Cells In Aplastic Anemia Research Articles

Gene Expression Profiling Identifies HOXB4 as a Direct Downstream Target of GATA-2 in Human CD34+ Hematopoietic Cells

Aplastic anemia is characterized by a reduced hematopoietic stem cell number. Although GATA-2 expression was reported to be decreased in CD34-positive cells in aplastic anemia, many questions remain regarding the intrinsic characteristics of hematopoietic stem cells in this disease. In this study, we identified HOXB4 as a downstream target of GATA-2 based on expression profiling with human cord blood-derived CD34-positive cells infected with control or GATA-2 lentiviral shRNA. To confirm the functional link between GATA-2 and HOXB4, we conducted GATA-2 gain-of-function and loss-of-function experiments, and HOXB4 promoter analysis, including luciferase assay, in vitro DNA binding analysis and quantitative ChIP analysis, using K562 and CD34-positive cells. The analyses suggested that GATA-2 directly regulates HOXB4 expression through the GATA sequence in the promoter region. Furthermore, we assessed GATA-2 and HOXB4 expression in CD34-positive cells from patients with aplastic anemia (n = 10) and idiopathic thrombocytopenic purpura (n = 13), and demonstrated that the expression levels of HOXB4 and GATA-2 were correlated in these populations (r = 0.6573, p<0.01). Our results suggested that GATA-2 directly regulates HOXB4 expression in hematopoietic stem cells, which may play an important role in the development and/or progression of aplastic anemia.

Functional characterization of CD4+ T cells in aplastic anemia

AbstractThe role of CD4+ T cells in the pathogenesis of aplastic anemia (AA) is not well characterized. We investigate CD4+ T-cell subsets in AA. Sixty-three patients with acquired AA were studied. Th1 and Th2 cells were significantly higher in AA patients than in healthy donors (HDs; P = .03 and P = .006). Tregs were significantly lower in patients with severe AA than in HDs (P < .001) and patients with non-severe AA (P = .01). Th17 cells were increased in severe AA (P = .02) but normal in non-severe AA. Activated and resting Tregs were reduced in AA (P = .004; P = .01), whereas cytokine-secreting non-Tregs were increased (P = .003). Tregs from AA patients were unable to suppress normal effector T cells. In contrast, AA effector T cells were suppressible by Tregs from HDs. Th1 clonality in AA, investigated by high-throughput sequencing, was greater than in HDs (P = .03). Our results confirm that Th1 and Th2 cells are expanded and Tregs are functionally abnormal in AA. The clonally restricted expansion of Th1 cells is most likely to be antigen-driven, and induces an inflammatory environment, that exacerbate the functional impairment of Tregs, which are reduced in number.

Impaired immunomodulatory ability of bone marrow mesenchymal stem cells on CD4+ T cells in aplastic anemia

Aplastic anemia (AA) is a marrow failure syndrome mediated by aberrant T-cell subsets. Mesenchymal stem cells (MSCs) play an important role in maintaining immune homeostasis through modulating a variety of immune cells. However, little is known about the immunomodulation potential of bone marrow MSCs (BM-MSCs) in AA. Here, we reported that BM-MSCs from AA patients were reduced in suppressing the proliferation and clonogenic potential of CD4+ T cells and the production of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), which was associated with decreased prostaglandin E2 (PGE2). Meanwhile, BM-MSCs from AA patients were defective to promote CD4+CD25+FOXP3+ regulatory T cells expansion through reduced transforming growth factor-β (TGF-β). No significant difference between AA and normal BM-MSCs was observed in affecting the production of interleukins (IL)-4, IL-10 and IL-17. Our data indicate that BM-MSCs were impaired in maintaining the immune homeostasis associated with CD4+ T cells, which might aggravate the marrow failure in AA.

Laboratory aspects of PNH

A flow cytometric assay demonstrating the loss of GPI-linked proteins on at least two haemopoietic lineages is now the only appropriate laboratory test to diagnose paroxysmal nocturnal haemoglobinuria (PNH). The Ham’s test (1939), sugar water test (1966) and sucrose haemolysis test are no longer considered appropriate. Guidelines have recently been released by the Australasian Flow Cytometry Group and the Clinical Laboratory Standards Institute and a proficiency testing module has been commenced by the Royal College of Pathologists of Australasia (RCPA) Hae-matology Quality Assurance Program (QAP). In the 2011 QAP survey, 21 of 30laboratories used FLAER as a marker. Laboratories should report results in a format that is clear and satisfies the requirements of drug regulatory authorities. It is recommended that lineage gating of granulocytes using either CD15/SS or CD33/SS shouldbe followedbythe useofCD24/FLAER for granulocytes and CD14/FLAER for monocytes. CD59 (clone MEM43) antibodies should be used to identify Type I, II or III red cells. Anti-glycophor-yin-A (CD235a) may be used to identify red cells but needs to be titrated to avoid formation of aggregates. Careful planning can ensure that all necessary leukocyte markers can be included in a single tube for diagnosis of PNH and the detection ofsmall numbers of PNH cells in aplastic anaemia.

Th17 immune responses contribute to the pathophysiology of aplastic anemia

AbstractT helper type 17 (Th17) cells have been characterized based on production of interleukin-17 (IL-17) and association with autoimmune diseases. We studied the role of Th17 cells in aplastic anemia (AA) by isolating Th17 cells from patients blood (n = 41) and bone marrow (BM) mononuclear cells (n = 7). The frequency and total number of CD3+CD4+IL-17–producing T cells were increased in AA patients at presentation compared with healthy controls (P = .0007 and .02, respectively) and correlated with disease activity. There was an inverse relationship between the numbers of Th17 cells and CD4+CD25highFoxP3+ regulatory T cells (Tregs) in the blood of AA patients. Concomitant with the classical Th1 response, we detected the presence of CD4+ and CD8+ IL-17-producing T cells in a mouse model of lymph node infusion–induced BM failure. Although anti–IL-17 treatment did not abrogate BM failure, early treatment with the anti–IL-17 antibody reduced the severity of BM failure with significantly higher platelet (P < .01) and total BM cell (P < .05) counts at day 10. Recipients that received anti-IL-17 treatment had significantly fewer Th1 cells (P < .01) and more Treg cells (P < .05) at day 10 after lymph node infusion. Th17 immune responses contribute to AA pathophysiology, especially at the early stage during disease progression.

Contrasting Roles of Th1 and Th17 Cells in Aplastic Anaemia (AA) and Myelodysplastic Syndrome (MDS).

Abstract 1090Poster Board I-112 IntroductionAutoimmunity is believed to play an important role in the pathogenesis of both idiopathic aplastic anaemia (AA) as well as low-risk MDS (L-MDS). While there is a significant overlap in some of the clinical features between AA and L-MDS, there is a striking difference in response to immunosuppressive therapy (IST) (70-80% in AA vs 20% in L-MDS).We have previously shown that Tregulatory cells are significantly reduced in L-MDS (Kordasti Blood 2008), while other groups have suggested that Tregs are decreased in AA. We hypothesised that differences in the immune regulatory profile as well as the cytokine environment, may differentially influence the pathogenesis of AA and MDS. In a prospective study we examined the immunological and cytokine profiles within a cohort of newly diagnosed AA and L-MDS patients. Patients and Methods33 Acquired aplastic anaemia(22 at diagnosis and pre-treatment and 11 post IST), 18 low risk MDS (IPSS=0) and 5 healthy controls were recruited.The percentage and absolute number of different CD4+ T cell subsets (Th1, Th2, Th17, TNF-αa producing CD4+ T cells and Foxp3+ Tregs) in peripheral blood, were investigated by flow cytometry. T cells were stimulated first and then stained intracellularly for IFN-γ, IL-4, IL-17 & TNF-αa. The serum level of 30 different cytokines was also measured by 30 Plex bead analysis (Luminex). NK cells were defined as CD3– CD56+. B cells were defined as CD3-CD19+. CD3+ CD4+ T-cell subsets were defined as CD45RO–CD27+ naïve, CD45RO+ CD27+ CD62L+ central memory,CD45RO+ CD27+ CD62L– effector memory, CD45RO+CD27– effectors and CD45RO– CD27– terminal effectors. CD4 regulatory T cells were defined as CD4+ CD25highFoxp3+. ResultsThe absolute number of Th1 cells and TNF-αa producing CD4+ T cells were significantly higher in AA patients compared to healthy controls(42 × 107/L v 29 × 107/L) (p=0.001, p=0.005). Although the number of Th17 cells was the same as healthy controls, this number was significantly lower than low risk MDS patients (0.86 × 107/L v 2.7 × 107/L) (p=0.009). Amongst AA patients, the number of Th2 cells and Foxp3+ Tregs were not significantly different from healthy controls. Following IST, the number of Tregs was significantly higher in responders than non responders (0.02 × 107/L v 0.001 × 107/L) (p=0.009). Interestingly the ratio of Th17/ Tregs was higher in non-responsive patients (15.2 v 2.8)(p=0.01). The number of T cell subsets, NK and B cells were not significantly different from healthy controls in our cohort of AA patients.The serum levels of proliferative cytokines, EGF (p=0.01), HGF(p=0.01), VEGF (p=0.01) and pro-inflammatory cytokines IL-13 (p=0.02), IL-8 (p<0.001) were significantly higher in AA patients than healthy controls and this was different from low risk MDS patients in whom the levels of IL-12 (P < 0.01), IL-7 (P < 0.005), IFN-γ (P < 0.01) and RANTES (P < 0.005) were elevated. ConclusionsOur data suggests that despite the presence of autoimmunity in both low risk MDS and AA, the involved mechanisms are significantly different. In AA the main mechanism is a Th1 derived (IFN-γ and TNF-αa production) response whereas in low risk MDS, Th17 cells may play an important role in creating a more indolent inflammatory environment. The serum cytokine profiles in these diseases are different too. These data also suggests a significant role for Tregs (absolute number as well as ratio of Th1 and Th17 to Tregs) in the prediction and evaluation of response to IST. DisclosuresNo relevant conflicts of interest to declare.

Antibodies to glycosylphosphatidyl-inositol anchored proteins (GPI-AP) in antithymocyte and antilymphocyte globulin: possible role for the expansion of GPI-AP deficient cells in aplastic anemia

Antithymocyte globulin (ATG) and antilymphocyte globulin (ALG) are currently used successfully for immunosuppressive treatment of aplastic anemia. In this study we have investigated whether commercial ATG/ALG preparations contain antibodies against glycosylphosphatidyl-inositol anchored proteins (GPI-AP), which could be responsible for emergence of GPI-deficient populations in aplastic anemia after ATG/ALG therapy. We analyzed four commercial ATG/ALG preparations by competitive binding assays using flow cytometry. Quantification was achieved by calculating the concentration of ATG/ALG required to give 50% inhibition of binding the specific fluorochrome-labeled monoclonal antibody (EC50). High concentrations of antibodies against the GPI-anchored protein CD52 were found in all preparations (Lymphoglobulin Genzyme, Thymoglobulin Genzyme, ATGAM. Pharmacia & Upjohn, and ATG-Fresenius S Fresenius). Antibodies against the GPI-anchored protein CD48 are present in significant concentrations except in the preparation ATGAM. CD16 antibodies were found in lower concentrations. We could not detect significant concentrations of antibodies against the GPI-anchored proteins CD157 and CD14. Campath-1H, a monoclonal antibody against the GPI-anchored protein CD52, has been used as immunosuppressive tool for T-cell depletion. CD52 antibodies in ATG/ALG preparations might contribute in the same way to the immunosuppressive effects in treatment of aplastic anemia. It is known that in a substantial proportion of patients with aplastic anemia GPI-deficient cells are present in a low level at diagnosis or emerge after immunosuppressive therapy. GPI-anchored antibodies in ATG/ALG preparations might lead to a relative advantage for pre-existing GPI-deficient cells caused by an escape from the antibody-mediated attack.

Screening of aplastic anaemia-related genes in bone marrow CD4+T cells by suppressive subtractive hybridization

CD4(+) T cells play a crucial role in the pathogenesis of aplastic anaemia. However, the mechanisms of over-proliferation, activation, infiltration of bone marrow and damage to haematopoietic cells of CD4(+) T cells in aplastic anaemia are unclear. Therefore, we screened differentially expressed genes of bone marrow CD4(+) T cells of aplastic anaemia patients and normal donors by suppressive subtractive hybridization to investigate the pathogenesis of aplastic anaemia. The bone marrow mononuclear cells of a first visit aplastic anaemia patient and a healthy donor of the same age and sex were isolated using lymphocyte separating medium by density gradient centrifugation. With the patients as "tester" and donor as "driver", their CD4(+) T cells were separated with magnetic bead sorting and a cDNA library established by suppressive subtractive hybridization. Then 15 of the resulting subtracted cDNA clones were randomly selected for DNA sequencing and homological analysis. With semiquantitative RT-PCR, bone marrow samples from 20 patients with aplastic anaemia and 20 healthy donors assessed the expression levels of differentially expressed genes from SSH library. PCR detected 89 clones in the library containing an inserted fragment of 100 bp to 700 bp. Among 15 sequenced clones, 12 were known genes including 3 repeated genes. Compared with normal donors, there were 9/12 genes over-expressed in bone marrow CD4(+) T cells of patients with aplastic anaemia. The effects of these genes included protein synthesis, biology oxidation, signal transduction, proliferative regulation and cell migration. Not all these genes had been reported in the mechanisms of haematopoietic damage mediated by CD4(+) T cells in aplastic anaemia. Screening and cloning genes, which regulate functions of CD4(+) T cells, are helpful in elucidating the mechanisms of over proliferation, activation, infiltrating bone marrow and damaging haematopoietic cells of CD4(+) T cells in aplastic anaemia.

Quantitative analysis of bone marrow CD34 cells in aplastic anemia and hypoplastic myelodysplastic syndromes

Aplastic anemia (AA) and hypoplastic myelodysplastic syndromes (hMDS) are often difficult to distinguish. However, an accurate diagnosis is important because the prognosis and treatment of these diseases may differ. CD34+ hematopoietic progenitors are central to the pathogenesis of both disorders; they are the targets of the autoimmune attack in AA and neoplastic transformation in MDS. The aim of this study was to assess whether bone marrow CD34+ cell numbers could be used in differentiating between AA and hMDS. The percentage of bone marrow CD34+ cells was normal or increased (mean -3.5+0.5%, range 1-7%) in 15 of 35 patients studied, and low (mean -0.13 +/- 0.02%, range 0.02-0.36%) in 20 of 35 patients. All patients with a normal or increased percentage of CD34+ cells were ultimately diagnosed with hMDS based on the detection of clonal cytogenetic abnormalities or progression to refractory anemia with excess blasts/acute myeloid leukemia. All patients with low marrow CD34+ cell numbers met standard clinical criteria for AA and have not demonstrated neoplastic transformation with follow-up. Quantification of marrow CD34+ cells may serve as an important tool for distinguishing between AA and hMDS.

A Murine Model of Bone Marrow Failure Mediated by Disparity in Minor Histocompatibility Antigens.

Destruction of hematopoietic cells in aplastic anemia and other hematologic diseases is mediated in most patients by effector cells of the immune system. We have developed a mouse model of immune-mediated bone marrow failure which employs parental lymph node cell infusion into F1 recipients, resulting in bone marrow hypercellularity, pancytopenia, and death from infection and bleeding, without other organ involvement by graft-versus-host disease (Bloom, ML et al. Exp. Hematol . 32:1163, 2004; Chen, J et al. Blood 104:1671, 2004). Because major histocompatibility antigens are not the targets in acquired aplastic anemia, we now have developed a model of murine marrow failure based on disparity of minor histocompatibility antigens. Lymph node cells from C57BL/6 mice were infused into sublethally-irradiated, MHC-matched, C.B10-H2b/LilMce recipients. Animals developed severe pancytopenia and marrow hypoplasia within two-three weeks. CD8+ T lymphocytes were expanded in the blood and infiltrated bone marrow, becoming detectable at day 7 and reaching peak levels at days 10–12. There were no inflammatory responses observed in the skin, intestines, or other visceral organs by gross or microscopic pathological examination. In our experiments we observed a time-dependent expansion followed by contraction of CD8+ T cells specific for a minor histocompatibility antigen H60 as measured by flowcytometry using an H60-specific tetramer. H60 is an antigen peptide derived from a glycoprotein, a known ligand for stimulatory NKG2D receptors, and is immunodominant over other minor antigens in stem cell transplantation. The proportion of H60-specific CD8+ T cells was strongly negatively correlated with peripheral blood white cell, neutrophil, and platelet counts. Isolated H60-specific T-cells from bone marrow of affected animals induced apoptosis in vitro of normal C57BL/6 bone marrow cells in co-culture. The degree of apoptosis was further increased by addition of CD4 T-cells from same affected donors, suggesting a helper lymphocyte effect. The role of H60-specific T cells was demonstrated in further transplant experiments. Infusion of 5 × 106 C57BL/6 lymph node cells that had been depleted of H60-specific T cells was unable to induce marrow failure in C.B10 recipients, while the same number of cells with the addition of 20–90 × 103 H60-specific CD8+ T cells led to thrombocytopenia and leucopenia in recipients. H60-specific T cells thus appear to be key effectors, responsible at least for the initiation of marrow cell destruction. Mice could be treated with cyclosporine at 50 mg/g/day for five days, if treatments were begun at the time of lymph node cell infusion. Immunosuppressive therapy abolished H60-specific CD8+ T cell expansion and attenuated the development of peripheral pancytopenia, effectively rescuing animals. In conclusion, we demonstrate in this model of immune-mediated bone marrow failure that a single clone of peptide-specific T-cells is capable of efficient marrow destruction and the production of aplastic anemia.

Expression of membrane-bound IL-15 by bone marrow fibroblast-like stromal cells in aplastic anemia

In order to explore the relationship between IL-15 and aplastic anemia (AA), bone marrow (BM) fibroblast-like stromal cells (BMFSCs) were obtained from BM samples of 23 AA patients by density centrifugation and primary culturing in vitro. Indirect immunofluorescence labeling as well as flow cytometry and confocal laser scanning microscopy analysis were used to determine the expression of membrane-bound IL-15 (mIL-15) on the surface of BMFSCs derived from AA patients (AA-BMFSCs). The effects of IFN-gamma and cyclosporin A (CsA) on the expression of mIL-15 were also investigated. [(3)H]thymidine incorporation test as well as specific antibody inhibition and Transwell separation experiment was adopted to functionally evaluate the expression of mIL-15 on the surface of AA-BMFSCs. mIL-15 was found to be over-expressed on the surface of AA-BMFSCs. IFN-gamma further significantly up-regulated its expression, which, however, was inhibited by CsA. Interestingly, a tight correlation was found between the expression of mIL-15 and IL-15Ralpha on the surface of AA-BMFSCs. AA-BMFSCs had the capability to stimulate the proliferation of T lymphocytes, which was partly or completely inhibited by using neutralizing anti-IL-15Ralpha antibody, neutralizing anti-IL-15 antibody, blocking anti-IL-2/15Rgamma(c) mAb or Transwell chambers with a 0.3-mum pore size membrane to block the direct cell-to-cell contact between AA-BMFSCs and T cells. Apparently, BMFSCs as the most important component of BM hematopoietic microenvironment usually over-express mIL-15 in AA patients. Therefore, AA-BMFSCs may indirectly participate in the T cell-mediated destruction of hematopoietic progenitors in AA by recruiting T cells to BM and stimulating them in situ.

Granulocyte Colony Stimulating Factor Preferentially Causes Proliferation of Pre-Existing Monosomy 7 Cells in Aplastic Anemia and Myelodysplastic Syndrome Because of Abnormal Truncated GCSF-Receptors on These Cells.

Administration of granulocyte colony stimulating factor (GCSF) has been linked to development of monosomy 7 in some patients with aplastic anemia (AA) and congenital neutropenia. We assessed the effect of GCSF on monosomy 7 to determine if this chromosomal abnormality developed de novo or if GCSF simply favored expansion of a pre-existing clone. Bone marrow mononuclear cells (BMMNC) were co-cultured for 14 days with pharmacological doses of GCSF and examined by fluorescent in situ hybridization (FISH). No karyotypically healthy control, AA, or myelodysplastic syndrome (MDS) bone marrow showed development of monosomy 7. In BMMNC cultures from thirteen patients with MDS and monosomy 7, all developed substantial increases in numbers of monosomy 7 cells after prolonged exposure to GCSF. Experiments on stored AA samples obtained six months prior to the development of abnormal karyotype showed monosomy 7 on FISH ,with expansion of that clone on co-culture with GCSF. GCSFR mRNA was increased in BM from monosomy 7 patients as measured by real time PCR, and monosomy 7 cells expressed more GCSFR on the cell surface than did normal cells. As a truncated GCSFR isoform is associated with a hyperproliferative response to G-CSF and prolonged activation of signal transducer and activator of transcription (STAT) complexes, we sequenced the GCSFR DNA of monosomy 7 cells and measured the expression of the truncated GCSFR mRNA relative to full length GCSFR in patients with monosomy 7. While genomic GCSFR DNA showed no abnormalities, mRNA demonstrated increased proportions of the truncated isoform IV in all six patients tested. We examined GCSF-mediated GCSFR signal transduction of the Jak/Stat system in monosomy 7 CD34 cells. STAT-1 was increased and the STAT 5: STAT 3 rato was increased by ten-fold in all patients with monosomy 7 compared to normals and to MDS with normal cytogenetics. In conclusion, pharmacologic doses of GCSF appear to increase the proportion of monosomy 7 cells; this heightened sensitivity to GCSFmay be related to altered amounts of the truncated form of the receptor, with changes in GCSF signal transduction resulting in expansion of the monosomy 7 clone.

Apoptosis Related Gene Expression in Marrow Cells from Aplastic Anemia Patients.

Increased apoptosis is a possible pathogenetic mechanism for the damage of hematopoietic stem cells in aplastic anemia (AA), but the regulation of apoptotic machinery remains unclear. We investigated the parallel mRNA expression of specific pro-apoptotic and anti-apoptotic genes in AA patients and controls. We also examined the presence of apoptosis in the same populations with flow cytometry (FC) analysis. Bone marrow was obtained from 11 aplastic anemia patients (6 male, 5 female) of median age 38 years (range 21–77) and 8 healthy volunteers of similar age. Seven AA patients had active disease (untreated or refractory) and eight patients had long term complete or partial hematological remission after immunosuppressive therapy. BMMNC from three patients and CD34+ cells from two patients were examined in active disease and then in remission. We investigated the apoptosis-related gene mRNA expression (Fas, FasL, TRAIL, bcl-xl, bcl-2, bax, caspase 3, caspase 8 and GAPDH as an internal control) by RT-Multiplex PCR in total bone marrow mononuclear cells (BMMNC) or via MACS (magnetic cell sorting) selected CD34+ cells, when possible, from aplastic anemia patients and controls. Two-or three-color FC analysis was used for quantitative measurement of cell surface expression of Apo-1/Fas receptor in CD34+ cells and Annexin+/PI− total BMMNC and CD34+ cells. We also estimated the CFCs and LTC-IC derived CFCs values from five AA patients, which were very low, even in two patients who were in hematological remission, compared to controls. Two-color FC analysis revealed a significant difference in Fas expression on BMMNC and on CD34+ in active disease patients compared to controls, whereas in patients in remission this difference remains on CD34+ cells only. CD34+/Annexin+/PI− cells are significantly increased in both active disease (p=0.02) and in remission patients (p=0.05) compared to controls. The patients in remission had significantly higher proportion of early apoptotic total BMMNC than the patients with active disease. The results from patients' BMMNC and CD34+ cells' mRNA gene expression are presented in the table. No mRNA expression of any of the above apoptosis-related genes was detected in CD34+ cells from normal controls whereas their expression in normal total BMMNC was variable. There is a variability of gene expression, especially in patients' BMMNC. Apart from Fas mRNA expression in BMMNC and/or CD34+ cells, which is a characteristic finding during active disease, TRAIL mRNA expression was also found in almost all samples. The BMMNC's TRAIL mRNA expression remains a constant finding even in patients in remission. The expression of anti-apoptotic bcl-xl and/or bcl-2 in BMMNC and CD34+ cells from patients with active disease seems unable to inhibit the TRAIL or Fas induced apoptosis, pointing to the possibility that the ratio of pro-apoptotic and anti-apoptotic signals is of importance. In conjunction with our in vitro data (Ref: abstract 1293, ASH 2003), along with Fas, TRAIL expression may be of importance in the pathogenesis of AA.Expression of apoptosis related genes (I)Disease status/cellsTotal ptsFasFasLCasp8TRAILGAPDHActive/ BMMNC541155CR or PR/ BMMNC510035Active/ CD34+410224CR or PR/ CD34+410114Expression of apoptosis related genes (II)Disease status/cellsTotal ptsBcl-xlCasp 3BaxBcl-2GAPDHActive/ BMMNC532235CR or PR/ BMMNC522215Active/ CD34+411004CR or PR/ CD34+411004

Adhesion molecule expression by bone marrow CD34-positive cells in aplastic anemia before and after immunosuppressive therapy.

Appropriate adhesion between bone marrow stem cells and the marrow microenvironment is necessary for hematopoiesis, since signals that promote maturation or apoptosis are transmitted from stromal cells to stem cells. In aplastic anemia (AA), interferon-gamma produced by stromal cells has more influence on the pathogenesis of marrow failure than interferon-gamma produced by lymphocytes. We evaluated the expression of cell adhesion molecules, such as very late antigen-4 (CD49d), and -5 (CD49e) or c-kit receptor (CD117), by CD34-positive bone marrow cells in patients with AA who achieved hematological complete remission after immunosuppressive therapy. Before treatment, CD34-positive cells showed markedly higher expression of CD49d and CD49e than cells from healthy controls, indicating the strong adhesion of stem cells to the bone marrow stroma. Expression of CD49d and CD49e was significantly decreased, reaching normal levels, after hematological recovery. These findings suggest that changes in adhesion molecule expression by stem cells are important in the pathology of AA.

Direct binding of antithymoctye globulin to haemopoietic progenitor cells in aplastic anaemia.

Antithymocyte globulin (ATG) is widely used in the treatment of aplastic anaemia (AA) and a response occurs in 60-80% of patients. However, its exact mechanism of action in the treatment of AA has yet to be determined. Previously, we have shown that ATG increases colony growth from purified bone marrow CD34+ cells of AA patients in vitro, and decreases stem cell apoptosis and the expression of soluble Fas receptor after ATG therapy in vivo. The aim of this study was to further examine the association of ATG with AA haemopoietic progenitor cells. We describe here that ATG bound directly to CD34+ cells. Forty-six patients and 20 normal control subjects were studied. ATG bound to CD34+ cells in normal control subjects (mean 90.38%) as determined by flow cytometry. The mean percentage of CD34+ cells binding to ATG was 59.90% in untreated aplastic patients, 83.24% in partial responders, 58.3% in non-responders and 62.73% in relapsed patients. In completely recovered patients, ATG binding was indistinguishable from control subjects. The functionality of AA patients' haemopoietic progenitor cells was assessed using colony assays. These results demonstrate the direct binding of ATG to CD34+ cells and suggest that differences in its binding to AA CD34+ cells could reflect functional differences in the haemopoietic stem cell compartment throughout the disease process.

Clinical relevance of the TNF-alpha promoter/enhancer polymorphism in patients with aplastic anemia.

Immune-mediated inhibition of hematopoiesis has been suspected as a major cause of the suppressed growth of progenitor cells in aplastic anemia (AA). Overproduction of TNF-alpha by bone marrow and peripheral blood-derived cells was shown to be of pathogenetic importance. Genetic factors have been suggested by higher specific histocompatibility antigen frequencies among AA patients as a group and among those unresponsive to immunosuppressive therapy with cyclosporine. In the present work we expand on the evidence for the contribution of the TNF system to therapeutic responses in patients with AA. The response to immunosuppressive therapy at 3 months was found to be significantly more frequent among carriers of the TNF2 (TNF -308 A) gene (TNF2 homozygotes and heterozygotes of the TNF-alpha promoter/enhancer polymorphism) than among those patients not carrying the TNF2 gene. The allelic distribution of the LT-alpha (TNF-beta) NcoI and IL-1 receptor antagonist variable number tandem repeat (VNTR) polymorphisms did not differ among the subgroups of patients. The association of the TNF-alpha genotype with a response to immunosuppressive therapy points to an immunogenetic association that may contribute to the pathogenesis and therapeutic response of aplastic anemia.

Decreased expression of transcription factor GATA-2 in haematopoietic stem cells in patients with aplastic anaemia.

Aplastic anaemia is characterized by reduced haematopoiesis resulting in pancytopenia. It has been speculated that there is an injury in haematopoietic stem cells in the bone marrow; however, the precise nature of the injury has not been elucidated. In this study, the levels of expression of mRNAs for three transcription factors, GATA-2, SCL and AML1, which function in the early stages of haematopoiesis, were examined by quantitative polymerase chain reaction in patients with aplastic anaemia, idiopathic thrombocytopenic purpura (ITP) and normal subjects. Among these factors, expression of GATA-2 mRNA in purified CD34-positive cells was markedly decreased in aplastic anaemia compared with that in ITP and in normal subjects. The expression levels of SCL and AML1 mRNA in CD34-positive cells in aplastic anaemia were not different from those in normal subjects. When the expression of GATA-2 protein in CD34-positive cells was examined by immunocytochemical analysis, the percentage of GATA-2-positive cells in aplastic anaemia was lower than that in normal subjects. These findings strongly suggest that there is an aberrant expression of transcription factors in stem cells in aplastic anaemia, which may be responsible for the development of the disease.

Effects of antithymocyte globulin on bone marrow CD34+ cells in aplastic anaemia and myelodysplasia

The mechanism of action of antithymocyte globulin (ATG) in the treatment of aplastic anaemia (AA) and myelodysplastic syndromes (MDS) is poorly understood and may involve many different mechanisms. The aim of this in vitro study was to investigate further the effect of ATG on haemopoietic progenitor cells. A total of 16 patients (10 AA and 6 MDS) and 12 normal control subjects were studied. Purified bone marrow (BM) CD34+ cells were cultured in committed progenitor assay in the presence of ATG and autologous serum, then scored on day 14 for granulocyte-monocyte colony-forming units (CFU-GM) and erythroid colonies. ATG was found to be inhibitory to haemopoietic progenitor cells at high concentrations (1000 microg/ml and 100 microg/ml). This was confirmed by CD34-FITC and 7AAD staining of purified normal CD34+ cells after overnight incubation with ATG. In contrast, at lower doses (0.1-10 microg/ml), ATG produced an increase in colony growth in most normal, MDS and AA BM CD34+ cells. The greatest effect was in patients with non-severe AA, in whom the greatest increase in CFU-GM was seen at 0.5 microg/ml (P < 0.02) and 0.1 microg/ml (P = 0.02) and erythroid colonies at 0.1 microg/ml (P < 0.05). Serum ATG levels peaked during infusion to levels that were found to be toxic to haemopoietic progenitor cells in vitro and fell thereafter to levels that were associated with the highest colony numbers (0.1 and 0.5 microg/ml) in vitro. These results suggest that an increase in haemopoietic progenitor cells by ATG may be one of several important mechanisms for haematological recovery in AA and MDS.

IMPLICATIONS OF RECENT INSIGHTS INTO THE PATHOPHYSIOLOGY OF PAROXYSMAL NOCTURNAL HAEMOGLOBINURIA

IMPLICATIONS OF RECENT INSIGHTS INTO THE PATHOPHYSIOLOGY OF PAROXYSMAL NOCTURNAL HAEMOGLOBINURIA

Failure of engraftment of lenograstim mobilised allogeneic peripheral blood progenitor cells in aplastic anaemia

Failure of engraftment of lenograstim mobilised allogeneic peripheral blood progenitor cells in aplastic anaemia