Favored use of immunoglobulin V(H)4 Genes in AIDS-associated B-cell lymphoma.

We examined the lg heavy chain variable region genes (Ig V(H) genes) expressed in biopsy specimens of 10 patients with acquired immunodeficiency syndrome (AIDS)-associated lymphoma. Eight expressed Ig V(H) genes of the V(H)4 group, indicating a bias toward expression of Ig V(H) genes of this subgroup. Sequence analyses of Ig V(H) genes isolated from any one lymphoma did not reveal evidence for intraclonal diversity. However, some lymphomas express Ig V(H) genes that apparently have undergone somatic diversification and selection. In addition, we found that the sequence encoding each examined third complementarity determining region most likely resulted from D-D fusion, a process that ordinarily contributes to the generation of a relatively small proportion of the Ig heavy chain genes expressed by normal adult B cells. The noted restriction in the use of Ig V(H) genes by AIDS-associated B-cell lymphomas suggests that antigenic stimulation contributes to lymphomagenesis in patients with AIDS.

Common clonal origin of chronic lymphocytic leukemia and high-grade lymphoma of Richter's syndrome.

Patients with B-cell chronic lymphocytic leukemia (CLL) infrequently may develop high-grade B-cell lymphoma, or Richter's syndrome lymphoma (RS lymphoma). Such lymphomas differ from the original leukemia in both histology and clinical behavior. Studies seeking to define the clonal relationship between the cells of the two malignancies in any one patient have yielded conflicting reports. We examined the clonal relationship between the early and late neoplastic cells of a patient who underwent Richter's transformation. In contrast to the original leukemia cells, the secondary high-grade lymphoma was CD5-. However, both the leukemia cells and the evolved RS lymphoma expressed surface IgM lambda reactive with Lc1, a murine monoclonal antibody specific for a supratypic cross-reactive idiotype encoded by a subset of human Ig variable region genes of the VH4 subgroup. Nucleic acid sequence analyses of the heavy and light chain variable region genes expressed by both leukemia and lymphoma cells show that the CD5- B-cell lymphoma constitutes a clonal expansion of mutant cells derived from the original CD5+ B-cell leukemia. Moreover, certain sets of somatic mutations distinguish the Ig variable region genes used by RS lymphoma from those expressed by the CLL B cells. This is the first study to establish the clonal relationship between CLL and RS lymphoma through primary structural analyses of the expressed Ig genes.

[Natural autoantibodies of neonatal rats detectable by a hybridoma technic]. / Estestvennye autoantitela neonatal'nykh krys, vyiavliaemye gibridomnoi tekhnikoi.

Neonatal rat hybridomas were tested for natural autoantibodies (NAA) production, using different screening procedures. NAA were discovered in 35% of immunoglobulins producing hybridomas. Radioimmunoassay (RIA) on brain and liver homogenates and immunocytochemistry on brain sections are the procedures of choice revealing the major part of the identified NAA. On the contrary, only a small portion of NAA could be detected with indirect immunofluorescence on fixed fibroblasts and with RIA on individual autoantigens. All the NAA revealed proved to be of the IgM type and almost all of them possessed neither organ nor species-specificity. In spite of that, most of the NAA reacted with definite cell populations of nervous tissue such as glia, neurons, ependyma or brain vessel cells. The studied panel of NAA from neonatal rats has common features with similar panels from newborn and old mice, though some species-specific characteristics do exist.

Allelic exclusion frequency analysis and molecular characteristics of immunoglobulins secreted by the hybridomas expressing both allelic genes.

The investigation of 750 B-lymphocyte hybridoma clones obtained by fusion of mouse myeloma and newborn heterozygous Igk-1a/Igk-1b rat splenocytes has revealed that 9.8% of Ig kappa-chain loci are rearranged productively. Seventeen hybridomas secrete kappa-chains of both allelic variants. The analysis of IgM molecules of 9 such clones demonstrated that in 6 cases only one L-chain allotype, either 1a or 1b, is present in IgM. Thus for the first time the high frequency of selective association of H and L chains in Ig-producing cells was shown. Evidently this selectivity may function as one of allelic exclusion mechanisms at the Ig assembly stage.

Monoclonal antibodies to dog heart sarcoplasmic reticulum. Antibodies that inhibit Ca2+-pump systems of cardiac and skeletal muscles.

Purified sarcoplasmic reticulum (SR) vesicles from dog heart were used as an antigen to produce monoclonal antibodies (mAbs) to the Ca2+-ATPase. Nine of twelve clones of hybridoma cells produce mAbs which cross-react with seven SR preparation isolated from cardiac and skeletal muscles of various species. Three mAbs of IgM type interact with the 45-kDa tryptic fragment of rabbit skeletal muscle Ca2+-ATPase and markedly inhibit Ca2+ uptake (by 95%) and ATPase activity (by 80%) and decrease (by 30-50%) the steady-state level of the Ca2+-ATPase phosphoenzyme. The ATPase activity could be completely blocked by one of these mAbs if the incubation medium was supplemented with 2 microM orthovanadate. On the other hand, when SR vesicles were treated with increasing concentrations of a nonionic detergent C12E8, the inhibiting effect of mAb 4B4 is diminished. It is concluded that the mAbs inhibit the Ca2+-ATPase only if the enzyme exists in an oligomeric form. The inhibition of the SR activities is due to an effect of the mAbs on the whole active center of the enzyme, rather than on a single partial reaction.

Monoclonal antibodies to dog heart sarcoplasmic reticulum. Application of the mAbs for studies of the structure and function of Ca2+-pumps.

One of monoclonal antibodies (mAbs) raised against purified dog heart sarcoplasmic reticulum (SR) efficiently decreases Ca2+-pump and Ca2+-ATPase activities of various SR preparations. The ATPase activity that is insensitive to the mAb (10-20% of the initial value) is present both in light and heavy fractions of rabbit skeletal muscle SR. The residual activity is completely blocked by 2 microM vanadate. The inhibition of the ATPase by the mAb is prevented in the presence of a nonionic detergent C12E8. It is concluded that the inhibiting effect of the mAb takes place when the Ca2+-ATPase exists in an oligomeric form. Another mAb does not affect SR functions and is specific only for Ca2+-ATPase from cardiac and slow muscle cells. Decrease in the Ca2+-pump activity of SR fractions from ischemic myocardium is accompanied by a diminished binding of both mAbs with the antigen. The mAbs described could be employed for differentiating endoplasmic reticulum and plasma-lemmal calcium pump systems, visualization of SR in the cells and estimating its amount in membrane preparations and tissue homogenates.

[Allelic exclusion in hybridomas. II. Characteristics of the synthesis of 2 allelic variants of rat immunoglobulin kappa-chains in a cloned hybridoma line]. / Allel'noe iskliuchenie v gibridomakh. II. Osobennosti sinteza dvukh allel'nykh variantov kappa-tsepei immunoglobulinov krysy klonirovannoi gibridomnoi liniei.

Production of Igk-1a and Igk-1b allelic variants of rat Ig kappa-chains in hybridoma clone 4C2 was studied. Gel-filtration of the culture fluid demonstrates that the cells secrete isolated L-chains in monomeric and dimeric form. Different L-chain allotypes fail to associate in dimers. Immunofluorescence studies prove that both allelic variants are produced in a single cell. Cells with double allotype production loose the ability to produce one or both allotypes at an essentially heightened rate as compared to those producing Igk-1a or Igk-1b alone. The results fit well into the regulated allelic exclusion model.

[Analysis of the frequency of allelic exclusion of immunoglobulin light chain genes and molecular characteristics of immunoglobulins secreted by hybridomas with expression of both allelic genes]. / Analiz chastoty allel'nogo iskliucheniia genov legkikh tsepei immunoglobulinov i molekuliarnaia kharakteristika immunoglobulinov, sekretiruemykh gibridomami s ékspressiei oboikh allel'nykh genov.

The investigation of 750 B-lymphocyte hybridoma clones obtained by fusion of mouse myeloma and newborn heterozygous Igk-la/Igk-1b rat splenocytes has revealed that 9,8% of Ig kappa-chain genes are rearranged productively. Seventeen hybridomas secrete kappa-chains of both allelic variants. The analysis of IgM molecules of nine such clones demonstrated that in six cases only one L-chain allotype is present in IgM. Thus for the first time the high frequency of selective association of H and L chains was shown. Evidently this selectively may function as one of the allelic exclusion mechanisms at the Ig assembly stage.

[Use of monoclonal antibodies against the light-chain immunoglobulins of the rat in the radioimmune analysis of differentiated lymphocyte antigens of the mouse]. / Ispol'zovanie monoklonal'nykh antitel protiv legkikh tsepei immunoglobulinov krysy v radioimmunnom analize differentsirovannykh antigenov limfotsitov myshi.

Spleen cells from SJL mice immunized with rat immunoglobulin light chains were fused with X-63 myeloma. Seven hybridoma lines were obtained. Monoclonal antibodies (McAB) secreted by lines L1G9, L2B2 and L3E8 were purified and labeled by 125I. McAB L1G9 did not bind directly to murine lymphocytes but reacted in a different manner with rat splenocytes. McAb L2B2 and L3E8 but not L1G9 reacted with murine lymphocytes treated with rat McAb against Lyt-1, Lyt-2, C1-9, C4-20 antigens whatever the class of heavy chains of rat McAb used. The binding levels obtained with 125I-McAb reflect the quantitative differences in the expression of respective antigens.

[Allelic exclusion in hybridomas. I. Isolation and properties of a hybrid clone producing two allelic variants of light chain immunoglobulins in the rat]. / Allel'noe iskliuchenie v gibridomakh. I. Poluchenie i svoistva gibridomnogo klona, produtsiruiushchego dva allel'nykh varianta legkikh tsepei immunoglobulinov krysy.

Expression of RI-1a and RI-1b allelic genes controlling the production of rat lg kappa L-chains by hybridoma cells in vitro was studied. By fusing mouse myeloma cells with RI-1a/RI-1b heterozygous rat splenocytes the unique cloned hybrid cell line secreting both allelic variants has been established. This line may have appeared because cell hybridization made it possible to fix the rare case of correct rearrangement of the kappa chain gene segments on both homologous chromosomes.