Academic literature on the topic 'Human haemopoietic cells'

Abstract Background and Aims: Spleen is the largest secondary lymphatic organ. It acts as a graveyard for RBCs, is essential for immune responses, performs lymphopoiesis in adults and haemopoiesis in fetuses. The present study was conducted to assess the histogenesis of spleen in human fetuses in view of existing literature. Material and Methods: The study was carried out on 34 formalin preserved human fetuses procured from Dr Sushila Tiwari Government Hospital, Haldwani with due clearance from ethical committee. The 6 pm sections of the spleen were stained with Haematoxylin and Eosin and observed under light microscope. Results: At 14 tol5 weeks, spleen had extensive sinusoids filled with RBCs and few lymphocytes. At 16-18 weeks, trabecular arteries were noticed more towards centre along with extensive haemopoietic cells in the venous sinusoids. By 20th week lymphocytic aggregation had started around arterioles. By 24 weeks periarteriolar lymphatic sheath was clearly observed. At term (37-40 weeks), classical primary lymphoid follicle was present but germinal centers were not observed. Conclusion: During earlier differentiation, spleen symbolizes the function of haemopoietic activities and gradually during subsequent gestation; it establishes its identity as a principle lymphoid tissue.

Heparan sulphate proteoglycans (HSPGs) present on the surface of bone marrow stromal cells and in the extracellular matrix (ECM) have important roles in the control of adhesion and growth of haemopoietic stem and progenitor cells. The two main groups of proteoglycans which contain heparan sulphate chains are members of the syndecan and glypican families. In this study we have identified the main surface membrane and matrix-associated HSPGs present in normal human bone marrow stroma formed in long-term culture. Proteoglycans were extracted from the adherent stromal layers and treated with heparitinase and chondroitinase ABC. The core proteins were detected by Western blotting using antibodies directed against syndecans-1-4, glypican-1 and the ECM HSPG, perlecan. Stromal cell expression at the RNA level was detected by Northern blotting and by reverse transcription PCR. Glypican-1, syndecan-3 and syndecan-4 were the major cell-membrane HSPG species and perlecan was the major ECM proteoglycan. There was no evidence for expression of syndecan-1 protein. Syndecan-3 was expressed mainly as a variant or processed 50-55 kDa core protein and in lower amounts as the characteristic 125 kDa core protein. These results suggest that syndecan-3, syndecan-4 and glypican-1 present on the surface of marrow stromal cells, together with perlecan in the ECM, may be responsible for creating the correct stromal ‘niche’ for the maintenance and development of haemopoietic stem and progenitor cells. The detection of a variant form of syndecan-3 as a major stromal HSPG suggests a specific role for this syndecan in haemopoiesis.

Abstract Several studies have preciously shown improved engraftment of human haemopoietic stem cells (HSC) following co-infusion of human mesenchymal stem cells (MSC). However, these studies have been criticised for using xenogeneic recipients which might support human haemopoiesis through the production of species-specific cytokines and growth factors by human MSC. To further investigate the potential for MSC to support HSC engraftment we used a murine to murine transplant model to co-infuse purified murine MSC and murine HSC. The MSC were derived from collagenase-treated bone fragments of the Rosa26 murine strain, had the phenotype CD45- CD31-CD11b- Sca-1bright VCAM1+ and could differentiate into osteocytes, chondrocytes and adipocytes. Donor and recipient HSC were obtained from unmanipulated bone marrow of either the C57Bl/6J murine strain, CD45 isotype CD45.2, or the PEP3b murine strain, expressing CD45.1. Following 10Gy irradiation to the recipients HSC and MSC were co-infused. Donor and recipient haemopoietic engraftment in the peripheral blood was quantified by FACS analysis of the CD45.1/CD45.2 ratio. Successive transplant experiments persistently showed improved donor haemopoietic engraftment following co-infusion of 2 x 105 BM cells and 1 x 106 MSC (three seperate transplant series p=0.001, p=0.09 and p=0.13). The beneficial effect was maximal at the lower HSC dose of 1 x 105 BM cells (p=0.004, p=0.045 and uninterpretable in the third transplant series due to excessive death observed in the non-MSC recipients). Increasing the MSC dose to 2 x 106 cells showed a non-significant increase in survival and donor haemopoietic engraftment but further MSC escalation resulted in fatal emboli post-infusion. LacZ staining of tissue sections failed to show evidence of MSC outside the lungs but was detected at very low levels in the bone from one recipient. As MSC are osteoblast precursors and recent literature suggests a role of osteoblasts as the HSC niche we investigated the effect of pre-culturing our MSC population in osteogenic media prior to transplant. Following 16 days of culture approximately 40% of colonies showed ALP activity. At this time point osteogenic-stimulated MSC (O-MSC) were removed from culture, washed to remove any residual osteogenic media, and infused in our standard method. O-MSC recipients receiving 1 x 105 BM cells showed a mean engraftment of 79% compared to 21% in non-msc recipients receiving 2 x 105 BM cells (p=0.000017), and 42% in recipients receiving standard MSC and 1 x 105 BM cells (p=0.01). In conclusion we confirm improved haemopoietic engraftment in a non-xenogeneic model and does not require significant MSC engraftment. The beneficial effect is maximised through osteogenic stimulation of MSC suggesting the possibility that the mechanism may be through priming of the HSC niche.

Four purine arabinosides that inhibit varicella-zoster virus (VZV) replication in vitro were tested as inhibitors of colony formation by progenitor cells from normal human bone marrow. In general, erythroid burst forming cells (BFU-E) were more sensitive to inhibition by these compounds than were either erythroid colony forming cells (CFU-E) or granulocyte/macrophage colony forming cells (CFU-GM). A 50% reduction in colony formation (IC50) was observed for BFU-E in the presence of 8 μM 6-methoxypurine arabinoside. Adenine arabinoside and hypoxanthine arabinoside had IC50 values of 1 μM and 4 μM respectively, whereas 6-ethoxypurine arabinoside was not inhibitory (IC50 > 50 μM). Enzyme studies showed that both 6-methoxypurine arabinoside and adenine arabinoside were converted to hypoxanthine arabinoside by adenosine deaminase. 6-Ethoxypurine arabinoside was a much less efficient substrate. When the BFU-E assays were performed in the presence of an inhibitor of adenosine deaminase, 6-methoxypurine arabinoside became non-inhibitory. In contrast, adenine arabinoside became much more inhibitory (IC50 = 0.03 μM). The potency of hypoxanthine arabinoside was unaffected. Thus, incubation of 6-methoxypurine arabinoside and adenine arabinoside under conditions appropriate for the BFU-E assay resulted in the in situ conversion of these compounds to hypoxanthine arabinoside. Biotransformation of compounds must be considered in the assessment of toxicity in vitro.

Abstract NKT cells, a novel class of regulatory T cells, secrete haemopoietic cytokines (GM-CSF, IL-3, IL-6) upon engagement of their TCR. Because of this property we hypothesized that NKT cells are involved in the regulation of haemopoiesis. NKT cells constitute <0.1% of T cells in blood, bone marrow and cord blood and are restricted by the glycolipid-presenting, non-polymorphic MHC-like molecule CD1d. CD1d is expressed in antigen presenting cells, thymocytes and in a variety of epithelial tissues including keratinocytes and enterocytes, but its expression in haematopoietic stem cells (HSC) has not been studied. Therefore we studied first the expression and function of CD1d in haemopoietic stem cells. Using multi-colour flow cytometry, we show that 1% of MACS-selected cord blood CD34+ cells are CD1d+ (n=6, range 0.4–1.67%). CD1d+CD34+ HSC express a variety of surface markers indicative of primitive HSC: CD7: 36.1% (35.6–36.4%), CD133: 68% (46.2–81.54%), CD117:74.2% (51–85.5%) and CD90 (Thy-1): 32.3% (26.7–39.1%); moreover, 6,2% (1.9–10.5%) and 12,8% (10.1–16%) of CD1d+CD34+ are CD1d+CD34+HLADR− and CD1d+CD34+CD38− respectively, consistent with an immature HSC phenotype. Expression of these markers by CD1d−CD34+ were identical to CD1d+CD34+ cells. Consistent with this, in long-term colony initiating cell (LTC-IC) assays (n=4), highly purified, flow-sorted, lineage-depleted (Stem Cell Technologies) Lin-CD1d+CD34+ HSC displayed a LTC-IC frequency of 1 in 35.7 cells (range 24.4–38) equivalent to those of CD1d−CD34+ HSC: LTC-IC frequency of 1 in 25.4 cells (range 20–30.3). Short term CFC activity of Lin−CD34+CD1d+ is slightly lower than their Lin−CD1d−CD34+ counterparts: 1 CFC per 15.3 cells (7.7–37.7) versus 1 CFC per 4.2 cells (3.8–5), respectively. To investigate the effect of cord blood NKT cells on the CFC activity of CD34+ cells, NKT were first activated ex vivo for 10 days in the presence of the CD1d-presented glycolipid a-galactosylceramide and subsequently were purified by flow-sorting using mAbs specific to their TCR a and b chains, i.e., anti-TCR Va24 and Vb11. Purified NKT were co-cultured in a ratio of 10:1 with CD34+ cells. In the absence of exogenous cytokines NKT enhanced the clonogenic capacity of CD34+ cells by 3-fold: 1 CFC per 14 cells (range 10.4–21) in the presence of NKT vs 1 per 43 cells (range 37–55.5) in the absence of NKT (n=4; p=0.024). By contrast, activated or resting autologous T cells co-cultured with CD34+ cells at the same ratio (10:1) had no effect on the CFC frequency, indicating that this enhancing effect on haemopoiesis is a unique property of NKT cells. The effect of NKT in the long-term clonogenic capacity is currently being evaluated. In summary, we have shown that a) CD1d is a novel marker expressed in HSC with long- and short -term clonogenic ability and b) CD1d-restricted NKT cells promote haemopoiesis These findings reveal a novel link between haemopoiesis and the CD1d-NKT axis of immune regulation and set the scene for the study of the role of NKT cells in the processes of engraftment and rejection in HSC transplantation.

"December 2001." Includes bibliographical references (leaves 178-225) Focuses on the ex vivo growth of human haemopoietic progenitor cells with the objective of defining culture conditions for generating myeloid post-progenitor cells for therapy

The reduced engraftment potential of cytokine cultured haemopoietic stem/progenitor cells from adult mobilised peripheral blood has been associated with their defective homing to bone marrow niches. In this work, using established in vivo systems and a novel ex vivo model, an additional cytokine-induced attachment defect is described that reduces the retention of these cells in the bone marrow, post-transplantation. This defect was found to be related to specific niche ligands and was not caused by downregulation of their respective receptors on the expanded cells. CD26 is a protease that cleaves SDF-1 abrogating its chemotactic effect. CD26 inhibition on the transplanted cells was not sufficient to reverse the engraftment defect, although infusion of the inhibitor in immunodeficient animals, together with ex vivo treated cells, significantly increased engraftment. Finally, mobilised peripheral blood stem/progenitor cells were found to express neuroreceptors and their expression was altered after exposure to cytokines. Epinephrine pre-treatment of these cells rescued their adhesion to specific niche ligands, increased their short-term homing and improved their long-term engraftment in immunodeficient animals.

This thesis studied aspects of the interaction of hepatitis B virus (HBV) with haemopoietic cells and cell lines, to address the reported tropism of HBV for haemopoietic tissues. Emphasis was directed at demonstrating specific attachment of HBV to defined subpopulations of peripheral blood leucocytes (PBL) and bone marrow cells (BM), and the distribution of receptors for HBV on well-defined haemopoietic cell lines. Biochemical characterisation of the virus-cell interaction was also performed, and the question of infectivity of haemopoietic cell lines was addressed' Firstly, a quantitative assay of HBV binding to liver plasma membranes (PM) was adapted to show that isolated PBL PM bound serum-derived FIBV particles to a similar degree, based on their protein content. Using synthetic peptides representative of various amino acid sequences of the preSl and preS2 regions of L HBsAg to inhibit HBV binding to the pM, it was found that peptide pres l(12-32) inhibited binding to PBL PM by 60-80% and peptide preSl(21-47) inhibitedby 0-30% (depending on the source of PM), while peptides preSl(32-49) and preS2(120-145) did not inhibit binding. This contrasts with results obtained using liverPM, where peptide preSl(12-32) did not inhibit binding, while peptide preSl(21- 47) inhibited by 70%, and preSl(32-49) inhibited by approximately 12%. Peptide preS2(120- 145) had no effect on binding. Thus, different regions of the L surface protein appear to mediate attachment to PBL and hepatocytes. HBV particles isolated from serum are complexed with serum proteins including IgG. To test the involvement of receptors for IgG and complement fragments (opsonins) in the HBV-PM interaction, a panel of ligand-blocking monoclonal antibodies (MAbs) to opsonin receptors was used, and it was shown that the three classes of receptors for IgG (FcγRI, FcγRII and FcγRIII) and CR3, are not major receptors for HBV on PBL or hepatocytes, as MAbs to these did not inhibit HBV binding. It was also shown that HBV does not utilise the receptor for IgA, FcαR, for attachment to PBL, despite reported sequence homology between the large envelope protein of HBV and the Fc portion of human IgA. In contrast to a published report that IL-6 mediates binding of HBV to hepatocytes, IL-6 was shown not to mediate attachment to either liver or PBL PM, by virtue of pre-incubation with a blocking polyclonal anti-serum to IL-6. Glycosaminoglycans (GAGs) were found to influence HBV binding to PM: soluble heparin (HE) inhibited binding to liver PM by up to 80%, and to leucocyte PM by up to 40%; chondroitin sulphare C (CS-C) enhanced virus binding (approximately 1.5-fold) to leucocyte pM only. Chondroitin sulphate A and hyaluronate had no effect on binding to either PM, arguing that simple electrostatic properties of GAGs were not responsible for the observed effecrs. The incomplete inhibition by HE and enhancement by CS-C could indicate the presence of more than one class of binding site for HBV on the respective PM, and coupled with the differential pattern of inhibition in the presence of synthetic peptides, argues that receptors for HBV on pBL and hepatocytes may be either different, or altered forms of the same molecule(s). To extend these studies, whole cell binding assays were developed in order to accurately define which subsets of pBL and BM cells could bind HBV. Using purified HBV particles as the first stage in an immunofluorescence-based detection system, followed by detection of bound HBV using anti-preS1 MAbs F35.25 or MAl8/7, and a FlTC-conjugated third-stage antibody, specific membrane staining of peripheral blood monocytes from 8/9 donors was observed. In addition, binding of HBV to the erythroleukaemia cell line K562 was observed, while other myeloid cell lines did not appear to bind virus. This assay was then adapted to a suspension cell assay with analysis by flow cytometry, using phycoerythrin as the detecting fluorochrome. The parameters of binding were optimised for K562 cells and these were then applied to analyse HBV binding to PBL and BM cells obtained from healthy volunteers, whose serum was free of HBV markers. Based on their light scattel characteristics, monocytes and neutrophils were the only cell types in the peripheral blood that bound HBV' while binding to lymphocytes was not observed. This was confirmed by two-colour immunofluorescence to simultaneously detect bound HBV and subset-specific leucocyte markers. Similarly, in the BM, only monocytes bound HBV. Importantly, haemopoietic stem cells (cD34+) did not bind HBV. Binding was tested to 'activated' populations of lymphocytes (pHA-treated), monocytes (LPS-treated), and neutrophils (fMLP-treated). The pattern of HBV binding was not affected by these treatments. Monocytes cultured in vitro, bound significantly more virus than freshly isolated monocytes. Taken together, these results indicate that only monocytes, and to a lesser extent neutrophils, express potential receptors for HBV, and a differentiation-dependent upregulation of receptor sites for HBV is observed on Monocytes The distribution of potential HBV receptors was determined on a number of haemopoietic cell lines, representative of various haemopoietic lineages. K562 (erythroid), and the monocyte cell line THP-l, were the only haemopoietic cell lines which bound HBV, while binding was also observed to the human hepatoma cell line HepG2. A number of other erythroid and monocyte cell lines, as well as T and B tymphoid, and a megakaryocytic line, all failed to bind HBv. A comparison of the surface immunophenotypes of all the cell lines tested excluded all known CD-classified molecules (including opsonin receptors), as candidate HBV receptors. The biochemical characteristics of the interaction of HBV with all of these cell types were then examined. On K562 and THP-1, HBV binding was sensitive to the protease chymopapain but insensitive to trypsin, indicating that the molecule was a glycosylated protein. Pre-treatment of these cell lines with tunicamycin, to inhibit post-translational addition of N-linked carbohydrate to surface glycoproteins, did not influence HBV binding, indicating that these moieties are not important for virus attachment. Enzymatic removal of cell surface sialic acids with neuraminidase significantly enhanced HBV binding to K562 and THP-1 cells but did not confer binding to otherwise 'negative' cell lines. Binding of HBV to cultured monocytes and HepG2 cells was trypsin and chymopapain sensitive' and was not increased by neuraminidase pre-treatment. Cation chelation demonstrated that HBV binding of to all cell types was Ca²⁺/Mg²⁺-independent, and acid elution of cells showed that binding was not mediated by peripherally-bound molecules. Binding of HBV to monocytes and to HepG2 cells was significantly reduced by pre-treatment of the cells with PI-PLC, implying that the molecule responsible for binding to these cells is GPl-linked. In this case, a comparison with HBV binding to K562 was not informative due to the resistance of the GPI linkages on these cells, and possibly also on THP-I cells (based on CD59 cleavage), to hydrolysis by PI-PLC. Thus, cells expressing potential receptor(s) for HBV, whose characteristics do not correlate with any other proposed candidate, have been identified. Immunoprecipitation analysis using HBV particles covalently cross-linked to the surface of ¹²⁵I-labelled K562 cells, and anti-S MAb coupled to goat anti-mouse IgG-Sepharose, resulted in the identification of a 50 kDa species as a putative HBV receptor. Contrary to reports that HBV inhibits colony formation by myeloid cell lines in semi-solid media, no inhibitory effect by HBV was seen on clonal cell growth in liquid culture. K562 cells were found to be able to efficiently internalise HBV particles, which accumulated in a perinuclear compartment. In infection studies, K562 cells positive for HBsAg aftet 2-4 days post-infection became enlarged, and their numbers decreased steadily over an 11 day period. It is not clear whether these cells represent a transient or differentiated cell type. Similarly, it appears that the level of HBV DNA in these cells declines steadily during the infection course.
Thesis (Ph.D.) -- University of Adelaide, Dept. of Microbiology and Immunology, 1998

BK virus are human polyomaviruses associated with BK virus-associated nephropathy (BKVAN) in post-transplant recipients on immunosuppression. Primary infection is usually asymptomatic, and the virus will remain in a latent phase. Reactivation usually occurs due to a number of risk factors, namely target organ damage and a reduction in the host immune function. BK virus can also cause haemorrhagic cystitis in haemopoietic stem cell transplant recipients. Diagnosis of BKVAN can range from visualization of decoy cells in urine microscopy (low sensitivity and high specificity) to serum and urine polymerase chain reaction (PCR) (high sensitivity and high specificity). Screening for BKVAN is generally recommended post transplantation. There is currently no specific antiviral therapy and the mainstay of treatment involves reduction in immunosuppressive agents.