Expression of a public idiotype by human monoclonal IgM directed to myelin-associated glycoprotein and characterization of the variability subgroup of their heavy and light chains.

Most studies using rabbit or mouse antisera failed to detect CRI between human IgM directed to MAG. We show here that 9 of 10 such IgM express a public CRI as defined by a nonhuman primate antiserum. Shared idiotype is likely involved in (or close to) the combining site of those IgM since antiidiotypic serum inhibited the binding of IgM to MAG and reacted with IgM having different variable regions of light and heavy chains. Partial aminoterminal sequence of heavy and light chains showed that anti-MAG IgM use either lambda chains (one IgM) or kappa light chains (six IgM) of different variability subgroups (V kappa IV in three instances, V kappa I in two, and V kappa II in one), whereas heavy chains belong to the VHIII (six IgM) or to the VHII (1 IgM) subgroup. These features distinguish these IgM from other human monoclonal IgM with a defined antibody activity, such as rheumatoid factors or cold agglutinins.

Interleukin 2-induced proliferation of leukemic human B cells.

The proliferative responses of purified leukemic human B cells from nine B cell chronic lymphocytic leukemias to recombinant interleukin 2 (IL-2), spontaneously, and after preactivation by Staphylococcus aureus Cowan I (SAC) or anti-mu antibodies were studied. Three patterns of response were observed: (a) no response (three cases); (b) a moderate spontaneous response enhanced by anti-mu (one case); (c) a high proliferative response after preactivation by anti-mu and/or SAC (five cases). IL-2 could also trigger normal B cells, purified from spleen, to proliferative after preactivation by anti-mu or SAC. These results provide evidence that IL-2 is a lymphokine that acts physiologically on both B and T cells.

Role of interleukin 10 in the B lymphocyte hyperactivity and autoantibody production of human systemic lupus erythematosus.

Interleukin-10 (IL-10) is produced at a high level by B lymphocytes and monocytes of patients with systemic lupus erythematosus (SLE). In the present work, we analyzed whether this increased production of IL-10 contributed to the abnormal production of immunoglobulins (Ig) and of autoantibodies in SLE. The role of IL-10 was compared with that of IL-6, another cytokine suspected to play a role in these abnormalities. The spontaneous in vitro production of IgM, IgG, and IgA by peripheral blood mononuclear cells from SLE patients was weakly increased by recombinant IL (rIL)-6, but strongly by rIL-10. This production was not significantly affected by an anti-IL-6 mAb but was decreased by an anti-IL-10 mAb. We then tested the in vivo effect of these antibodies in severe combined immunodeficiency mice injected with PBMC from SLE patients. The anti-IL-6 mAb did not significantly affect the serum concentration of total human IgG and of anti-double-stranded DNA IgG in the mice. In contrast, the anti-IL-10 mAb strongly inhibited the production of autoantibodies, and, to a lesser extent, that of total human IgG. These results indicate that the Ig production by SLE B lymphocytes is largely IL-10 dependent, and that the increased production of IL-10 by SLE B lymphocytes and monocytes may represent a critical mechanism in the emergence of the autoimmune manifestations of the disease.

Interleukin-6 antisense oligonucleotides inhibit the growth of human myeloma cell lines.

IL-6 has been shown to be a plasmacytoma growth factor in mice and is believed to play a key role in the development of human multiple myeloma. We investigated the IL-6 requirements for the growth of two human myeloma cell lines, U 266 and RPMI 8226. These cell lines secreted minute amounts of IL-6 (20 U/ml) and featured IL-6 mRNA. IL-6 receptors were detectable at the surface of malignant cells by immunofluorescence. Antibodies to IL-6 did not alter the proliferation of these myeloma cells. There was a dose-dependent decrease, however, in [3H]-thymidine uptake in the presence of IL-6 antisense (and not sense) oligodeoxynucleotides; in the presence of 20 microM IL-6 antisense, an 80 and 95% inhibition of the proliferation of U 266 and RPMI 8226 cells was observed, respectively. These results provide strong evidence for an IL-6 autocrine proliferation of myeloma cells which may occur via internal interaction between IL-6 and the IL-6 receptor.

Cross-idiotypic antigens among monoclonal immunoglobulin M from patients with Waldenström's macroglobulinemia and polyneuropathy.

The monoclonal immunoglobulin (Ig)M from 5 to 16 patients with Waldenström's macroglobulinemia and a polyneuropathy shared cross-idiotypic antigenic determinants as demonstrated by hemagglutination and hemagglutination inhibition experiments as well as by precipitin reactions. This reactivity was located to the Fab (and not Fc) fragment of the protein. The IgM from 73 patients with macroglobulinemia but without neuropathy all gave negative reactions. In contrast, the monoclonal IgG from a patient with polyneuropathy also possessed similar idiotypic determinants. Since cross-idiotypic determinants are usually related to the combining site of a monoclonal Ig, this finding suggests that the monoclonal Ig of these patients may mediate the nerve injury via their antibody activity, which could be directed either to a nerve antigen or to some component involved in the pathogenesis of the neuropathy.

Interleukin-10 prevents spontaneous death of germinal center B cells by induction of the bcl-2 protein.

In this study, we show that IL-10 enhances in vitro the viability of purified splenic B cells. There was a two- to threefold increase in recovery of viable cells during a 15-d culture period in the presence of IL-10. This effect was abolished by neutralizing antibodies to IL-10. The survival of large splenic B cells, which mostly represent follicular center cells, was similarly increased. The in vitro rescue from spontaneous death of the latter cells is known to involve a bcl-2-dependent pathway. We therefore investigated whether IL-10 might affect bcl-2 expression. Unseparated B cells as well as large splenic B cells displayed a strong expression of bcl-2 protein by immunofluorescence at days 2-7 of culture in the presence of IL-10. Other lymphokines such as IL-2 and IL-4 were able to trigger only a transient and faint expression of bcl-2; moreover, this effect was abolished by anti-IL-10 mAb. Inasmuch as activated B cells can produce their own IL-10, this lymphokine may play a crucial role in relieving from apoptosis those B cells that encounter their antigen in B cell follicles.

A molecular defect in thrombasthenic platelets.

An IgG antibody found in the serum of a thrombasthenic patient reacted in complement fixation with platelets from 350 normal individuals but was nonreactive with platelets from eight other thrombasthenic patients. ADP-induced aggregation of normal platelets was inhibited by the patient's antibody. Family studies using the quantitative complement fixation test showed that healthy heterozygotes were easily distinguishable from normal or thrombasthenic individuals since their platelets had an intermediate amount of the reactive antigen. Indirect immunoprecipitation tests using this serum and soluble membrane antigens labeled with iodine-125 that had been extracted from normal platelets by the detergent Nonidet P-40 gave a single radioactive peak at 120,000 mol wt in sodium dodecyl sulfate polyacrylamide gel electrophoresis. A similar estimate of the molecular weight was obtained from Sephadex G-200 filtration of the soluble antigens extracted from normal platelets by spontaneous release or chaotropic agents and tested in complement fixation with the patient's serum. These findings strongly suggest that the molecule recognized by this antibody is absent or structurally modified in thrombasthenia cases and that it may be involved in platelet aggregation.

Cryoprecipitation of an anti-Pr2 monoclonal IgM cold agglutinin in the presence of GM3 ganglioside.

The mechanism of cryoprecipitation of a monoclonal IgM kappa cryoglobulin (Mou) with a cold agglutinin activity of Pr2 specificity has been studied. By immunodiffusion this cryoglobulin reacted (by its Fab' fragment) with micellar GM3, a ganglioside bearing the Pr2 antigenic determinant. In contrast to previous reports that indicated a possible temperature dependent self-association of IgM molecules via an immunological interaction leading to cold precipitation, we could not detect any affinity of this cryoglobulin for IgM when we used passive hemagglutination or an indirect enzyme-linked immunosorbent assay (ELISA). However, a GM3-like ganglioside could be extracted, by drastic methods, from the cryoglobulin studied at 22 degrees C, whereas no GM3 was extracted from two control cryoglobulins. Some minor gangliosides (representing less than 25% of total amount of bound gangliosides) were also extracted from Mou cryoglobulin and these gangliosides were shown to crossreact with GM3, as they specifically bind to Mou cryoglobulin by ELISA. After cryoprecipitation the serum still contained a monoclonal anti-Pr2 IgM kappa. A GM3-like ganglioside could be extracted from this purified IgM, and cryoprecipitability could be induced by the addition of a minute amount of micellar GM3. These results suggest that Mou cryoglobulin circulates as an immune complex and that cryoprecipitation may depend on unique IgM-GM3 (or IgM-GM3 cross-reacting gangliosides) complexes.

Cytogenetic study of a European Burkitt's lymphoma cell line.

The chromosomes of an Epstein-Barr virus-negative European Burkitt's lymphoma cell line were studied. All the cells carried the t(8;14) translocation. One clone had 51 chromosomes and was (+1,+7,+16,+15,+21), whereas another clone also had 51 chromosomes but was (+1q+,+7,+16,+15,+21). A third clone had 46 chromosomes (4q+/-).

Abnormal expression of vimentin intermediate filaments in human lymphoid cell lines with deletion or translocation of the distal end of chromosome 8.

The expression of vimentin, the major polypeptide of the intermediate filament (IFM) cytoskeleton of lymphoid cells, was studied in normal and malignant human lymphoid cell lines. Cells from 24 of 27 Burkitt's lymphoma cell lines (BLCL) were found to have an absent (16 lines) or decreased (8 lines) expression of vimentin IFM. In contrast, non-Burkitt's malignant lymphoid cell lines (5 lines) and lymphoblastoid cell lines (LCL) derived from normal B-cells (45 lines) exhibited a well-developed vimentin IFM network. However, low expression of vimentin was also found in 3 LCL derived from patients with the Langer-Giedion syndrome, which is characterized by a deletion of the distal end of chromosome 8. Treatment of vimentin-negative BLCL and Langer-Giedion LCL with azacytidine led to a transient reexpression of vimentin.

Acute lymphoblastic leukemia with Burkitt's lymphoma cells: membrane markers and serum immunoglobulin.

Blast cells from 24 patients with acute lymphoblastic leukemia of Burkitt's cell type were studied for lymphocyte surface markers. The leukemia cells were of B-cell origin in 23 cases and showed a non-B, non-T phenotype in 1 case. Surface immunoglobulins on blast cells were monoclonal, with a striking predominance of cases with light chains of lambda type. They consisted most often of high-density IgM usually without associated IgD. However, 3 patients had cells with surface IgG and 1 had surface IgA. The blast cells lacked detectable IgG Fc receptors in more than half the patients. Serum immunoglobulins were studied in 15 cases: A monoclonal IgM was found in 5 patients (whose blast cells had surface IgM) and a Bence Jones protein was found in 2 others, both of whom had blasts with surface IgG lambda.

Membrane markers in "histiocytic" lymphomas (reticulum cell sarcomas).

Neoplastic cells from 9 patients affected with a "histiocytic" lymphoma were studied with 5 membrane markers of B or T lymphocytes. In 2 patients a monoclonal B-cell proliferation was found; they had been affected previously with well documented B-cell proliferations: chronic lymphocytic leukemia or Waldenström's macroglobulinemia. The blast cells of 2 other patients had T-cell features; in a fifth case, the abnormal cells carried only a strong receptor for the Fc fragment of IgG, which suggested their truly monocytic origin. In 4 patients, the cells had no detectable surface markers. These findings demonstrated that this group of lymphomas is heterogenous, that the term "histiocytic" appears to be wrong in most instances, and that the cellular origin of the malignant cells frequently remains unidentified and thus prevents a satisfactory new classification.

Characterization of intermediate filaments expressed by Ewing tumor cell lines.

The histogenesis of Ewing sarcoma is still controversial; we therefore studied the expression of intermediate filaments (IF) in cell lines derived from Ewing tumors since identification of IF in tumor cells is considered a reliable marker of tissue origin and differentiation. All nine lines studied expressed vimentin IF; in addition, a small number of Ewing cells from three lines expressed keratin filaments. After treatment with phorbol esters, a high percentage of cells from these three lines synthesize keratin IF identified by immunoblotting as keratin 8 and 18 polypeptides, which are expressed by single epithelia and epithelial cells in early embryonic development. Furthermore cells from a fourth line synthesize keratins after transplantation in nude mice. These data indicate that, under certain conditions, undifferentiated Ewing cells may acquire an IF phenotype related to that of epithelial cells.

Arsenic trioxide and melarsoprol induce apoptosis in plasma cell lines and in plasma cells from myeloma patients.

Recent data have renewed the interest for arsenic-containing compounds as anticancer agents. In particular, arsenic trioxide (As2O3) has been demonstrated to be an effective drug in the treatment of acute promyelocytic leukemia by inducing programmed cell death in leukemic cells both in vitro and in vivo. This prompted us to study the in vitro effects of As2O3 and of another arsenical derivative, the organic compound melarsoprol, on human myeloma cells and on the plasma cell differentiation of normal B cells. At pharmacological concentrations (10(-8) to 10(-6) mol/L), As2O3 and melarsoprol caused a dose- and time-dependent inhibition of survival and growth in myeloma cell lines that was, in some, similar to that of acute promyelocytic leukemia cells. Both arsenical compounds induced plasma cell apoptosis, as assessed by 4',6-diamidino-2-phenylindole staining, detection of phosphatidylserine at the cell surface using annexin V, and by the terminal deoxynucleotidyl transferase-mediated nick end labeling assay. As2O3 and melarsoprol also inhibited viability and growth and induced apoptosis in plasma-cell enriched preparations from the bone marrow or blood of myeloma patients. In nonseparated bone marrow samples, both arsenical compounds triggered death in myeloma cells while sparing most myeloid cells, as demonstrated by double staining with annexin V and CD38 or CD15 antibodies. In primary myeloma cells as in cell lines, interleukin 6 did not prevent arsenic-induced cell death or growth inhibition, and no synergistic effect was observed with IFN-alpha. In contrast to As2O3, melarsoprol only slightly reduced the plasma cell differentiation of normal B cells induced by pokeweed mitogen. Both pokeweed mitogen-induced normal plasma cells and malignant plasma cells showed a normal nuclear distribution of PML protein, which was disrupted by As2O3 but not by melarsoprol, suggesting that the two arsenical derivatives acted by different mechanisms. These results point to the use of arsenical derivatives as investigational drugs in the treatment of multiple myeloma.

Association of the human type C retrovirus with a subset of adult T-cell cancers.

To determine whether the human T-cell lymphoma-leukemia virus (HTLV) is associated with particular cancers, patient sera were surveyed for HTLV-specific antibodies. An association was seen with aggressive cancers of mature T-cells, specifically Japanese adult T-cell leukemia (ATL) and T-cell lymphosarcoma cell leukemia (TLCL), a similar cancer of Caribbean blacks. Ninety to 100% of these patients possessed HTLV-specific antibody. Forty-seven and 20% of relatives of ATL and TLCL patients, respectively, and 12 and 4% of healthy donors from ATL and TLCL endemic areas were also antibody positive. Visceral organ involvement, hypercalcemia, and skin manifestation, features of ATL and TLCL, were often seen in other antibody-positive patients. Childhood cancers, most cutaneous T-cell and all non-T-cell leukemias and lymphomas, myeloid leukemias, Hodgkin's disease, and solid tumors were not associated with HTLV. Healthy United States donors and European patients with non-malignant diseases were antibody negative. HTLV is thus associated with a subtype of adult T-cell leukemia-lymphoma, clustered in viral endemic areas, with apparent racial and geographic predilection.

Molecular analysis of a t(9;14)(p11;q32) translocation occurring in a case of human alpha heavy chain disease.

We performed the cloning and sequencing of the der(14) breakpoint of a new chromosomal translocation involving the 14q32 immunoglobulin locus. This t(9;14)(p11;q32) translocation was found in a case of malignant lymphoma occurring in human alpha heavy chain disease. A rearranged alpha 1 gene fragment was cloned and shown to contain chromosome 9 information by Southern blotting on sorted chromosomes and by in situ hybridization. Sequence analysis of the junction point region established that the breakage occurred 3' to the heavy chain joining region. In contrast to the data obtained in other translocations affecting 14q32 immunoglobulin locus, the recombination did not involve the immunoglobulin heavy chain locus specific recombination signals on chromosome 14, or homologous sequences on chromosome 9. In the present case, the existence of two almost perfect inverted repeats flanking the junction point suggests that the translocation originated from a local pairing of the two chromosomes 9 and 14. Chromosome 9 fragments sequenced in the vicinity of the breakpoint did not share significant homology with sequences listed in GenBank and EMBL data bases.

Heterogeneity of the breakpoint localization in malignant cells with a 9p11 chromosomal abnormality.

The p11 band of the short arm of chromosome 9 is involved in various cytogenetic alterations occurring in several malignant diseases. Using probes isolated from the 9p11 band in the study of a case of alpha-heavy-chain disease (MAL) with t(9;14)(p11;q32), we studied the DNA from seven malignant cell samples, including four cases of acute lymphoblastic leukemia with tdic(9;12)(p11;p12). Using pulsed-field electrophoresis analysis we demonstrated that the breakpoints were 3-300 kb distant from the original MAL breakpoint without clustering within the subset of leukemias with the tdic(9;12).

T cell-derived blast crisis in chronic myelocytic leukemia.

We describe herein the occurrence of a T cell-derived blast crisis of chronic myelocytic leukemia which presented as a typical T cell leukemia on clinical (thymic mass and high white blood cells) and immunological grounds (T6+ T4+ T8+), with rearranged T cell receptor genes and germ line immunoglobulin genes. A Philadelphia chromosome was detected in all blast cell mitoses. The significance of T cell involvement during the chronic and acute phases of chronic myelocytic leukemia is discussed.

mu-chain disease. Report of two new cases.

We report two cases of mu-heavy-chain disease. Both patients were affected with a lymphoproliferative disease that shared several suggestive features with the previously reported cases of mu-chain disease: the presence of vacuolated plasma cells in bone marrow, a small amount of alpha 2 moving abnormal mu-chain protein, and urinary kappa Bence Jones protein in one case.

Evaluation of bone mineral density and fat-lean distribution in patients with multiple myeloma in sustained remission.

To study the usefulness of bone mineral density (BMD) in the follow-up of myeloma (MM) patients, BMD was evaluated in 44 MM patients in sustained remission for at least 2 years (35.4 +/- 10.5 months) after high-dose or conventional chemotherapy in a retrospective study. Patients never received bisphosphonates before or during the follow-up. Patients underwent lumbar spine (LS) BMD and a whole body (WB) BMD testing before therapy and at least once in the remission period. At baseline, mean LS BMD was 0.863 +/- 0.026 g/cm2, mean lumbar Z-score was -1.45 SD. LS BMD significantly increased from baseline by 5 +/- 1.8%, 9.3 +/- 1.7%, and 14 +/- 1.9% at 1, 2, and 3 years, respectively. The percentage of patients with a T-score below 2.5 SD decreased from 39% at baseline to 18.5% at 3 years. Compared with baseline, WB BMD decreased by -2.8 +/- 0.5%, -2.6 +/- 0.7%, and -1.7 +/- 0.6% at 1, 2, and 3 years, respectively. Mean percentage change of the fat compartment increased from baseline by +28.4 +/- 7.1% at the trunk, and +17.1 +/- 5% in peripheral areas at 3 years. In conclusion, in MM patients in remission after chemotherapy, LS BMD progressively increased after a mean follow-up of 3 years. These patients never received bisphosphonates, so this increase was related to the anti-myeloma treatment. The major effect on BMD was observed at the LS, which is primarily composed of trabecular bone containing the bone marrow. Interestingly, a drastic increase of the fat content was also observed. These results underlined that BMD and fat-lean evaluation could be of interest in the follow-up of MM patients.