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 in parvoviruses, as the latter has been incorporated into the former due to the departure of Dr. Kevin Brown. 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. Multiple studies are in progress. While we observed some responses in first presentation aplastic anemia to the monoclonal antibody CAMPATH, this arm of our three-arm randomized protocol was discontinued early when it became clear that anti-CD52 was unlikely to be as effective as standard therapy. A comparison of horse and rabbitt ATG continues in this patient population. In AA refractory to standard horse ATG plus cyclosporine, both rabbit ATG and CAMPATH show activity about 30% salvage rate. We are also testing CAMPATH in the treatment of relapsed AA and in MDS. Our participation in a mulit-center trial of eculuzimab was completed; in addition to leading to FDA approval for this agent, now marketed as Soliris, for the indications of symptoms and/or severe anemia due to intravassular hemolysis in PNH, further analysis of end points has shown that the monoclonal antibody is also strikingly effective in reducing the rate of thrombosis, the major cause of death in PNH. In the laboratory, studies of immune destruction of hematopoietic cells has continued in several directions. We have examined the mechanism of action of the ATGs, especially as horse and rabbit products do not appear to be equally effective in the treatment of aplastic anemia. In cell culture and molecular studies, we have confirmed that rabbit ATG in vitro induces T-regulatory cells from CD25-FoxP3-CD4 cells while horse ATG lacks this activity. In order to study T-cells intensively, lymphocytopheresis in now being performed in patients who consent to this procedure prior to therapy and at the completion of a six month course in order to perform molecular studies of cytokine gene expression. Preliminary data suggests that T-cell clones that persist in patients who have fully recovered hematopoietic capacity lack production of gamma-interferon and other type-1 cytokines and therefore may be tolerant. In studies of the genetics of acquired AA with a focus of telomere repair, we have extended our findings from a large mennonite kindred bearing a novel TERT mutation, which disclosed a relationship to both acute myeloid leukemia in the proband's father and to severe hepatic disease in the proband's aunt and two more distant female relatives all of whom bear the mutation. In two separate cohert studies, one of 100 Brazilian patients with AML and the other of 92 patients collected at MD Anderson Cancer Center, we found a rate of approximately 6% of TERT mutations in this leukemia. In vitro studies have suggested that these are loss of function alterations; germline inheritance by study of non-bone marrow tissues and of family members. Similarly, preliminary data among samples collected of patients who have undergone liver transplant have also show several with TERT mutations. These results implicate telomere repair in the initiation of leukemia and also in severe liver disease, characterized at this point as a combinnation of fibrosis and inflammation. In our studies of the mechanism of action of male hormones in hematologic disease, we have demonstrated in vitro that synthetic and natural androgens and also estrodiol increase transcription of the TERT gene and of telomerase activity. The inhibitory activity of tamoxofin and of both anti-estrogen and anti-androgen receptor agents indicates that both aromatization of androgens to estrodiol and the estrogen and androgen receptors are involved in this effect on hematopoietic cells. We are pursuing our hypothesis that telomere shortening results in aneuploidy due to chromosome fusion and genomic instability, as has been demonstrated in murine models. In studies from the Virus Discovery Group related to parvoviruses, there have been a number of signal advances. First, a recombinant B19 parvovirus vaccine is now in its second phase 1 trial among human volunteers to determine the appropriate dose of empty capsids needed to elicit neutralizing antibodies, prior to a planned phase III trial in children with sickle cell disease with the goal of preventing transient aplastic crisis. Second, we have developed a practical infectious clone of B19 parvovirus, which has been useful to define by molecular studies and confocal microscopy the role of specific viral genes in infection of human cells. Finally, we have utilized our ability to obtain CD34 cells in bulk and to induce erythroid differentiation to develop a highly productive cell culture system for B19 parvovirus. CD34 cell-derived CD36 cells recapitulate normal erythropoeisis, including expression of the glogoside receptor, and they are useful also as a much more sensitive method to measure neutralizing antibodies, of use in our vaccine trials, and for molecular and cell biology studies of virus interaction with its known pathogenic target cell.
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