ABSTRACT. Although microsatellite instability (MSI), usually detected by DNA length polymorphisms, has been implicated in the induction of solid tumors in both humans and animals, its role in leukemogenesis is unclear. The goal of this study was to investigate whether there is an association between MSI and radiation leukemogenesis in CBA/Ca mice. Microsatellite lengths at 55 loci, mapped to eight different mouse chromosomes, were examined in two groups of DNA samples: 1) 10 normal DNA samples collected from the bone marrow cells of control male CBA/Ca mice, and 2) 17 DNA samples isolated from the spleens of mice that developed myeloid leukemia (ML) after exposure to neutrons, or X rays, or gamma rays. Microsatellite markers were amplified using the non-radioisotopic multiplex-touchdown PCR protocols developed in our laboratory, and the sizes of amplicons were examined on 6% non-denaturing polyacrylamide gels. Although no correlation between microsatellite length polymorphisms and radiation leukemogenesis was observed at the 55 CBA/Ca mouse loci tested in this study, an uncommon MSI, manifested as the absence of DNA bands after PCR amplification at 2 loci (D2MIT140 and D4MIT104), was observed in both control and ML samples. However, the frequency of ML samples showing this type of MSI is statistically significant (p<0.05). Although there is no direct evidence that this type of MSI predisposes mice to the development of leukemia, the results suggests that genes flanking the D2MIT140 and D4MIT104 are susceptible to spontaneous mutation and perhaps to damage caused by ionizing radiation.

Keywords: Microsatellites, instability, leukemia, radiation, PCR.

Reprint requests to: K. Noy Rithidech, Medical Department, Brookhaven National Laboratory, 30 Bell Avenue, Upton, NY 11973, phone: (516) 344-2672, fax: (516) 344-5311, e-mail address: rithidech@bnlarm.bnl.gov.

ABSTRACT. Aplastic anemia (AA) is characterized by multilineage bone marrow failure of unknown etiology. In order to assess the role of immune-mediated mechanisms in hematopoietic suppression, we examined the diversity of T lymphocyte repertoire in terms of variable (V) gene segment usage of the T cell receptor (TCR) beta chain in bone marrow and peripheral blood of six patients with severe untreated AA. Expression of transcripts encoding Vbeta1- Vbeta24 subfamilies was analyzed by reverse transcription - polymerase chain reaction (RT-PCR). The results revealed that T lymphocytes in AA utilize highly diverse segments of the beta chain loci. Over the heterogenous Vbeta expression background, transcripts encoding Vbeta3, Vbeta20, Vbeta21, and Vbeta22 subfamilies were enhanced by at least threefold in 5 of 6 patients as compared to normal samples, but a different transcript species was over expressed in each patient. To evaluate clonality of T cells, size diversity within the complementarity determining region 3 (CDR3) and usage of TCRbeta joining (J) gene segments were analyzed in PCR products specific for each of the 24 Vbeta subfamilies. We found that the majority of transcripts display normal CDR3 size patterns, as is characteristic of polyclonal populations. Nevertheless, one or two predominating junctional rearrangements were observed in each patient. They were identified in Vbeta5, Vbeta7, Vbeta8, Vbeta13, Vbeta15, Vbeta16, and Vbeta23 transcripts, which differed from patient to patient and did not correspond to transcripts with an abnormally high expression level. Our results demonstrate that T cell repertoire in AA is random with respect to the TCR beta chain. Unique rearrangements detected in the CDR3 region are suggestive of a limited process of an antigen-driven (oligo)clonal T cell expansion which may take place over the overwhelmingly polyclonal repertoire of T lymphocytes at the onset of severe AA.

Keywords: Aplastic anemia, T cell receptor, Vbeta chain, complementarity determining region 3.

Reprint requests to: Aleksandra Wodnar-Filipowicz, Department of Research, University Hospital Basel, Hebelstrasse 20, 4031 Basel, Switzerland, phone: 41 61 2652333, fax: 41 61 2652350, e-mail: filipowicz@ubaclu.unibas.ch.

ABSTRACT. We have solved the crystal structure of the Fab fragment of NMC-4, a mouse monoclonal antibody that binds to the A1 domain of von Willebrand factor (vWF). Two Asp and three Tyr residues in the complementarity determining regions 1 and 3 of the heavy chain exhibited a spatial orientation suggestive of a dominant role in establishing contact with the antigen. A cluster of Asp and Tyr residues occurs also in a region of the platelet glycoprotein (GP) Ib alpha amino terminal domain known to be critically involved in vWF binding. Thus, the structural information obtained with NMC-4 may prove relevant to understand the stereochemical bases of the GP Ib alpha-vWF interaction essential for thrombus formation at sites of vascular lesion.

Keywords: Platelet, adhesion, aggregation, hemostasis, thrombosis, von Willebrand factor, A1 domain, Fab structure.

Reprint requests to: Kottayil I. Varughese, Ph.D., The Scripps Research Institute, Department of Molecular and Experimental Medicine, NX-1, 10550 N. Torrey Pines Road, La Jolla, CA 92037, phone: (619) 784-7945, fax: (619) 784-7966, e-mail: kiv@scripps.edu.