congenital neutropenia) show functional G-CSF (50 ng/mL)-induced differentiation into the myeloid lineage. However, G-CSF failed to induce terminal granulocytic differentiation of SCN iPSC derived myeloid progenitors resulting in differentiation arrest at the promyelocyte/myelocyte stage. The hematopoietic cells derived from SCN iPSC line show reduced frequency of CFU-G and CFU-GM (CFU-G+CFU-GM, 22±1.15 in 104 embryoid body cells from non SCN iPSC vs 4±2 in 104 embryoid body cells from ELANE mutated SCN iPSC). Moreover, SCN iPSC promyelocytes show significantly increased apoptosis over controls as determined by Annexin-V analysis (31% Annexin-V+ in SCN iPSC derived promyelocyte vs 3% in control iPSC). Surviving SCN promyelocytes showed decreased reactive oxygen species activity and no phagocytic activity. In contrast, suprapharmacological doses of G-CSF (1000 ng/mL) rescued SCN iPSC-derived differentiation. It has been shown that NE, expressed in promyelocytes, can cleave recombinant G-CSF and CSF3R. We evaluated the possibility that inadequate G-CSF signaling in SCN iPSC was due to abnormal elastase activity. To this end, we supplemented 50 ng/mL G-CSF cultures with a specific, cell-permeant neutrophil elastase inhibitor (Sivelastat, 230 nM) to find rescued granulocyte differentiation arrest (31% neutrophil in myeloid differentiation culture of SCN iPSC derived hematopoietic progenitors in presence of Sivelastat vs 2% mature neutrophils without Sivelastat). In conclusion, SCN modeling through patient iPSC derived myelopoiesis and G-CSF driven granulocytic recapitulation unveil a mechanism of resistance to G-CSF therapy with important translational implications for therapy. Combination of low-dose G-CSF and NE inhibitory therapy may result in an efficacious, safer approach in SCN therapy. Disclosures: No relevant conflicts of interest to declare.

Background: The immunogold labeling technique and transmission electron microscopy were used to demonstrate the expression and position of the intermediate filament vimentin in rat osteoblast and osteocyte cell bodies and cell processes. Conventional light and transmission electron microscopic studies of bone cells demonstrated adjacent cell linkage to be mediated by osteoblast and osteocyte processes present within the canalicular system traversing the bone matrix. The cell processes were filled with densely packed filaments, many of which have been shown previously to be actin microfilaments. The appearance, however, of 10 nm diameter filaments in some cell processes and the fact that the intermediate filament vimentin has been defined in many cells of mesenchymal origin raised the possibility that some of these filaments might be vimentin. The ultrastructural colloidal gold immunochemical technique allowed for demonstration in situ of the expression of vimentin filaments plus accurate definition of their position. Methods: The studies were performed in newborn rat femoral and tibial diaphyseal cortical bone and in 1-week-old repair bone from 2.4 mm diameter defects made through the lateral cortex in 6-week-old rat femurs and tibias. The bone tissues for the immunochemical study were fixed in 1% glutaraldehyde, 4% paraformaldehyde, and 0.1 M phosphate buffer (pH 7.4) for 2 days. Decalcification was performed in 6% EDTA for 2–3 days. Infiltration involved use of Lowicryl resin K4M, and the embedding and curing processes were performed in a cryostat with temperatures −30°C. An antivimentin monoclonal antibody was used for labeling using the postembedding technique. Effective antibody dilutions ranged from 1:10 to 1:200, with the dilutions of 1:25 and 1:100 showing the best combination of filament labeling with the least matrix background. The grids were exposed to 10 nanometer gold colloid conjugated goat anti-mouse IgM for demonstration of binding. Results: Vimentin immunolabeling was defined clearly in relation to filaments within the osteoblast and osteocyte cell body cytoplasm, throughout the entire length of the osteoblast and osteocyte cell processes, and in close relationship to the intercellular gap junctions which were present within the cell processes both close to the cell bodies and within the canaliculi well away from them. Conclusions: Immunogold labeling demonstrates the presence of the intermediate filament vimentin in osteoblast and osteocyte cell bodies and processes of rat bone. Vimentin distribution is not concentrated to specific areas, is present throughout the extent of the bodies and processes, and is seen immediately adjacent to gap junctions. © 1995 Wiley-Liss, Inc.

Abstract 113 The Kruppel-like factor 5 (Klf5) is a transcription factor that regulates cellular signaling and is involved in cell proliferation and oncogenesis. Recent studies have implicated Klf5 in the self-renewal and maintenance of pluripotent stem cells. In this project, we examined the role of Klf5 in hematopoietic stem/progenitor cells (HSC/P) activity. Using a murine model of inducible deficiency of Klf5 in hematopoietic tissue (Mx1-Cre/Klf5flox/flox, Klf5-deficient), we observed a severe engraftment deficiency in competitive repopulation assays in primary and secondary recipients, although it did not cause any significant hematopoietic defects basally or after chemotherapy stress in primary mice. Engraftment failure of Klf5-deficient HSC was associated with a severe defect in homing (1.8±0.8% vs 6.0±2.0% for Klf5-deficient and WT HSC/P, respectively, p Disclosures: No relevant conflicts of interest to declare.