Hemopoietic Progenitor Research Articles

Hemopoietic Progenitor Cells from the Bone Marrow of Serpinb3A-/- Mice Are Radioresistant

Introduction: Serpins are a superfamily of serine proteases that regulate proteolytic pathways by utilizing a conformational change to bind to and inhibit multiple peptidases. Currently 36 human serpins have been identified, but the roles of most are not fully understood (Silverman et al., J Biol Chem. 285(32):24299-305, 2010). A majority of serpins inhibit serine proteases but serpins that inhibit lysosomal cytosine peptidases, granzymes, serine protease and calpains. Previous studies have shown that serpins play a critical role in cell survival by blocking lysosomal induced necrosis injury. Serpinb3A (Antichymotrypsin) has been identified as an inhibitor of the serine peptidases, chymotrypsin and cathepsin G. We now report that hematopoietic progenitor cells in the bone marrow of Serpinb3A-/- mice are radioresistant.Materials & Methods: Serpinb3A-/- and control Balb/c mice were obtained from Charles River and were housed five per cage according to University of Pittsburgh Institutional Animal Care and Use Committee (IACUC) regulations. All protocols were approved by the University of Pittsburgh IACUC. Veterinary care was provided by the Division of Laboratory Animal Research of the University of Pittsburgh. Whole bone marrow was obtained by flushing the marrow from the femurs of sacrificed Serpinb3A-/- and Balb/c mice. Single cell suspensions were irradiated to doses ranging from 0 to 8 Gy using a at 70 cGy/min using a Shepherd Mark 1 137Cs γ-ray source (J.L. Shepherd, San Fernando, CA, USA). The cells were plated in 0.8% methylcellulose containing hematopoietic growth supportive medium (StemCell Technology product #3134) at varying densities in quadruplicate in Linbro plates (Fisher Scientific, Pittsburgh, PA, USA) and incubated at 37°C and 5% CO2 for 9-11 days at which time the number of colony forming unit granulocyte-macrophage (CFU-GM) colonies of 50 cells or greater were counted. The data from 5 experiments was analyzed using linear quadratic and single-hit, multi-target models.Results: CFU-GM from Serpinb3A-/- mouse marrow showed an increase in radiation resistance with an increase in the shoulder of the irradiation survival curve compared to Balb/c marrow (ň = 4.9 ± 1.4 and 1.4 ± 0.1, p = 0.0451, respectively). There was no significant difference in the Do for CFU-GM of the two mouse strains (Do = 1.48 ± 0.19 Gy for Serapinb3A-/- mice and 1.56 ± 0.06 Gy for Balb/c mice, p = 0.0997.Conclusions: The data support a possible role for the loss of Serpinb3a in providing a cell survival advantage for hematopoietic progenitor cells exposed to lethal doses of ionizing irradiation. This would suggest that the Serpinb3a target protease helps repair the cellular injury induced by ionizing radiation. Alternatively, the germ line loss of Serpinb3a may up-regulate the expression of other clade B serpins known to protect cells from injury. Ongoing studies will help determine the nature of this protective effect against radiation-induced cell death. DisclosuresNo relevant conflicts of interest to declare.

Treatment of aplastic anaemia with lower-dose anti-thymocyte globulin produces similar response rates and survival as per standard dose anti-thymocyte globulin schedules.

Aplastic anaemia (AA) is a rare acquired bone marrow failure syndrome resulting from the immune-mediated destruction of haemopoietic stem cells. For adults in whom first-line haemopoietic progenitor cell transplantation is not feasible, combination anti-thymocyte globulin (ATGAM) plus cyclosporine A is standard therapy; however, there are minimal data available regarding the optimal ATGAM dosage in terms of efficacy and survival. Our institutions have historically used different dosing protocols of ATGAM in the treatment of AA. We aimed to review the outcome of AA patients treated with these protocols and compare them to the published literature. We conducted a retrospective study of 31 adults who received first-line ATGAM for AA and compared response rates and survival between cohorts who received standard (40 mg/kg/day D1-4) versus lower-dose (15 mg/kg/day D1-5) ATGAM schedules. There were similar rates of response (64 vs 71%, P = 1.0), relapse (33 vs 33%, P = 1.0), transformation (14 vs 24%, P = 0.66) or infection (43 vs 47%, P = 1.0), respectively, between standard and lower-dose cohorts. At a median follow up of 24 months, there was no statistical difference between standard and lower-dose cohorts in either event-free (42.2 vs 64.7%, P = 0.91) or overall survival (73.1 vs 88.2%, P = 0.75). Our experience suggests that lower-dose ATGAM at 15 mg/kg/day D1-5 as treatment of AA produces similar responses and outcomes as per standard-dose ATGAM schedules. Prospective trials comparing ATGAM dose schedules in AA are warranted.

CHRONIC MYELOID LEUKEMIA IN PATIENT WITH THE KLINEFELTER SYNDROME

Genetic inborn along with acquired diseases arise due to the lesions in genome of multipotent hematopoietic stem cells. The aim was to study an influence of constitutional anomaly, Klinefelter syndrome, and additional structural rearrangements on the BCR-ABL tyrosine kinase inhibitor targeted therapy efficacy. We describe a 32-year-old male patient with chronic myeloid leukemia (CML) who was detected to have sex chromosomal abnormality during evaluation for Philadelphia chromosome. At diagnosis of CML, two clones were detected by standard cytogenetic investigation of bone marrow cells: 1) clone with translocation t(9;22)(q34;q11), with two sex X chromosomes and absence sex chromosome Y; 2) clone with t(9;22) and unbalanced t(Y;20)(q11;q13). Analysis of blast transformed lymphocytes from peripheral blood showed karyotype 47,XXY. Monitoring of targeted therapy with second generation inhibitor of BCR-ABL tyrosine kinase indicated a cytogenetic remission and absence of BCR-ABL1 fusion signals after 11 months. Absence of translocation t(9;22)(q34;q11) in blast transformed T-lymphocytes at diagnosis of CML evidences that this translocation may appear not only at the level of multipotent haemopoietic cell progenitors but also may have oligo lineage myeloid origin. Presence of additional structural chromosomal abnormality in the clone with t(9;22)(q34;q11) does not affect the efficacy of therapy with the use of second generation BCR-ABL tyrosine kinase inhibitor.

Therapeutic apheresis

Apheresis is a procedure to separate and collect or remove a particular blood component. Apheresis may be used for collecting a therapeutic dose of component, remove the circulating amount of harmful component/substances, or collect a particular blood cell/precursor from a patient for reinfusion. Therapeutic plasma exchange (TPE) is a therapeutic procedure in which the plasma is removed and replaced with a replacement solution. It is used primarily for the removal of pathological immunoglobulin. Red cell exchange is a procedure in which the patient's red cells are removed and replaced with the donor red cells. It is usually used in the case of sickle cell disease to prevent stroke. Mononuclear cell collection is a procedure used primarily to collect peripheral blood haemopoietic progenitor cells (HPC) for autologous or allogeneic transplantation. As the most frequently performed procedures, they will be described in some detail in this article. Therapeutic leucapheresis and thrombocytapheresis are less frequently performed and will be mentioned briefly.

61 - Effect of Prolonged Transportation on Haemopoietic Progenitor Cells for CD34 Positive Selected Allogeneic Transplants in Paediatrics: Evaluation of Cell Viability and Transplant Outcomes

Introduction: Haematopoietic progenitor cells (HPC) from matched unrelated donors (MUD) frequently require transportation from their site of collection, often internationally. Due to geographical isolation, allogeneic translant recipients in Australia often receive cells from international donors with transit times in excess of 24 hours.

Therapeutic plasma exchange performed in tandem with hemodialysis without supplemental calcium in the apheresis circuit.

Therapeutic plasma exchange (TPE) and hemopoietic progenitor cell (HPC) collection are apheresis procedures that can safely be performed in tandem with hemodialysis. Despite the return of citrate-anticoagulated blood to the patient during HPC collection, it is not necessary to administer supplemental calcium during these procedures because the ionized calcium concentration is restored as the returning blood passes through the dialyzer. It is not known whether this applies to TPE, in which a mixture of blood and pharmaceutical albumin, an avid binder of plasma ionized calcium, is returned to the patient through the dialyzer. We report on three dialysis-dependent patients who required TPE and underwent tandem treatments without supplemental calcium in the apheresis circuit. Overall, ionized calcium fell 4-12% (P = 0.0.024) and patients reported no symptoms of hypocalcemic toxicity. Tandem hemodialysis/TPE can be performed without supplemental calcium in the apheresis circuit. J. Clin. Apheresis 32:154-157, 2017. © 2016 Wiley Periodicals, Inc.

Preclinical Evaluation of Flt3 Ligand to Improve T Cell Adaptive Immune Response During Sepsis

BackgroundSepsis is a leading cause of death in the United States. Severely burned patients are highly susceptible to opportunistic infections and sepsis, leading to multi‐organ failure and death. Studies by Hotchkiss and colleagues show that T cell dysfunction significantly contributes to impaired immune response during sepsis in non‐burn subjects. However, the changes in T cell adaptive immunity after burn wound sepsis have not been investigated in detail. We used a clinically relevant mouse model of burn wound infection to assess adaptive immune system function. Furthermore, we evaluated the cytokine, Fms‐like tyrosine kinase‐3 ligand (Flt3L), to improve T cell adaptive immunity post burn wound sepsis. Flt3L is known to stimulate the expansion of hemopoietic progenitors, particularly dendritic cells.Methods8–10 weeks old male BALB/c mice were subjected to 35% total body surface area full thickness burn and the wound was infected with Pseudomonas aeruginosa on day 4 post‐burn. Flow cytometry was used to characterize the cell counts and phenotype of CD4+ and CD8+ T and dendritic cells in spleen and wound draining lymph nodes. Flt3L (10 μg, i.p.) was administered daily for 4 days following burn injury.ResultsWound infection led to severe sepsis in the mice as evidenced by detection of bacteria in blood and lungs, and multi‐organ injury (increased markers of liver and kidney injury) on day 2 after infection and mortality rate of approximately 80% by seven days. We observed a significant decline (>80%) in blood absolute lymphocyte count and CD4+ and CD8+ T cell counts in the spleen and burn‐draining lymph nodes of wound infected mice as compared to non‐infected burn mice on day 2 post infection. Flt3L significantly attenuated the infection induced decline in CD4+ and CD8+ T cell counts in the spleen. Wound infection also caused a decline in the CD11c+ dendritic cell numbers in lymph nodes. Flt3L stimulated the expansion of both the splenic and lymph node dendritic cells and protected against the dendritic cell loss in the lymph nodes. Furthermore, wound infection caused an increase in the expression of Programmed Death‐Ligand 1 (PD‐L1) expression on dendritic cells, F4/80+ macrophages and Ly6C+ inflammatory monocytes in the spleen. PD‐L1 is known to interact with its binding partner PD‐1 on T cell surface leading to T cell inhibition. Flt3L attenuated the increase in PD‐L1 expression and thus indirectly improves T cell function.ConclusionBurn wound sepsis causes significant adaptive immune system dysfunction and Flt3L represents a novel therapeutic cytokine to protect T cell function during sepsis and merits further investigation.Support or Funding InformationSupported by NIH R01 GM66885

Differentiation of haemopoietic stem cells and progenitor cells from human pluripotent stem cells

BackgroundInduced pluripotent stem cells hold great promise as an unlimited source of clinical grade immunologically matched haemopoietic stem cells (HSCs) for transplantation. However, there are currently no robust, reproducible differentiation protocols for the production of engraftable HSCs. We aimed to characterise the production of haemopoietic progenitor cells (HPCs) and HSCs during differentiation of human embryonic stem cells and induced pluripotent stem cells. MethodsWe used a serum-free, feeder-free, good manufacturing practice-compliant differentiation protocol established by the Novosang Consortium for the production of red blood cells for transfusion. Cell phenotype was assessed by flow cytometry for markers of HPCs and HSCs. Cell function was assessed in vitro by the colony forming units in culture (CFU-C) assay and in vivo with a long-term transplantation assay. FindingsAfter preliminary studies, the work focused on days 6, 8, and 10 of the differentiation protocol. In seven independent experiments, HPCs were reproducibly produced at high levels with over 90% of cells expressing CD43 by day 10. Almost all CD41+ cells coexpressed CD43 at all timepoints (day 10, 65% CD43+CD41+). CFU-Cs emerged in large numbers from day 6 and increased by day 10 (mean 1000 colonies per 105 cells plated [SD 478], six experiments). Cytospin and flow cytometry confirmed production of erythroid, granulocyte and macrophage, macrophage, and mixed colonies. The human embryonic HSC markers—vascular endothelial cadherin and CD45—were coexpressed at low levels (mean 3·6% [SD 2·5], seven experiments). No human engraftment was detected when cells were injected into immune-deficient NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice. InterpretationThis differentiation protocol reproducibly gave a high yield of HPCs. However, there was no evidence of engraftable HSCs being produced. This finding could indicate inadequate extrinsic signalling during differentiation. Further work will therefore focus on co-culturing human pluripotent stem cells with human stromal cells in an attempt to mimic the embryonic HSC niche. FundingWellcome Trust.

Human Hemopoietic Progenitor Cell Toll-like and Thymic Stromal Lymphopoietin Receptor Expression and Function in Allergic Asthmatic Subjects

Human Hemopoietic Progenitor Cell Toll-like and Thymic Stromal Lymphopoietin Receptor Expression and Function in Allergic Asthmatic Subjects

Donald Metcalf 1929–2014

Donald Metcalf was one of Australia's most distinguished medical researchers and is acknowledged internationally as the father of the modern field of haemopoietic growth factors. He defined the hierarchy of haemopoietic progenitor cells, purified and cloned the major molecular regulators of their growth and maturation, determined their mechanisms of action and participated in their development for clinical use in cancer patients. He received numerous awards and distinctions during his career, but was most pleased by the fact that his life's work improved human health.

Dysregulation of Vascular Endothelial Progenitor Cells Lung-Homing in Subjects with COPD.

Chronic obstructive pulmonary disease (COPD) is characterized by fixed airflow limitation and progressive decline of lung function and punctuated by occasional exacerbations. The disease pathogenesis may involve activation of the bone marrow stimulating mobilization and lung-homing of progenitor cells. We investigated the hypothesis that lower circulating numbers of vascular endothelial progenitor cells (VEPCs) are a consequence of increased lung-sequestration in COPD. Nonatopic, current or ex-smokers with diagnosed COPD and nonatopic, nonsmoking normal controls were enrolled. Blood and induced sputum extracted primitive hemopoietic progenitors (HPCs) and VEPC were enumerated by flow cytometry. Migration and adhesive responses to fibronectin were assessed. In sputum, VEPC numbers were significantly greater in COPD compared to normal controls. In blood, VEPCs were significantly lower in COPD versus normal controls. There were no differences in HPC levels between the two groups in either compartment. Functionally, there was a greater migrational responsiveness of progenitors from COPD subjects to stromal cell-derived factor-1alpha (SDF-1α) compared to normal controls. This was associated with greater numbers of CXCR4+ progenitors in sputum from COPD. Increased migrational responsiveness of progenitor cells may promote lung-homing of VEPC in COPD which may disrupt maintenance and repair of the airways and contribute to COPD disease pathogenesis.

Donald Metcalf AC. 26 February 1929 — 15 December 2014

Donald Metcalf was one of Australia's most distinguished medical researchers and is acknowledged internationally as the father of the modern field of haemopoietic growth factors. He defined the hierarchy of haemopoietic progenitor cells, purified and cloned the major molecular regulators of their growth and maturation, determined their mechanisms of action and participated in their development for clinical use in cancer patients. He received numerous awards and distinctions during his career, but was most pleased by the fact that his life's work improved human health.

Alpha-Particle-Induced Complex Chromosome Exchanges Transmitted through Extra-Thymic Lymphopoiesis In Vitro Show Evidence of Emerging Genomic Instability.

Human exposure to high-linear energy transfer α-particles includes environmental (e.g. radon gas and its decay progeny), medical (e.g. radiopharmaceuticals) and occupational (nuclear industry) sources. The associated health risks of α-particle exposure for lung cancer are well documented however the risk estimates for leukaemia remain uncertain. To further our understanding of α-particle effects in target cells for leukaemogenesis and also to seek general markers of individual exposure to α-particles, this study assessed the transmission of chromosomal damage initially-induced in human haemopoietic stem and progenitor cells after exposure to high-LET α-particles. Cells surviving exposure were differentiated into mature T-cells by extra-thymic T-cell differentiation in vitro. Multiplex fluorescence in situ hybridisation (M-FISH) analysis of naïve T-cell populations showed the occurrence of stable (clonal) complex chromosome aberrations consistent with those that are characteristically induced in spherical cells by the traversal of a single α-particle track. Additionally, complex chromosome exchanges were observed in the progeny of irradiated mature T-cell populations. In addition to this, newly arising de novo chromosome aberrations were detected in cells which possessed clonal markers of α-particle exposure and also in cells which did not show any evidence of previous exposure, suggesting ongoing genomic instability in these populations. Our findings support the usefulness and reliability of employing complex chromosome exchanges as indicators of past or ongoing exposure to high-LET radiation and demonstrate the potential applicability to evaluate health risks associated with α-particle exposure.

449. Multipathogen-Specific T Cells for Immune Reconstitution – A Decade of Manufacturing Development and Clinical Use

Since 2003 we have treated 101 patients with antigen specific T cells for treatment or prevention of opportunistic infections in the context of haemopoietic progenitor cell (HPC) and solid organ transplant. Over this time, we have modified laboratory procedures to progressively incorporate T cell therapy into standard transplant practice. We have increased the number of pathogen targets, utilised HPC products and streamlined culture techniques. We are now able to produce a multipathogen product within 3 weeks of HPC harvest in a manner consistent with good manufacturing practice and within the scope of systems established for HPC processing. Our approach could be used by most accredited transplant laboratories.We have developed robust, simple and inexpensive methods that use marrow or G-CSF mobilised HPC products as starting material for T cell expansion. This has major practical importance in minimising repeated donor screening, blood draws and apheresis. Our current procedure uses monocyte derived dendritic cells (mo-DC) isolated using a modified CD14 selection method that requires minimal quantities of CD14 selection reagent. Maturation of mo-DCs utilises GM-CSF, IL-4, PG-E2, IL-6 and IL-1β. Multiple antigens are presented to T cells by mo-DCs including peptide mixes for cytomegalovirus (CMV), Epstein Barr virus (EBV), BK virus and adenovirus, vaccines (Varicella Zoster and Influenza virus) and mycelial lysate (Aspergillus). Activated antigen specific T cells are expanded using IL-2. We use no T cell selection/depletion procedures, feeder layers, cytokine, tetramer capture or clinical grade sorting methodologies. The overall cost of manufacture of each product, including reagents, staffing and ongoing infrastructure costs is approximately AU$2400 per dose.The phenotypic composition of multipathogen products is mainly CD3 (mean 97%, range 90-97), with variable CD4:8 ratio, minimal contaminating B cells (mean 0.1%), monocytes (mean 0.3%) and NK cells (mean 3%). We have demonstrated antigen specificity using intracellular cytokine staining, MHC multimer analysis (CMV and EBV), interferon-γ Elispot and chromium (Cr51) release cytotoxicity assay. Products do not show alloreactivity by Cr51 release assay (Mean 0.8% specific lysis at E:T ratio of 20:1, range 0-5.3).Clinical results of the first 50 patients treated with CMV specific T cells have previously been reported and showed infusion to be safe and to reduce the need for CMV-directed antiviral therapy. Clinical outcomes of the remaining patients treated with multi-pathogen T cells specific for up to 8 pathogens will be reported when data is mature.Future areas of development will involve extension of the range of infectious pathogens targeted, addition of cells targeting malignant antigens and combination of these therapies with CD34+ stem cell selection. Developing progressively greater selectivity through graft engineering and immunotherapy will improve outcomes of transplantation in the future.

Umbilical cord blood for autologous transfusion in the early postnatal ontogeny: analysis of cell composition and viability during long-term culturing.

Changes in cell composition and viability as well as the content and functional activity of hemopoietic progenitor cells were analyzed during long-term (up to 1 month at 4°C) storage of human umbilical cord blood cells. No significant quantitative changes in erythrocytes were found during this period. The total content and viability of leukocytes changed, which resulted in the prevalence of mononuclear cells (lymphocytes and monocytes). Analysis of functional activity of hemopoietic stem cells in semisolid culture revealed a decrease in the relative content of CFU during the first week of storage [corrected] and inability of cells to colony formation after 2 weeks.

Effect of SIRT6/NF-κB signal axis in delaying hematopoietic stem/progenitor cell senescence with ginsenoside Rg1

To investigate the effect of SIRT6/NF-κB signal axis in delaying hematopoietic stem/progenitor cell senescence with ginsenoside Rg1, in order to provide theatrical and experimental basis for looking for methods for delaying HSC senescence. Sca-1 + HSC/HPC was isolated by magnetic cell sorting (MACS) and divided into five groups: the normal control group, the aging group, the positive control group, the Rg1 anti-senescence group, and the Rg1-treated group. Senescence-associated β-galactosidase (SA-β-Gal) staining, cell cycle analysis and hemopoietic progenitor cell mix (CFU-Mix) were adopted to determine the effect Rg1 in delaying or treating Sca-1 + HSC/HPC senescence biology. The mRNA and protein of senescence regulation molecules SIRT6 and NF-KB were examined by realtime fluorescence quantitative PCR (FQ-PCR) and western blotting. Compared with the senescence group, the Rg1 anti-senescence group and the Rg1-treated group showed lower percentage in SA-β-Gal-stained positive cells, decreased cell proportion in G1 phase, increased number of CFU-Mix, up-regulated in SIRT6 mRNA and protein expression, down-regulation in NF-KB mRNA and protein expression. The Rg1 anti-senescence group showed more evident changes in indexes than the Rg1-treated group. Rg, may inhibit Sca-1 + HSC/HPC senescence induced by t-BHP by regulating SIRT6/NF-KB signal path.

Protective effect of HS-6101 on rhesus monkeys with severe hematopoietic acute radiation sickness

This study was purposed to investigate the protective effects of lipoprotein HS-6101(6101) on rhesus monkey total body irradiated with 7.0 Gy ⁶⁰Coγ-ray. A total of 30 health adult rhesus monkeys were randomly divided into symptomatic therapy (ST), WR2721 and HS-6101 30, 90 and 270 mg/kg groups (n = 6), the rhesus monkeys of each groups were injected with physiological saline 0.3 ml/kg, WR-2721 30 mg/kg, or HS-6101 30, 90 and 270 µg/kg, respectively. All agents were once intramuscularly injected at 1 hr prior irradiation. General observation, peripheral blood cell counts, colony forming unite assay of bone marrow hemopoietic progenitor cells, and histopathological examination were performed. The results showed that animals in symptomatic therapy group begin to die on the 13(th) day and 4 animals died within 24 days, the average survival time was 18.2 ± 4.3 days; 2 animals in WR-2717 groups died on day 15.8 and day 18.5 post irradiation respectively. 1 animal in HS-6101 270 mg/kg group died on day 35.8, all other animals survived. Nadirs of peripheral blood white blood cells, neutrophils and platelets of animals in HS-6101 treatment groups were significantly higher than those in other 2 groups including ST and WR-2721 groups, and the hemopoietic recovery were also significantly speeding up(P < 0.05 and 0.01). In vitro results showed that HS-6101 obviously promoted 7.0 Gy ⁶⁰Coγ irradiated monkey's bone marrow mononuclear cells to form various hematopoietic progenitor cell colonies (P < 0.05 and 0.01) . Compared with symptomatic therapy and WR-2717 groups, bone marrow histopathological changes in HS-6101 treatment groups showed more active hemopoietic cell proliferation and higher density structure. It is concluded that HS-6101 90 µg/kg treatment can promote the bone marrow recovery of 7.0 Gy ⁶⁰Coγ irradiated monkey, alleviate their animal symptom, simplify the treatment measures and improve the animal survival rate. The HS-6101 shows remarkable radioprotective effects as compared with the currently internationally acknowledged radioprotectant of WR-2721.

Response of hemopoietic, progenitor, and multipotent mesenchymal stromal cells to administration of ketanserin during pulmonary fibrosis.

We studied the effect of ketanserin on hemopoietic progenitor cells (Lin(-)Sca-1(+)c-Kit(+)CD34- and Lin(-)Sca-1(+)c-Kit(+)CD34(+)), progenitor hemopoietic cells (Lin(-)Sca-1(+)c-kit(+)), and multipotent mesenchymal stromal cells (CD45(-)CD73(+)CD106(+)) in C57Bl/6 mice during pulmonary fibrosis. It was shown that the blocker of 5-HT2A receptors lowers the activity of bleomycin-induced inflammation in the lungs and prevents the infiltration of alveolar interstitium and alveolar ducts by hemopoietic stem and hemopoietic progenitor cells; in this case, they are more numerous in the bone marrow of sick animals. Ketanserin reduces the capacity for self-renewal of lung multipotent mesenchymal stromal cells in the fibrotic phase of the disease and inhibits their differentiation into stromal cell lines (adipocytes, chondrocytes, and fibroblasts) simultaneously with the decrease in the percentage of connective tissue in the lung parenchyma.

Astrocyte-like cells differentiated from a novel population of CD45-positive cells in adult human peripheral blood.

We have previously reported a novel CD45-positive cell population called peripheral blood insulin-producing cells (PB-IPCs) and its unique potential for releasing insulin in vitro. Despite the CD45-positive phenotype and self-renewal ability, PB-IPCs are distinguished from hemopoietic and endothelial progenitor cells (EPCs) by some characteristics, such as a CD34-negative phenotype and different culture conditions. We have further identified the gene profiles of the embryonic and neural stem cells, and these profiles include Sox2, Nanog, c-Myc, Klf4, Notch1 and Mash1. After treatment with all-trans retinoic acid (ATRA) in vitro, most PB-IPCs exhibited morphological changes that included the development of elongated and branched cell processes. In the process of induction, the mRNA expression of Hes1 was robustly upregulated, and a majority of cells acquired some astrocyte-associated specific phenotypes including anti-glial fibrillary acidic protein (GFAP), CD44, Glutamate-aspartate transporter (GLAST) and S100β. In spite of the deficiency of glutamate uptaking, the differentiated cells significantly relaxed the regulation of the expression of brain-derived neurotrophic factor (BDNF) mRNA. This finding demonstrates that PB-IPCs could be induced into a population of astrocyte-like cells and enhanced the neurotrophic potential when the state of proliferation was limited by ATRA, which implies that this unique CD45+ cell pool may have a protective role in some degenerative diseases of the central nervous system (CNS).

20: Neutralising anti-G-CSFR monoclonal antibody blocks neutrophil-driven inflammatory joint disease

Granulocyte colony-stimulating factor (G-CSF) is a haemopoietic cytokine first identified for its role in steady state granulopoiesis but is now also recognised as a proinflammatory mediator of mature neutrophil activation and function. G-CSF binds to the G-CSF receptor (G-CSFR), which is present on neutrophils and haemopoietic progenitors. G-CSF-deficient mice are protected from disease in several models of rheumatoid arthritis, and blockade of G-CSF is also protective in those models. To further investigate the actions of blocking G-CSF/G-CSFR signalling in inflammatory disease, we developed a neutralising monoclonal antibody to mouse G-CSFR which prevents the binding of G-CSF and inhibits G-CSFR signalling. In the anti-collagen antibody induced arthritis model, anti-G-CSFR treatment rapidly halted the progression of established disease. This therapeutic effect was observed at low dose and was associated with transient suppression of peripheral blood neutrophilia. Anti-G-CSFR treatment significantly inhibited neutrophil accumulation in the joints without rendering animals neutropenic, suggesting an effect of G-CSFR blockade on neutrophil homing to inflammatory sites. Consistent with this, neutrophils in the blood and arthritic joints of anti-G-CSFR treated mice showed alterations in homing receptors, with reduced CXCR2 and increased CD62L expression. Microarray analysis of neutrophils from joints and peripheral blood showed a common up-regulation of 6 genes and down-regulation of 22 genes following anti-G-CSFR treatment. Blocking neutrophil traffic to joints with anti-G-CSFR suppressed local production of proinflammatory cytokines (IL-1b and IL-6) and chemokines (KC and MCP-1) known to drive tissue damage. These data show for the first time the effect of blockade of G-CSFR in a therapeutic model of inflammation and provide further support for utilising this approach in the treatment of inflammatory diseases such as rheumatoid arthritis.