No description available.

Thesis (doctoral)--Rijksuniversiteit te Groningen.

Tesis-Universitas Indonesia. / Summary in English. Includes bibliographical references (p. 225-240).

Aplastic anemia (AA) is a rare, life-threatening disorder characterized by immune mediated loss of hematopoietic stem cells (HSC) that results in bone marrow hypoplasia, pancytopenia and its complications. Majority of AA are acquired and idiopathic. Cytotoxic T cell-mediated autoimmune destruction to the HSC is the main pathophysiological mechanism. Intrinsic defects in the hematopoietic stem cell, alterations in bone marrow microenvironment, acquired clonal abnormalities and drug/viral direct injury are other described etiologies. Improvement in molecular studies also led to better understanding of cytogenetic abnormalities, somatic mutations, telomere attrition in AA which may affect treatment response, prognosis and survival. HSCT or immunosuppression therapy (IST) can restore normal hematopoiesis. HSCT is curative, but eligibility depends on age, comorbidities, and available donors. HSCT use is limited by matched donor availability. The alternative option is IST. The standard today is intensive triple therapy with anti-thymocyte globulin (ATG), cyclosporin (CsA) and thrombopoietin receptor agonist, eltrombopag (EPAG) or with ATG and CsA in patients who require less intense IST. 72 year-old male presented with pancytopenia (WBC 1.7K/uL, Hgb 12.9 g/dL, Platelet count 6 K/uL, and ANC 600, absolute reticulocyte count 18x10*9. Bone marrow biopsy revealed markedly hypocellular marrow ( Response rates for first line IST is about 60–70%. The response and its duration are strong surrogate markers for prolonged survival. Triple therapy with hATG, CsA and EPAG improved response rate to 90% in AA in 6 months. Bone marrow evaluation in relapse setting is essential to rule out clonal evolution into MDS/AML or subtle adult inherited bone marrow syndromes. Survival is limited after second relapse in elderly. Our patient had accelerated response and CR with sustained survival after alemtuzumab, CsA and romiplostim. This report highlights a rare response to alemtuzumab and an alternate TPO mimetic, romiplostim. Given his exposure to various methods of IST and the bone marrow showing clonal evolution, he is at risk of myeloid malignancies.

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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.

We sought to evaluate the individual contributions of Notch1 and PKC-ζ to disease progression in a mouse model of immune-mediated bone marrow failure and to define a mechanism for their potential cellular cooperation. We transferred parental bulk splenocytes into F1-hybrid recipients to induce a robust immune-mediated bone marrow failure (BMF) that we could partially rescue by administering a pharmacological inhibitor of Notch activation. Transferring splenocytes from PKC--ζ-/- animals did not induce disease, and treating animals with a pharmacological inhibitor of PKC-ζ also provided full protection from disease. We found that inhibiting Notch1 resulted in PKC-ζ down-regulation, and blocking PKC-ζ reduced Notch1 activation, possibly within a positive feedback loop. Our data suggest that both Notch1 and PKC-ζ contribute to disease progression in our mouse model of immune-mediated bone marrow failure. Furthermore, additional findings from the lab demonstrated physical interactions between Notch1, members of the T cell signalosome and PKC-ζ that are essential to mediating full activation of T cells following signaling through the TCR and CD28. Notch1 and/or PKC-ζ may represent novel therapeutic targets in the treatment of bone marrow failure.