The contribution of the bone marrow microenvironment in myelodysplastic syndrome is controversial. We therefore analyzed the functional properties of primary mesenchymal stromal cells from patients with myelodysplastic syndrome in the presence or absence of lenalidomide. Compared to healthy controls, clonality and growth were reduced across all disease stages. Furthermore, differentiation defects and particular expression of adhesion and cell surface molecules (e.g. CD166, CD29, CD146) were detected. Interestingly, the levels of stromal derived factor 1-alpha in patients’ cells culture supernatants were almost 2-fold lower (P<0.01) than those in controls and this was paralleled by a reduced induction of migration of CD34+ hematopoietic cells. Co-cultures of mesenchymal stromal cells from patients with CD34+ cells from healthy donors resulted in reduced numbers of cobblestone area-forming cells and fewer colony-forming units. Exposure of stromal cells from patients and controls to lenalidomide led to a further reduction of stromal derived factor 1-alpha secretion and cobblestone area formation, respectively. Moreover, lenalidomide pretreatment of mesenchymal stromal cells from patients with low but not high-risk myelodysplastic syndrome was able to rescue impaired erythroid and myeloid colony formation of early hematopoietic progenitors. In conclusion, our analyses support the notion that the stromal microenvironment is involved in the pathophysiology of myelodysplastic syndrome thus representing a potential target for therapeutic interventions.

There is limited research on music therapy for transplant recipients, yet board-certified music therapists working in medical settings often encounter individuals from the transplant population. The purpose of this study was to examine the current practices of music therapists working with bone marrow and organ transplant recipients. A total of 68 board-certified music therapists completed an online survey, providing information related to goal areas addressed in music therapy sessions and interventions frequently used with bone marrow and organ transplant patients. The most frequently reported goal areas included: coping skills, mood, and self-expression. The most frequently reported interventions included: singing, songwriting, and improvisation. Survey respondents also shared opinions related to the need for additional resources for music therapists working with transplant recipients, as well as the most rewarding and challenging aspects of working with transplant patients. Study limitations, suggestions for future research, and implications for clinical practice are included.

Thesis (MCurr)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Haematopoietic stem cell transplantation has become an increasingly popular treatment option for persons with life-threatening blood related diseases such as leukemia, lymphoma, myeloma and certain forms of anaemia. Due to this new therapy the use of bone marrow from a healthy individual also called a living donor for transplantation is inevitable. These living donors can experience psychological and economic issues and these components needs to be addressed in the transplant protocol. The researcher described the psychosocial factors that influenced sibling donors during allogeneic bone marrow transplantation at a public sector hospital in Cape Town, whether the transplant team members explained the administrative process of the transplant in an understandable manner and language and the effect of the psychosocial factors and administrative process of the allogeneic bone marrow transplantation on the sibling donors. A quantitative research approach with a descriptive design was used in this study. The sample was selected by means of full population sampling. The final sample size of (n=64) stem cell sibling donors over 18 years of age participated in the study. A self-reporting questionnaire was used to gather data, inclusive of four open-ended questions to establish an in depth sense of what the donor experiences during the bone marrow donation process. Descriptive statistics used to describe the variables included frequency distributions in the form of histograms and frequency tables. The Pearson chi-square statistical analysis test was used to test for relationships amongst groups. The study drew on the Roy Adaptation Model (RAM) as the theoretical framework to explain the phenomena surrounding the psychosocial and administrative effect of the transplantation process on the sibling donor. Based on the findings the haematopoietic stem cell donors coped with the psychosocial impact of the donation process by making use of their coping mechanism to adapt to their situation according to the Roy Adaptation Model. This model also offers guidance to the nurses to apply this model to nursing practice. Results revealed that sibling donors developed feelings of anxiety in relation to the invasive procedures that cause them to experience physical pain. Most respondents claimed that they were not psychologically affected by the donation process. The moral obligation the sibling donor has towards his sister or brother outweighed the physical pain or discomfort experienced during the donation process. Results revealed that the responding donors claimed they were well informed regarding the donation process and understood the treatment plan of the recipient. However, results revealed that there was a lack in visual donor information such as books, pamphlets as well as internet information. Results concerning the demographics revealed that (n=29) respondents had no schooling and some respondents had some schooling which can give an indication of how to bridge the knowledge and information gap between them and the donor in terms of language. Statistical significance results regarding the emotional state and economic situation of the donors was found. Some of the respondents were responsible for their own transport and their own accommodation, some of those that are employed were responsible for leave without pay. An organ donation policy needs to be developed to prevent live organ donors from losing valuable working hours that could result in loss of salary and should provide other financial incentives. Furthermore, a lack in a post-donation follow-up medical to alleviate and detect post-donation complications was identified. Further nursing research can help nurses to understand living donation for transplantation, also how the nurses that practice in organ transplant units experience and deal with the psychosocial factors that influence them particularly. / AFRIKAANSE OPSOMMING: Hematopoïetiese stamseloorplanting het ’n toenemend gewilde-behandelingsopsie vir persone met lewensgevaarlike bloedverwante siektes soos leukemie, limfoom, miëloom en sekere soorte anemie geword. Vir hierdie tipe terapie word die beenmurg van ’n gesonde individu, ook bekend as ’n lewende skenker, vir oorplanting gebruik. Lewende skenkers kan sielkundige en ekonomiese probleme ervaar en hierdie kwessies moet in die oorplantingsprotokol hanteer word. In hierdie studie is ondersoek ingestel na die psigososiale faktore wat bloedverwante skenkers tydens allogeneïese beenmurgoorplanting by ’n openbare hospitaal in Kaapstad beïnvloed, of die oorplantingspan die administratiewe proses van die oorplanting op ’n verstaanbare manier en in verstaanbare taal verduidelik het, en wat die uitwerking wat die psigososiale faktore en administratiewe proses is op die bloedverwante skenkers tydens allogeneïese beenmurgoorplanting. ’n Kwantitatiewe benadering met ’n beskrywende navorsingsontwerp is in hierdie studie gebruik. Die steekproef is op grond van volledige populasiesteekproefneming gekies. ’n Finale steekproefgrootte van stamselskenkers (n=64) ouer as 18 jaar het aan die navorsing deelgeneem. ’n Selfverslaggewende vraelys is gebruik om data in te samel, wat vier oop vrae ingesluit het om grondige begrip te verkry van wat die skenker tydens die beenmurgskenkingsproses ervaar. Beskrywende statistiek wat gebruik is om die veranderlikes te beskryf, sluit in frekwensie-verspreidings in die vorm van histogramme en frekwensie-tabelle. Die Pearson chi-kwadraat- statistieseanalise is gebruik om die verwantskappe onder groepe te toets. Die Roy Adaptation Model (RAM) is as die teoretiese raamwerk vir die studie gebruik om die verskynsels betrokke by die psigososiale en administratiewe ervaring van die oorplantingsproses vir die bloedverwante skenker te verklaar. Op grond van die bevindinge het die hematopoïetiese stamselskenkers die psigososiale impak van die skenkingsproses hanteer deur gebruik te maak van hulle hanteringsmeganisme om by hulle situasie aan te pas, wat met die RAM ooreenstem. Hierdie model bied ook leiding aan verpleegkundiges om dit in die verplegingspraktyk toe te pas. Resultate het getoon dat bloedverwante skenkers gevoelens van angs ontwikkel het vanweë die indringende prosedures, wat fisiese pyn veroorsaak het. Die meeste deelnemers het aangedui dat hulle nie sielkundig deur die skenkingsproses geraak is nie. Die morele verpligting wat die bloedverwante skenker het teenoor sy of haar broer of suster het die fisiese pyn of ongemak gedurende die skenkingsproses oortref. Resultate het getoon dat die deelnemende skenkers aangedui het dat hulle goed ingelig was oor die skenkingsproses en die behandelingsplan van die ontvanger verstaan het. Die resultate dui egter daarop dat daar ’n gebrek was aan visuele skenkersinligting soos boeke, pamflette en internet-inligting. Resultate rakende die demografie het bewys dat van die deelnemers (n=29) ongeskoold en sommige deelnemers laag geskoold is, wat ’n aanduiding kan gee van hoe die kennis- en inligtingsgaping tussen hulle en die skenker ten opsigte van taal oorbrug kan word. Statisties beduidende resultate rakende die emosionele toestand en ekonomiese situasie van die skenkers is gevind. Sommige deelnemers was verantwoordelik vir hulle eie vervoer en verblyf. Diegene wat werk, het verlof sonder betaling geneem. ’n Orgaanskenkingsbeleid moet ontwikkel word om te verhoed dat lewende orgaanskenkers kosbare werksure verloor, wat kan lei tot ’n verlies aan salaris. Ander finansiële aansporings behoort ook gegee te word. Voorts is ’n gebrek aan opvolg mediese behandeling vir skenkers om skenkingskomplikasies vas te stel en te verlig, geïdentifiseer. Voortgesette navorsing kan verpleegkundiges help om begrip te verkry van die implikasies van lewende orgaanskenking. Verpleegkundiges wat in hierdie orgaanoorplantings- eenhede werksaam is, kan ‘n beter begrip kry van die psigososiale faktore wat hierdie skenkers spesifiek beïnvloed.

published_or_final_version / abstract / Medicine / Doctoral / Doctor of Philosophy

CBL proteins are E3 ubiquitin ligases and adaptor proteins. The mammalian homologs – CBL, CBL-B and CBL-3 show broad tissue expression; accordingly, the CBL proteins play roles in multiple cell types. We have investigated the function of the CBL-B protein in hematopoietic cells and fibroblasts. The causative agent of chronic myeloid leukemia (CML) is BCR-ABL. This oncogenic fusion down-modulates CBL-B protein levels, suggesting that CBL-B regulates either the development or progression of CML. To assess the involvement of CBL-B in CML, bone marrow transduction and transplantation (BMT) studies were performed. Recipients of BCR-ABL-infected CBL-B(-/-) cells succumbed to a CML-like myeloproliferative disease with a longer latency than the wild-type recipients. Peripheral blood white blood cell numbers were reduced, as were splenic weights. Yet despite the reduced leukemic burden, granulocyte numbers were amplified throughout the animals. As well, CBLB(-/-) bone marrow (BM) cells possessed defective BM homing capabilities. From these results we concluded that CBL-B negatively regulates granulopoiesis and that prolonged latency in our CBL-B(-/-) BMT animals was a function of perturbed homing.To develop an in vitro model to study CBL-B function we established mouse embryonic fibroblasts (MEFs) deficient in CBL-B expression. Transduction of the wild-type and CBL-B-deficient MEFs with BCR-ABL did not confer transformation; nevertheless, the role of CBL-B in fibroblasts was evaluated. The CBL-B(-/-) MEFs showed enhanced chemotactic migration toward serum in both Transwell migration and time-lapse video microscopy studies. The biochemical response to serum was extensively evaluated leading to the development of a model. We predict that CBL-B deficiency either: (a) augments GRB2-associated binding protein 2 (GAB2) phosphorylation leading to enhanced extracellular signal-regulated kinase (ERK) and protein kinase B (PKB / Akt) signaling, or (b) alleviates negative control of Vav3 resulting in stimulation of Rho effectors. In either case, our results reveal a negative regulatory role for CBL-B in fibroblast migration. The two studies detailed herein expand our knowledge of CBL-B function. They strongly suggest that CBL-B can modulate granulocyte proliferation and point toward a role for CBL-B in the motility of numerous cell types.

Bone marrow oedema is a term used to define the appearance of regions of low signal on T1 weighted and high signal on T2 weighted fat-suppressed magnetic resonance images. The potential association between bone marrow oedema and prognosis in pathologies such as osteoarthritis is becoming increasingly recognised through clinical studies. A limited number of clinical studies have linked bone marrow oedema to altered bone density or altered bone marrow perfusion. The principal aims of this study were to investigate these findings in vitro, using the equine forelimb. The presence of bone marrow oedema within the equine forelimb was initially confirmed by undertaking magnetic resonance imaging scans. Bone samples were selected from 10 animals, 5 exhibiting the presence of bone marrow oedema-type abnormalities (BMOA) at the distal metacarpal. Raman microspectroscopy was used to determine the chemical composition of bone and projection radiography to provide a measure of bone density. Micro computed x-ray tomography was undertaken on a subset of three bone samples exhibiting BMOA. A second component of the study utilised contrast enhanced magnetic resonance imaging to enable comparison of perfusion to bone marrow with and without evidence of oedema. A saline flushing agent containing Evan’s blue was used so that subsequent sectioning of the bone would enable visualisation of the distribution of contrast agent as part of a histological examination of the oedematous region. An initial observation was that the majority of bone marrow oedema that was observed in the distal metacarpal appeared in a consistent location, namely the postero-inferior aspect of the bone, corresponding to the point of greatest load thereby suggesting a potential relationship to forces upon the joint. The principal observations were that there appears to be increased bone volume densities in those bone samples with evidence of bone marrow oedema. The Raman microspectroscopy did not demonstrate any statistically significant differences in the chemical composition of bone. Hence the overall impression is that bone marrow oedema is associated with a greater volume of bone, although of similar maturity and composition. There was limited evidence of increased perfusion (suggestive of increased vascularity and / or hyperpermeability) in those samples with bone marrow oedema. This work suggests that these particular bone marrow oedema lesions are associated with bone changes and potentially vascular changes although the aetiology is currently unclear. Further work is needed to determine the clinical significance and prognosis associated with these particular lesions, and whether these findings can be replicated for bone marrow oedema demonstrated at other anatomical locations.

A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine. / Bone marrow transplants (BMT, also hematopoietic stem cell transplants or HSCT/SCT) are one of the greatest medical achievements of the 20th century. They offer a treatment for a host of malignant and nonmalignant hematopoietic disorders, genetic diseases and solid tumors that could otherwise be fatal. Studies have found that 60% of patients undergoing BMT develop pulmonary complications (PC), and 1/3 of those require intensive care after transplantation. Despite the potential pneumotoxicity of induction agents, to date there have been no longitudinal studies following pulmonary function in this high‐risk patient population. This study reviewed patient who underwent autogeneic bone marrow transplant for multiple myeloma at Banner University Medical Center – Tucson (formerly University of Arizona Health Network) from January 1, 2003 through December 31, 2013. Pretransplant evaluatin and pulmonary function testing data were obtained and stratified between high dose (standard) Melphalan (200 mg/ms2) and reduced dose (140 mg/ms2). Statistically significant differences were present between the 2 groups at baseline for DLCO but disappeared at 6 and 12‐month followup, while a statistically significant difference for FEV1/FVC ratio was seen at baseline and 6 months but disappeared at 12‐month follow‐up. There were no statistically significant differences seen with FEV1 between the two groups. Given there is no difference in mortality and relapse outcomes between the groups, the standard of care dosing for Melphalan is not associated with an increase in pulmonary morbidity.

Advances in haploidentical bone marrow transplantation (h-BMT) have drastically broadened the treatment options for patients requiring BMT. The possibility of significantly reducing the complications resulting from graft-versus-host disease (GvHD) with the administration of post-transplant cyclophosphamide (PT-CY) has substantially improved the efficacy and applicability of T cell-replete h-BMT. However, higher frequency of disease recurrence remains a major challenge in h-BMT with PT-CY. There is a critical need to identify novel strategies to prevent GvHD while sparing the graft-versus-leukaemia (GvL) effect in h-BMT. To this end, we evaluated the impact of bendamustine (BEN), given post-transplant, on GvHD and GvL using clinically relevant murine h-BMT models. We provide results indicating that post-transplant bendamustine (PT-BEN) alleviates GvHD, significantly improving survival, while preserving engraftment and GvL effects. We further document that PT-BEN can mitigate GvHD even in the absence of Treg. Our results also indicate that PT-BEN is less myelo-suppressive than PT-CY, significantly increasing the number and proportion of CD11b(+)Gr-1(hi) cells, while decreasing lymphoid cells. In vitro we observed that BEN enhances the suppressive function of myeloid-derived suppressor cells (MDSCs) while impairing the proliferation of T-and B-cells. These results advocate for the consideration of PT-BEN as a new therapeutic platform for clinical implementation in h-BMT.

Hematologic malignancies are among the most often diagnosed forms of cancers. Treatment regimens often utilise various combination of cytostatic drugs and total body irradiation and subsequent transplantation of hematopoietic stem cells. One of the most common combinations includes ionising radiation with the antineoplastic alkylating agent cyclophosphamide. In this study we used congenic Ly5.2 and L5.1 mouse strains that express different isoforms of CD45 antigen to evaluate the effects of various time interval between cyclophosphamide and irradiation treatments on the viability of hematopoietic stem cells and their viability. This was done by competitive repopulation assay. The results revealed that level of engraftment and subsequent reconstitution of hematopoiesis can significantly vary and depend on the time interval between cyclophosphamide and total body irradiation administrations. The results indicate that patients with hematologic malignancies could possibly benefit from the treatment especially if they received transplants after being irradiated five or seven days after cyclophosphamide because at that time point their own stem cells would be least competitive. Key words: bone marrow transplantation, cyclophosphamide, chimerism, hematopoietic stem cells, ionising radiation

Researches on Hematopoietic Stem Cell (HSC) have been expanding that leads to an increase in our understanding of HSC normal behaviors and abnormal alterations. One of the most important issues in the research on HSCs is to understand the mechanism of the homing process of these cells to settle in their niche in the bone marrow and establish the production of various blood cell types after bone marrow transplantation. The cells first must come in contact with the endothelial cells. This contact is known as adhesion and occurs through a multi-step paradigm ending with transmigration to the bone marrow niche. The initial step of the homing, tethering and rolling of HSC, is mediated by P- and E-Selectins present on endothelial cell surface through their interactions with the ligands expressed on the surface of HSC. Thus, understanding the adhesion process and its contribution for efficient HSCs homing will have great impact on HSC therapy. The selectin – ligands interaction has been intensively studied using in vivo and in vitro approaches. However, the molecular mechanism involved by HSCs at single molecule level is poorly understood. Here in this study, a novel experimental method to unravel the molecular mechanisms of the Selectin-ligands interactions in vitro at the single molecule level is developed by combining microfluidics, epi-fluorescence microscopy and live cells. In this work, the new single-molecule imaging technique enabled us to directly visualize the nanoscale spatiotemporal dynamics of the membrane protein-ligand interactions under conditions of shear stress acting on the cells at the molecular level in real time. Using this method, we revealed that selectin ligands on membrane-tethers and slings show unique spatiotemporal dynamics that is distinct from those on the cell body. We demonstrated that the membrane tethers are formed from single microvilli on the cells, which provides a mechanism to spatially localize selectin ligands, PSGL-1 and CD44 on the tethers and slings. We also demonstrated that the selectin ligands show fast diffusional motion along the tethers and slings compared with that on the cell body due to the detachment of cell membranes from actin cytoskeleton during the formation of the tethers. Our results suggest that the spatial confinement of the selectin ligands together with the fast scanning of a large area by the selectin ligands increase the efficiency of selectin-ligands interaction during the rolling, resulting in slow and stable rolling of the cell on selectin. Our findings contribute significantly to molecular level understanding of the initial step of HSCs. This single-molecule imaging technique that we developed in this study will find wide applications in the molecular-level studies on cell-cell interactions including cancer cell metastasis.