Hematopoietic Tissue Cells Research Articles

Preclinical Safety and Efficacy of Human CD34+ Cells Transduced With Lentiviral Vector for the Treatment of Wiskott-Aldrich Syndrome

Gene therapy with ex vivo-transduced hematopoietic stem/progenitor cells may represent a valid therapeutic option for monogenic immunohematological disorders such as Wiskott-Aldrich syndrome (WAS), a primary immunodeficiency associated with thrombocytopenia. We evaluated the preclinical safety and efficacy of human CD34+ cells transduced with lentiviral vectors (LV) encoding WAS protein (WASp). We first set up and validated a transduction protocol for CD34+ cells derived from bone marrow (BM) or mobilized peripheral blood (MPB) using a clinical grade, highly purified LV. Robust transduction of progenitor cells was obtained in normal donors and WAS patients' cells, without evidence of toxicity. To study biodistribution of human cells and exclude vector release in vivo, LV-transduced CD34+ cells were transplanted in immunodeficient mice, showing a normal engraftment and differentiation ability towards transduced lymphoid and myeloid cells in hematopoietic tissues. Vector mobilization to host cells and transmission to germline cells of the LV were excluded by different molecular assays. Analysis of vector integrations showed polyclonal integration patterns in vitro and in human engrafted cells in vivo. In summary, this work establishes the preclinical safety and efficacy of human CD34+ cells gene therapy for the treatment of WAS.

Recombinant medaka (Oryzias melastigmus) pro-hepcidin: Multifunctional characterization

Recently, two hepcidin variant genes (Om-hep1 and Om-hep2) were identified in a model fish marine medaka and both were highly induced in vivo with bacterial challenge, suggesting that the medaka hepcidin may have a similar function to other reported teleostean hepcidins. In the present study, the antibacterial, antiviral and antitumor activities of Om-hep1 were determined using its synthetic and recombinant pro-peptides. The recombinant pro-hepcidin1 was expressed in Escherichia coli and an effective method to produce recombinant Pro-Omhep1 was developed in order to obtain a right folded structure. The results showed that both the synthetic mature peptide and recombinant pro-peptide had similar antibacterial activity against Gram-positive and negative bacteria. In particular, both the synthetic mature Om-hep1 and recombinant Pro-Omhep1 inhibited the viral replication of white spot syndrome virus in the hematopoietic tissue cells of the crayfish Cherax quadricarinatus. Om-hep1 also presented antitumor activity on the cultured human hepatocellular carcinoma cells. In addition, the antimicrobial mechanism of Om-hep1 was measured and it was found that Om-hep1 was likely to be non-membranolytic. The recombinant Pro-Omhep1 performed better biological activity compared to the synthetic mature Om-hep1. This study suggested that Om-hep1 was likely to be an important multifunction protein involved in various resistance actions in the marine medaka immune system.

Uncaria tomentosa stimulates the proliferation of myeloid progenitor cells

Ethnopharmacological relevanceThe Asháninkas, indigenous people of Peru, use cat's claw (Uncaria tomentosa) to restore health. Uncaria tomentosa has antioxidant activity and works as an agent to repair DNA damage. It causes different effects on cell proliferation depending on the cell type involved; specifically, it can stimulate the proliferation of myeloid progenitors and cause apoptosis of neoplastic cells. Neutropenia is the most common collateral effect of chemotherapy. For patients undergoing cancer treatment, the administration of a drug that stimulates the proliferation of healthy hematopoietic tissue cells is very desirable.It is important to assess the acute effects of Uncaria tomentosa on granulocyte-macrophage colony-forming cells (CFU-GM) and in the recovery of neutrophils after chemotherapy-induced neutropenia, by establishing the correlation with filgrastim (rhG-CSF) treatment to evaluate its possible use in clinical oncology. Materials and methodsThe in vivo assay was performed in ifosfamide-treated mice receiving oral doses of 5 and 15mg of Uncaria tomentosa and intraperitoneal doses of 3 and 9μg of filgrastim, respectively, for four days. Colony-forming cell (CFC) assays were performed with human hematopoietic stem/precursor cells (hHSPCs) obtained from umbilical cord blood (UCB). ResultsBioassays showed that treatment with Uncaria tomentosa significantly increased the neutrophil count, and a potency of 85.2% was calculated in relation to filgrastim at the corresponding doses tested. An in vitro CFC assay showed an increase in CFU-GM size and mixed colonies (CFU-GEMM) size at the final concentrations of 100 and 200μg extract/mL. ConclusionsAt the tested doses, Uncaria tomentosa had a positive effect on myeloid progenitor number and is promising for use with chemotherapy to minimize the adverse effects of this treatment. These results support the belief of the Asháninkas, who have classified Uncaria tomentosa as a ‘powerful plant’.

Corticotropin-Releasing Factor: A Key Element in Cancer Anorexia-Cachexia Syndrome

Myelodysplastic syndrome (MDS) is clonal disease of hematopoietic precursor cells that is characterized by pancytopenia. Patients with MDS have an increase in the risk of developing acute myeloid leukemia (AML), characterized by unlimited growth and accumulation of blast cells in hematopoietic tissues. Blast cells derived from a group of patients with AML produce and secrete tryptase. These patients exhibit increased expression of bone marrow tryptase and serum tryptase levels. There is evidence that PAR-2, cleaved and activated by tryptase, contributes to tumor progression and proliferation in various cancer cell types. However, the role of PAR-2 in the context of myeloid leukemia remains unknown. Aims. The aim of the present study was to determine the effect of PAR-2 activation on leukemic cell survival and proliferation. Methods. Protein expression of PAR-2 was examined in K562 cells, a human myeloid leukemia cell line, by RT-PCR. K562 cells were treated with the PAR-2 activating peptide SLIGRL-NH2 (1, 10 and 100 μM) for up to 72 hours in serumfree media. Media containing serum and serum free media were used as positive and negative controls, respectively. Cell proliferation in response to agonist peptide was evaluated by the MTT assay after exposure to serum-free media, SLIGRL-NH2 or serum-containing media. Caspase-3 and cleaved caspase-3 protein levels were determined by Western blots in the combined presence of PAR-2 activating peptide and cyclohexamide (50 μg/ml). Results. PAR-2 was detected in K562 cells. These cells showed a significant increase in cell growth as determined by MTT assay after treatment with serum-containing media. However, PAR2 activating peptide did not significantly alter cell growth as determined absorbance by MTT assay when compared to controls at any of the time points examined. Treatment with cyclohexamide alone significantly increased cleaved caspase-3. The PAR-2 activating peptide alone did not alter K562 cell apoptosis, as indicated by measurement of cleaved caspase-3. However, the effect of cyclohexamide on caspase-3 cleavage was significantly decreased in the present of PAR-2 activating peptide, suggesting anti-apoptotic activity. Conclusions. Our studies show that PAR-2 activation does not stimulate cellular proliferation in myeloid leukemia cells. Instead, PAR-2 activating peptide reduces leukemic cell response to cyclohexamide-induced apoptosis. The results imply that enhanced tryptase levels during MDS or AML may, via activating PAR-2, offer protection of malignant cells against chemotherapeutic agents thereby result in a survival advantage of the malignant clone.

Protease-Activated Receptor-2 Reduces Cyclohexamide-Induced Apoptosis in K562 Myeloid Leukemia Cells

Myelodysplastic syndrome (MDS) is clonal disease of hematopoietic precursor cells that is characterized by pancytopenia. Patients with MDS have an increase in the risk of developing acute myeloid leukemia (AML), characterized by unlimited growth and accumulation of blast cells in hematopoietic tissues. Blast cells derived from a group of patients with AML produce and secrete tryptase. These patients exhibit increased expression of bone marrow tryptase and serum tryptase levels. There is evidence that PAR-2, cleaved and activated by tryptase, contributes to tumor progression and proliferation in various cancer cell types. However, the role of PAR-2 in the context of myeloid leukemia remains unknown. Aims. The aim of the present study was to determine the effect of PAR-2 activation on leukemic cell survival and proliferation. Methods. Protein expression of PAR-2 was examined in K562 cells, a human myeloid leukemia cell line, by RT-PCR. K562 cells were treated with the PAR-2 activating peptide SLIGRL-NH2 (1, 10 and 100 μM) for up to 72 hours in serumfree media. Media containing serum and serum free media were used as positive and negative controls, respectively. Cell proliferation in response to agonist peptide was evaluated by the MTT assay after exposure to serum-free media, SLIGRL-NH2 or serum-containing media. Caspase-3 and cleaved caspase-3 protein levels were determined by Western blots in the combined presence of PAR-2 activating peptide and cyclohexamide (50 μg/ml). Results. PAR-2 was detected in K562 cells. These cells showed a significant increase in cell growth as determined by MTT assay after treatment with serum-containing media. However, PAR2 activating peptide did not significantly alter cell growth as determined absorbance by MTT assay when compared to controls at any of the time points examined. Treatment with cyclohexamide alone significantly increased cleaved caspase-3. The PAR-2 activating peptide alone did not alter K562 cell apoptosis, as indicated by measurement of cleaved caspase-3. However, the effect of cyclohexamide on caspase-3 cleavage was significantly decreased in the present of PAR-2 activating peptide, suggesting anti-apoptotic activity. Conclusions. Our studies show that PAR-2 activation does not stimulate cellular proliferation in myeloid leukemia cells. Instead, PAR-2 activating peptide reduces leukemic cell response to cyclohexamide-induced apoptosis. The results imply that enhanced tryptase levels during MDS or AML may, via activating PAR-2, offer protection of malignant cells against chemotherapeutic agents thereby result in a survival advantage of the malignant clone.

Invertebrate Hematopoiesis: An Astakine-Dependent Novel Hematopoietic Factor

A novel factor, named crustacean hematopoietic factor (CHF), was identified from a library of suppression subtractive hybridization with the aim to find downstream genes of an invertebrate cytokine, astakine 1, in the freshwater crayfish Pacifastacus leniusculus. CHF is a small cysteine-rich protein (∼9 kDa) with high similarity to the N-terminal region of vertebrate CRIM1 in containing an insulin growth factor binding protein variant motif with unknown function. CHF was found to be induced in primary cell cultures of crayfish hematopoietic tissue (Hpt) cells (precursors of crayfish blood cells) after treatment with astakine 1. Silencing of CHF did not affect the renewal of Hpt cells in vitro, but induced apoptosis of Hpt cells. CHF is exclusively expressed in the blood cell lineage of crayfish (Hpt cells and blood cells), and in vivo RNA interference experiments show that knockdown of this gene results in severe loss of blood cells and a higher apoptotic rate in Hpt. Our data further suggest that crayfish CHF is critical for the survival of hemocytes and Hpt cells by preventing their apoptosis, thus it plays an important role in hemocyte homeostasis in crayfish. Our study of CHF may also shed light on the function of this untypical insulin growth factor binding protein motif located in the N-terminal of vertebrate CRIM1.

Eosinophils in the zebrafish: prospective isolation, characterization, and eosinophilia induction by helminth determinants

AbstractEosinophils are granulocytic leukocytes implicated in numerous aspects of immunity and disease. The precise functions of eosinophils, however, remain enigmatic. Alternative models to study eosinophil biology may thus yield novel insights into their function. Eosinophilic cells have been observed in zebrafish but have not been thoroughly characterized. We used a gata2:eGFP transgenic animal to enable prospective isolation and characterization of zebrafish eosinophils, and demonstrate that all gata2hi cells in adult hematopoietic tissues are eosinophils. Although eosinophils are rare in most organs, they are readily isolated from whole kidney marrow and abundant within the peritoneal cavity. Molecular analyses demonstrate that zebrafish eosinophils express genes important for the activities of mammalian eosinophils. In addition, gata2hi cells degranulate in response to helminth extract. Chronic exposure to helminth- related allergens resulted in profound eosinophilia, demonstrating that eosinophil responses to allergens have been conserved over evolution. Importantly, infection of adult zebrafish with Pseudocapillaria tomentosa, a natural nematode pathogen of teleosts, caused marked increases in eosinophil number within the intestine. Together, these observations support a conserved role for eosinophils in the response to helminth antigens or infection and provide a new model to better understand how parasitic worms activate, co-opt, or evade the vertebrate immune response.

Ancient Cytokines, the Role of Astakines as Hematopoietic Growth Factors

Hematopoiesis is the process by which hemocytes mature and subsequently enter the circulation. Vertebrate prokineticins (PKs) are known to take part in this process, as are the invertebrate prokineticin domain proteins, astakines. In Pacifastacus leniusculus, astakine 1 is essential for the release of new hemocytes into the open circulatory system of these animals. In addition to astakine 1, we have now cloned a homologue of astakine 1 with an insert of 13 amino acids, named as astakine 2. Both crustacean astakines lack the N-terminal AVIT motif, which is present in vertebrate PKs, and hence receptor binding differs from that of vertebrate PKs. We have found astakine-like sequences in 19 different invertebrate species, and the sequences show that some motifs are conserved among invertebrate groups. Previously we showed that astakine 1 is directly involved in hematopoiesis, and now we show that astakine 1 and astakine 2 have different roles in hemocyte lineage differentiation. Astakine 1 can stimulate proliferation of hematopoietic tissue (Hpt) cells (precursor of hemocytes) as well as specifically induce differentiation of Hpt cells along the semigranular cell lineage, whereas astakine 2 plays a role in granular cell differentiation. Moreover, we discuss the impact of the putative structures of different astakines in comparison with the vertebrate prokineticins.

Peptidoglycan activation of the proPO-system without a peptidoglycan receptor protein (PGRP)?

Recognition of microbial polysaccharide by pattern recognition receptors triggers the prophenoloxidase (proPO) cascade, resulting in melanin synthesis and its deposition on the surface of invading pathogens. Several masquerade-like proteins and serine proteinase homologues have been shown to be involved in the proPO activation in insects. In this study, a novel serine proteinase homologue, Pl-SPH2, was found and isolated as a 30 kDa protein from hemocytes of the freshwater crayfish, Pacifastacus leniusculus, by its binding property to a partially lysozyme digested or TCA-treated insoluble Lysine (Lys)-type peptidoglycan (PGN) and soluble polymeric Lys-type PGN. Two other proteins, the Pl-SPH1 and lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP) were also found in the several different PGN-binding assays. However no PGRP homologue was detected. Neither was any putative PGRP found after searching available crustacean sequence databases. If RNA interference of Pl-SPH2, Pl-SPH1 or LGBP in the crayfish hematopoietic tissue cell culture was performed, it resulted in lower PO activity following activation of the proPO-system by soluble Lys-type PGN. Taken together, we report for the first time that Lys-type PGN is a trigger of proPO-system activation in a crustacean and that two Pl-SPHs are involved in this activation possibly by forming a complex with LGBP and without a PGRP.

A gC1qR Prevents White Spot Syndrome Virus Replication in the Freshwater CrayfishPacifastacus leniusculus

The gC1qR/p32 protein is a multiple receptor for several proteins and pathogens. We cloned a gC1qR homologue in a crustacean, Pacifastacus leniusculus, and analyzed the expression of P. leniusculus C1qR (PlgC1qR) in various tissues. The gC1qR/p32 transcript was significantly enhanced by white spot syndrome virus (WSSV) infection 6 h after viral infection both in vitro in a hematopoietic tissue cell culture (Hpt) and in vivo compared to appropriate controls. Moreover, PlgC1qR silencing in both the Hpt cell culture and live crayfish enhanced the WSSV replication. In addition, by making a recombinant PlgC1qR protein we could show that if this recombinant protein was injected in a crayfish, Pacifastacus leniusculus, followed by injection of WSSV, this significantly reduced viral replication in vivo. Furthermore, if the recombinant PlgC1qR was incubated with Hpt cells and then WSSV was added, this also reduced viral replication. These experiments clearly demonstrate that recombinant PlgC1qR reduce WSSV replication both in vivo and in vitro. The results from a far-Western overlay and glutathione S-transferase pull-down assays showed that PlgC1qR could bind to VP15, VP26, and VP28. Altogether, these results demonstrate a role for PlgC1qR in antiviral activity against WSSV.

Increased Mitochondrial Mass and Mitochondrial DNA Copy Number Are Biomarkers for Benzene Exposure in Hematopoietic Tissue and Leukemia Cells.

Abstract 4426 BackgroundExposure to benzene and its metabolites increases risk of marrow failure disorders, leukemia and other hematological diseases. However, biomarkers of benzene toxicity have not been comprehensively studied in hematopoietic cells and leukemia cells. We previously reported that benzene metabolites may impair electron chain transport and mitochondrial function (3rd WHO Conference on Children's Health and the Environment, 7-10 June 2009). Therefore, we hypothesized that alterations in mitochondrial mass and mitochondrial DNA (mtDNA) may occur in bone marrow cells and leukemia cells after benzene exposure to compensate for damaged mitochondria. Materials and MethodsTotal bone marrow cells from healthy individuals and leukemia cell lines (THP-1, Kasumi-1, K562, Molt-4 and HL-60) were cultured in RPMI media containing 10% fetal bovine serum for 5 days. Benzene was added in cell culture media with 0, 1 and 10mM concentration at 24 hour interval. Cell count was performed using an automated blood cell analyzer (ADVIA120, Siemens, Germany). Viability and apoptosis were assessed by tryptophan blue dye exclusion test and flowcytometry based annexin V staining protocol. Hydrogen peroxide content is measured using the commercial kit (Bioxytech® H2O2-560TM, OXIS International) according to the manufacturer's instructions. Mitochondrial mass, membrane potential and mtDNA copy number were measured using MitoTracker Green, MitoTracker Red probes (Invitrogen), and real time PCR using the QuantiTect SYBR Green PCR kit (Qiagen) and Rotor-Gene 3000 (Corbett Research), respectively. ResultsThe number of cells were gradually increased regardless of concentration of benzene in day 3, and then steadily maintained during 3 weeks culture. Interestingly, the growth of K562 cells showed no growth inhibition effect (three fold increase) after 5-day exposure to benzene. Overall viability of five leukemia cell lines disclosed significant decrease after two week treatment of benzene (about 60% of viability was observed in 3- week suspension culture). The proportion of apoptosis was increased in time and dose dependent manner after 2-week treatment of benzene. Interestingly, mitochondrial contents and membrane potentials were dramatically increased in 3-week suspension culture after benzene exposure at dose dependent manner. The level of hydrogen peroxide significantly elevated after two week treatment of benzene (4.4 ± 1.9 μM/mg protein) compared with non-benzene treatment group (1.2 ± 1.0 μM/mg protein; P = 0.001). The average mtDNA copy number was gradually increased after exposure to benzene. ConclusionsBenzene exposure caused increased mitochondrial mass and mtDNA copy number in response to oxidative stress induced by benzene. So, these mitochondrial changes can be used for biomarkers of benzene toxicity in hematopoietic tissue and leukemia cell. Disclosures:No relevant conflicts of interest to declare.

High sequence variability among hemocyte-specific Kazal-type proteinase inhibitors in decapod crustaceans

Crustacean hemocytes were found to produce a large number of transcripts coding for Kazal-type proteinase inhibitors (KPIs). A detailed study performed with the crayfish Pacifastacus leniusculus and the shrimp Penaeus monodon revealed the presence of at least 26 and 20 different Kazal domains from the hemocyte KPIs, respectively. Comparisons with KPIs from other taxa indicate that the sequences of these domains evolve rapidly. A few conserved positions, e.g. six invariant cysteines were present in all domain sequences whereas the position of P1 amino acid, a determinant for substrate specificity, varied highly. A study with a single crayfish animal suggested that even at the individual level considerable sequence variability among hemocyte KPIs produced exist. Expression analysis of four crayfish KPI transcripts in hematopoietic tissue cells and different hemocyte types suggest that some of these KPIs are likely to be involved in hematopoiesis or hemocyte release as they were produced in particular hemocyte types or maturation stages only.

Fetal-cell microchimerism, lymphopoiesis, and autoimmunity

During all human and murine pregnancies, fetal cells enter the maternal circulation and tissues and may persist there for decades. The immune consequences of this phenomenon have been explored for many years as a potential origin of autoimmunity or protection from cancer in women after pregnancy. The leading hypothesis, suggesting that semi-allogenic fetal T cells may trigger a graft-versus-host type of disease, has been supported by several studies showing an increased frequency of fetal-cell microchimerism (FMc) in women affected with systemic sclerosis. However, a large proportion of healthy women or women affected with non-immune disorders also display fetal T cells, challenging the direct pathogenic role of such cells. In addition, recent evidence showing the transfer of various fetal progenitor cells to the mother during gestation has shed new light on the interpretation of microchimerism in autoimmunity. This review discusses the functional capacity of fetal hematopoietic progenitors to form T and B cells in maternal hematopoietic tissues, where they undergo an educational process probably resulting in tolerance to maternal antigens. Therefore, hypotheses other than the transfer of fetal cells to the mother's circulation should be considered in explaining the observed association of FMc and autoimmune disorders.

Inhibition of Notch and tumor regression

e14623 Background: Notch signaling is an evolutionary-conserved pathway in vertebrates and invertebrates which is involved many developmental processes, including cell fate decisions, apoptosis, proliferation, and stem-cell self renewal. There is increasing evidence that the same molecular pathways regulating the self renewal of stem cells are also being employed in cancer progression. The Notch signal transduction pathway has been implicated in the self-renewal of stem cells in hematopoietic, skin, neural, germ and breast tissue. Increasing evidence suggests that the Notch signaling pathway is frequently up regulated in many forms of cancer including acute T-cell lymphoblastic leukemia, cervical, prostate, lung, breast and others. Thus,inhibition of the pathway could provide a novel treatment of cancer and cancer stem cells. Methods: We have genetically engineered a fusion protein, consisting of the Drosophila transcription factor Antennapedia (ANTP) and with the truncated version of Mastermind-like (MAML) that behaves in a dominant negative (DN) fashion and inhibits Notch activation (ANTP/DN MAML, TR4). This novel fusion protein has been tested for its ability to target tumor cells in vitro and in vivo. Results: Our data show that ANTP/DN MAML fusion protein, TR4 contains signals for proper cell targeting, internalization and nuclear transport. Furthermore, TR4 inhibits human mammary and colon xenograft tumor growth and metastases in immuno deficient mice.TR4 presence and activity was also detected in the brains of treated animals demonstrating that TR4 can cross the blood-brain barrier and potentially eliminate brain tumors and metastases, unlike other anticancer drugs and biological such as monoclonal antibodies that cannot cross the blood brain barrier. TR4 was found to be non- immunogenic following repeat administration in healthy animals. At very high doses (>10x therapeutic dose) it caused anorexia and weight loss in mice. Conclusions: The TR4 protein, a Notch inhibitor, can induce tumor regression and resolution of breast and colon cancer xenografts. It is non- immunogenic following repeat administration and has acceptable toxicity profile. No significant financial relationships to disclose.

A Novel Protein Acts as a Negative Regulator of Prophenoloxidase Activation and Melanization in the Freshwater Crayfish Pacifastacus leniusculus

Melanization is an important immune component of the innate immune system of invertebrates and is vital for defense as well as for wound healing. In most invertebrates melanin synthesis is achieved by the prophenoloxidase-activating system, a proteolytic cascade similar to vertebrate complement. Even though melanin formation is necessary for host defense in crustaceans and insects, the process needs to be tightly regulated because of the hazard to the animal of unwanted production of quinone intermediates and melanization in places where it is not suitable. In the present study we have identified a new melanization inhibition protein (MIP) from the hemolymph of the crayfish, Pacifastacus leniusculus. Crayfish MIP has a similar function as the insect MIP molecule we recently discovered in the beetle Tenebrio molitor but interestingly has a completely different sequence. Crayfish MIP as well as Tenebrio MIP do not affect phenoloxidase activity in itself but instead interfere with the melanization reaction from quinone compounds to melanin. Importantly, crayfish MIP in contrast to Tenebrio MIP contains a fibrinogen-like domain, most similar to the substrate recognition domain of vertebrate l-ficolins. Surprisingly, an Asp-rich region similar to that found in ficolins that is likely to be involved in Ca2+ binding is present in crayfish MIP. However, crayfish MIP did not show any hemagglutinating activity as is common for the vertebrate ficolins. A mutant form of MIP with a deletion lacking four Asp amino acids from the Asp-rich region lost most of its activity, implicating that this part of the protein is involved in regulating the prophenoloxidase activating cascade. Overall, a new negative regulator of melanization was identified in freshwater crayfish that shows interesting parallels with proteins (i.e. ficolins) involved in vertebrate immune response.

Identification and properties of a receptor for the invertebrate cytokine astakine, involved in hematopoiesis

We have recently isolated an invertebrate cytokine from a freshwater crayfish, which we named astakine 1. Interestingly this protein is expressed exclusively in hemocytes and hematopoietic tissue and is essential for the release of new hemocytes into the open circulatory system of these animals. This astakine has a prokineticin (PK) domain but lacks the N-terminal AVIT amino acids and hence receptor binding may differ from vertebrate PKs. Accordingly, here we report that a receptor for astakine 1 on hematopoietic tissue (Hpt) cells is identical to the β-subunit of F 1ATP synthase. In this study we have used several different methods to clearly demonstrate that ATP-synthase is located on the plasma membrane of a subpopulation of Hpt cells and there may function as a receptor for astakine, whereas mature blood cells (hemocytes) do not have any ATP-synthase on the outside of their plasma membranes. Our results clearly show that ATP synthase β subunits are present on the cell surface of Hpt cells and highlight the need for more detailed studies on intracellular traffic connections between mitochondria and other membrane compartments.

Transglutaminase activity in the hematopoietic tissue of a crustacean, Pacifastacus leniusculus, importance in hemocyte homeostasis

BackgroundTransglutaminases (TGases) form a group of enzymes that have many different substrates and among the most well known are fibrin for Factor XIIIa and the clotting protein in crustaceans. We also found that TGase is an abundant protein in the hematopoietic tissue (Hpt) cells of crayfish and hence we have studied the possible function of this enzyme in hematopoiesis.ResultsTGase is one of the most abundant proteins in the Hpt and its mRNA expression as well as enzyme activity is very high in the Hpt cells, lesser in the semi-granular hemocytes and very low in the granular cells. In cultured hematopoietic tissues, high activity was present in cells in the centre of the tissue, whereas cells migrating out of the tissue had very low TGase activity. RNAi experiments using dsRNA for TGase completely knocked down the transcript and as a result the cell morphology was changed and the cells started to spread intensely. If astakine, a cytokine directly involved in hematopoiesis, was added the cells started to spread and adopt a morphology similar to that observed after RNAi of TGase. Astakine had no effect on TGase expression, but after a prolonged incubation for one week with this invertebrate cytokine, TGase activity inside and outside the cells was completely lost. Thus it seems as if astakine addition to the Hpt cells and RNAi of TGase in the cell culture will lead to the same results, i.e. loss of TGase activity in the cells and they start to differentiate and spread.ConclusionThe results of this study suggest that TGase is important for keeping the Hpt cells in an undifferentiated stage inside the hematopoietic tissue and if expression of TGase mRNA is blocked the cells start to differentiate and spread.This shows a new function for transglutaminase in preventing hematopoietic stem cells from starting to differentiate and migrate into the hemolymph, whereas their proliferation is unaffected. Astakine is also important for the hematopoiesis, since it induces hemocyte synthesis in the Hpt but now we also show that it in some unknown way participates in the differentiation of the Hpt cells.

Hemocyte‐lineage marker proteins in a crustacean, the freshwater crayfish, Pacifastacus leniusculus

To identify proteins associated with development of different hemocyte types in the freshwater crayfish Pacifastacus leniusculus, 2-DE followed by MS analysis was carried out with hematopoietic tissue (Hpt) cells, semigranular cells (SGC) and granular cells (GC). Within the hemocyte lineages one two-domain Kazal proteinase inhibitor (KPI) was found to be specific for SGC, while a superoxide dismutase (SOD) was specific for GC at protein as well as at mRNA level. The proliferation cell nuclear antigen (PCNA) was detected at the mRNA level in Hpt cells only. We also provide evidence that SGC and GC most likely differentiate to maturation as separate lineages. We found that after laminarin or lipopolysaccharide (LPS) injection into crayfish, the transcript levels of PCNA and SOD increased in the Hpt cells, whereas the KPI transcript never was present in Hpt regardless of any challenge. RNA interference of PCNA in the Hpt cells led to that most of the cells did not spread or attach to the tissue culture dish. These results suggest that PCNA, KPI and SOD can be used as markers for Hpt cells, SGC and GC, respectively, and in conjunction with these results, a model is proposed how the Hpt responds to a microbial challenge by proliferation and release of Hpt cells.

Characterization and localization of side population (SP) cells in zebrafish kidney hematopoietic tissue

AbstractWe previously showed that side population (SP) cells, characterized by specific Hoechst dye efflux pattern in flow cytometric analysis, were present in teleost kidney hematopoietic tissue, and that kidney SP cells were enriched in hematopoietic stem cells (HSCs). ABCG2/Abcg2 is an ATP-binding cassette (ABC) transporter that is known to be associated with Hoechst dye efflux activity of mammalian HSCs. In the present study, we examined the expression and function of Abcg2 in kidney SP cells from zebrafish (Danio rerio). Although the zebrafish genome has 4 paralogous copies of ABCG2 (zAbcg2a, b, c, and d), zAbcg2a and zAbcg2c mRNA was expressed in kidney SP cells. Transfection of COS-7 cells with zAbcg2a and zAbcg2c showed that zAbcg2a was directly associated with the SP phenotype. These results indicate that zAbcg2a mRNA is a useful marker for zebrafish HSCs. In situ hybridization in kidney tissue showed that zAbcg2a-positive cells were sporadically localized on the surface of renal tubules, and tightly adhered to renal tubule epithelial cells. This result suggests that teleost HSCs adhere to the surface of renal tubules, and that renal tubule epithelial cells are a key component of HSC niche in teleosts.

Constitutive Expression of the ATP-Binding Cassette Transporter ABCG2 Enhances the Growth Potential of Early Human Hematopoietic Progenitors

The ATP-binding cassette transporter, ABCG2, is a molecular determinant of the side population phenotype, which is enriched for stem and progenitor cells in various nonhematopoietic and hematopoietic tissues. ABCG2 is highly expressed in hematopoietic progenitors and silenced in differentiated hematopoietic cells, suggesting a role of ABCG2 in early hematopoiesis. To test whether ABCG2 is involved in human hematopoietic development, we retrovirally transduced umbilical cord blood-derived early hematopoietic cells and analyzed hematopoiesis in vitro and in vivo. ABCG2 increased the number of clonogenic progenitors in vitro, including the most primitive colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte, by twofold (n = 14; p < .0005). Furthermore, ABCG2 induced a threefold increase in the replating capacity of primary colonies (n = 9; p < .01). In addition, ABCG2 impaired the development of CD19+ lymphoid cells in vitro. In transplanted NOD/SCID mice, the ATP-binding cassette transporter decreased the number of human B-lymphoid cells, resulting in an inversion of the lymphoid/myeloid ratio. ABCG2 enhanced the proportion of CD34+ progenitor cells in vivo (n = 4; p < .05) and enhanced the most primitive human progenitor pool, as determined by limiting dilution competitive repopulating unit assay (p < .034). Our data characterize ABCG2 as a regulatory protein of early human hematopoietic development.