This years annual report incorporates both the work of the Cell Biology Section in bone marrow failure diseases and also of the Virus Discovery Section. Aplastic anemia (AA) and other types of bone marrow failure have clinical and laboratory features consistent with an autoimmune pathophysiology, with a diversity of putative inciting antigens, including viruses, chemicals, medical drugs, and tumor antigens. Whatever its specific etiology, a majority of patients respond with hematologic improvement after immunosuppressive therapies. One important clinical feature of AA is its association with clonal hematologic diseases, especially paroxysmal nocturnal hemoglobinuria (PNH) and myelodysplastic syndromes (MDS). In the clinic, studies have been directed towards more effective immunosuppression in AA and the application of immunosuppressive regimens to related bone marrow failure syndromes. Among current clinical protocols, our major study is a direct comparison two commercially available antithymocyte globulins, horse ATG versus rabbit ATG; more than 70 patients have been enrolled and a preliminary glimpse at the data suggests that these agents are not equivalent. A component of this study is to determine if low dose cyclosporine will prevent relapse, a common problem after successful immunosuppressive therapy. In other protocols, we are continuing to examine the role of rabbit ATG and the monoclonal antibody anti-CD52 (campath), in aplastic anemia refractory to a single course of horse ATG. Campath also is now utilized in a research setting for relapsed aplastic anemia, avoiding the use of cyclosporine. Retrospective examination of our large experience with aplastic anemia has allowed us to determine a new predictive model for response to immunosuppressive therapy and long-term survival, based on blood counts at presentation. Also, we have noted a striking improvement in both short and long-term survival in patients with aplastic anemia, due in part to better supportive care and also to the employment of alternative therapies, such as repeat immunosuppression and stem cell transplantation. In the laboratory, our murine model of immune-mediated bone marrow failure continues to yield interesting results. In addition to demonstrating that a type 1 cytokine response dominates in these animals, we have also shown that T-regulatory cells can abrogate disease, parallel with the observation of low T-regs in patients with aplastic anemia and suggesting potential utility of these cells in autoimmune diseases. We also have utilized cross-breeding with knock-out animals to determine that the Fas-ligand cell death pathway, rather than perforin-granzyme, dominates in mediating bone marrow failure in our model. In studies of the human immune system in aplastic anemia, we have identified lesions in modulating signal transduction pathways, including decreased zeta chain expression and diminished sap protein expression, consistent with dysregulated immune response. In studies of telomere biology as they related to human disease, we have extended our original observations of TERT mutations in acute myeloid leukemia to include collaborative studies of an infrequent polymorphism in Canadian patients, again demonstrating a higher prevalence in AML. The relationship of TERT mutations to hepatic cirrhosis, observed initially in a large Mennonite kindred, has now been extended to other families, including detailed histopathology disclosing an unusual combination of inflammation and fibrosis. The relationship between telomere shortening and genomic instability is being pursued, with some evidence of early success, with several strategies. We utilize cells from patients with telomere repair complex mutations with disease as well as clinically healthy persons from the same families who also have mutations. Results to date include the detection of an increased rate of aneuploidy after in vitro tissue culture, as determined by fluorescent in situ hybridization (FISH), end-to-end chromosome joining by conventional cytogenetics as well as spectrakaryotyping (SKY), and the establishment of a method to perform comparative genomic hybridization (CGH) on limited numbers of human hematopoietic cells from individual colonies from normal and patient bone marrows. In our virus discovery group, we have developed an efficient method of propogating B19 parvovirus in normal human hematopoietic cells in suspension culture. An important new observation is that parvovirus infection hijacks the erythroid cells differentiation program. In studies involving microarray, real time PCR, and immunoblot, determined regulation of multiple significant erythropoietic genes as they are affected at early stages of viral infection, prior to apoptosis of target cells. E2F-mediated control gene expression appears to be critical in this dramatic alteration in the pattern of gene expression. Finally, we have subjected tissue samples from patients with seronegative hepatitis to high throughput sequencing and identified potential novel infectious agents that may be responsible for this disease as well as for hepatitis-associated aplastic anemia and fulminant hepatitis of childhood.
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