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Interactions between the haematopoietic stem cell and the myeloid microenvironment in aplastic anaemia
In patients with aplastic anaemia that respond to immunosuppressive therapy, quantitative, morphological and functional haematologic derangement have been reported. To explain these findings, abnormalities in the marrow stroma or the stem cell have been postulated. To define the relative contributio...
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| Format: | Thesis |
| Language: | English |
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Division of Clinical Haematology
2017
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| _version_ | 1867613904688906240 |
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| access_status_str | Open Access |
| author | Novitzky, Nicolas |
| author2 | Jacobs, Peter |
| author_browse | Jacobs, Peter Novitzky, Nicolas |
| author_facet | Jacobs, Peter Novitzky, Nicolas |
| author_sort | Novitzky, Nicolas |
| collection | Thesis |
| description | In patients with aplastic anaemia that respond to immunosuppressive therapy, quantitative, morphological and functional haematologic derangement have been reported. To explain these findings, abnormalities in the marrow stroma or the stem cell have been postulated. To define the relative contribution of each of the latter, the integrity of the bone marrow from sixteen patients that responded to anti-lymphocyte globulin and high dose methyl prednisolone was compared to normal individuals. Bone marrow mononuclear cells were divided into two fractions. From the first, stroma was cultured in aMEM containing 12.5% of both horse and foetal calf serum and 10-5 M hydrocortisone at 37° C in 5% CO2 in 90% humidity. The medium was changed weekly. Upon confluence, these stromal layers were studied morphologically and with cytospin preparations stained with Sudan black, 0 red oil, alkaline and acid phosphatases. The remainder was monocyte and lymphocyte depleted, CD 34+ progenitors were selected with paramagnetic beads and the population morphologically and immunophenotypically defined. To determine the functional status, control or patient CD 34+ progenitors, were suspended for two hours on normal or aplastic stroma for adherence to take place. The non-adhesive fraction was decanted by standardised washing and cultured for fourteen days in the presence of PHA-conditioned medium in the CFU-gm assay. Strama-adherent progenitors were covered with 0.3% agar and cultured for five days. Aggregates with more than twenty cells were scored (CFU-bl). The remaining CD 34+ cells were cultured in the mixed colony assay with combinations of recombinant cytokines belonging to the G protein super-family and the tyrosine kinase group in dose response studies. Light density cells from patients with treated aplasia contained significantly fewer CD 34+ cells than those present in the control suspensions (mean 0.65%, SD 0.35% vs 1.62%, SD 1.4%; p= 0.002). Normal and aplastic stroma became confluent at three and four weeks. There was no difference on the morphology or the cytochemical stains between the two groups. Functionally, aplastic bone marrow stroma supported CFU-bl formation no differently from normal layers. However, CD 34+ precursors from the patients cultured on control stroma resulted in significantly fewer CFU-bl (p= 0.0002,) and CFU-gm (p= 0.0009). This work provides original evidence supporting the reduced clonogenicity of the corresponding populations of CFU-bl from patients with aplasia is unrelated to attachment to the stroma, but intrinsic to the CD 34+ cells. Moreover, this study shows for the first time that exposure of these progenitors to growth factors belonging to the G protein and tyrosine kinase receptor families have defective responses, correctable only at supra physiological concentrations, while effects on combinations containing c-kit ligand, appear preserved. Following immunosuppressive therapy, the bone marrow is repopulated by a hypoproliferative progenitor cell population which responds suboptimally to physiological cytokine stimulation. This suggests that abnormal interactions between receptors and their ligands or alterations in the signal transduction for cell division by the cytokines belonging to the G superfamily lead to suboptimal growth. |
| format | Thesis |
| id | oai:open.uct.ac.za:11427/24943 |
| institution | University of Cape Town (South Africa) |
| language | eng |
| last_indexed | 2026-06-10T12:43:33.865Z |
| license_str | Not specified — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UCTD — University of Cape Town Open Access Repository |
| publishDate | 2017 |
| publishDateRange | 2017 |
| publishDateSort | 2017 |
| publisher | Division of Clinical Haematology |
| publisherStr | Division of Clinical Haematology |
| record_format | dspace |
| source_str | UCTD — University of Cape Town Open Access Repository |
| spelling | oai:open.uct.ac.za:11427/24943 Interactions between the haematopoietic stem cell and the myeloid microenvironment in aplastic anaemia Novitzky, Nicolas Jacobs, Peter Anemia, Aplastic - physiopathology Bone Marrow - pathology Hematopoietic stem cells Haematology In patients with aplastic anaemia that respond to immunosuppressive therapy, quantitative, morphological and functional haematologic derangement have been reported. To explain these findings, abnormalities in the marrow stroma or the stem cell have been postulated. To define the relative contribution of each of the latter, the integrity of the bone marrow from sixteen patients that responded to anti-lymphocyte globulin and high dose methyl prednisolone was compared to normal individuals. Bone marrow mononuclear cells were divided into two fractions. From the first, stroma was cultured in aMEM containing 12.5% of both horse and foetal calf serum and 10-5 M hydrocortisone at 37° C in 5% CO2 in 90% humidity. The medium was changed weekly. Upon confluence, these stromal layers were studied morphologically and with cytospin preparations stained with Sudan black, 0 red oil, alkaline and acid phosphatases. The remainder was monocyte and lymphocyte depleted, CD 34+ progenitors were selected with paramagnetic beads and the population morphologically and immunophenotypically defined. To determine the functional status, control or patient CD 34+ progenitors, were suspended for two hours on normal or aplastic stroma for adherence to take place. The non-adhesive fraction was decanted by standardised washing and cultured for fourteen days in the presence of PHA-conditioned medium in the CFU-gm assay. Strama-adherent progenitors were covered with 0.3% agar and cultured for five days. Aggregates with more than twenty cells were scored (CFU-bl). The remaining CD 34+ cells were cultured in the mixed colony assay with combinations of recombinant cytokines belonging to the G protein super-family and the tyrosine kinase group in dose response studies. Light density cells from patients with treated aplasia contained significantly fewer CD 34+ cells than those present in the control suspensions (mean 0.65%, SD 0.35% vs 1.62%, SD 1.4%; p= 0.002). Normal and aplastic stroma became confluent at three and four weeks. There was no difference on the morphology or the cytochemical stains between the two groups. Functionally, aplastic bone marrow stroma supported CFU-bl formation no differently from normal layers. However, CD 34+ precursors from the patients cultured on control stroma resulted in significantly fewer CFU-bl (p= 0.0002,) and CFU-gm (p= 0.0009). This work provides original evidence supporting the reduced clonogenicity of the corresponding populations of CFU-bl from patients with aplasia is unrelated to attachment to the stroma, but intrinsic to the CD 34+ cells. Moreover, this study shows for the first time that exposure of these progenitors to growth factors belonging to the G protein and tyrosine kinase receptor families have defective responses, correctable only at supra physiological concentrations, while effects on combinations containing c-kit ligand, appear preserved. Following immunosuppressive therapy, the bone marrow is repopulated by a hypoproliferative progenitor cell population which responds suboptimally to physiological cytokine stimulation. This suggests that abnormal interactions between receptors and their ligands or alterations in the signal transduction for cell division by the cytokines belonging to the G superfamily lead to suboptimal growth. 2017-08-23T13:04:35Z 2017-08-23T13:04:35Z 1993 2017-07-10T12:44:02Z Doctoral Thesis Doctoral PhD http://hdl.handle.net/11427/24943 eng application/pdf Division of Clinical Haematology Faculty of Health Sciences University of Cape Town |
| spellingShingle | Anemia, Aplastic - physiopathology Bone Marrow - pathology Hematopoietic stem cells Haematology Novitzky, Nicolas Interactions between the haematopoietic stem cell and the myeloid microenvironment in aplastic anaemia |
| thesis_degree_str | Doctoral |
| title | Interactions between the haematopoietic stem cell and the myeloid microenvironment in aplastic anaemia |
| title_full | Interactions between the haematopoietic stem cell and the myeloid microenvironment in aplastic anaemia |
| title_fullStr | Interactions between the haematopoietic stem cell and the myeloid microenvironment in aplastic anaemia |
| title_full_unstemmed | Interactions between the haematopoietic stem cell and the myeloid microenvironment in aplastic anaemia |
| title_short | Interactions between the haematopoietic stem cell and the myeloid microenvironment in aplastic anaemia |
| title_sort | interactions between the haematopoietic stem cell and the myeloid microenvironment in aplastic anaemia |
| topic | Anemia, Aplastic - physiopathology Bone Marrow - pathology Hematopoietic stem cells Haematology |
| url | http://hdl.handle.net/11427/24943 |
| work_keys_str_mv | AT novitzkynicolas interactionsbetweenthehaematopoieticstemcellandthemyeloidmicroenvironmentinaplasticanaemia |