ALBERT

Description: The mononuclear cells in the blood of myeloma patients have been reported to contain a high proportion of phenotypically abnormal myeloma B lymphocytes. These cells have been proposed to constitute the drug-resistant proliferative myeloma cell compartment. To determine the extent of B lymphocyte involvement, the proportion of clonotypic cells among the CD19-expressing cells from myeloma patients was estimated by quantitative polymerase chain reaction analysis of the third complementarity determining region (CDR3). The results indicate that the B lymphocytes constitute, on average, 6% of blood mononuclear cells, and that only a minor fraction of these are clonally related to the myeloma cells. While the small number of circulating clonal cells is not incompatible with their proposed role as a reservoir of proliferating myeloma progenitors, the majority of the B cells appear not to be clonally related to the myeloma cells.

Description: Although monoclonal antibody (MoAb) therapy of the human malignant lymphomas has shown success in clinical trials, its full potential for the treatment of hematologic malignancies has yet to be realized. To expand the clinical potential of a promising human-mouse chimeric antihuman B-cell MoAb (chCLL-1) constructed using the variable domains cloned from the murine Lym-2 (muLym-2) hybridoma, fusion proteins containing granulocyte-macrophage colony-stimulating factor (GM-CSF) (chCLL-1/GM–CSF) or interleukin (IL)-2 (chCLL-1/IL–2) were generated and evaluated for in vitro cytotoxicity and in vivo tumor targeting. The glutamine synthetase gene amplification system was employed for high level expression of the recombinant fusion proteins. Antigenic specificity was confirmed by a competition radioimmunoassay against ARH-77 human myeloma cells. The activity of chCLL-1/GM–CSF was established by a colony formation assay, and the bioactivity of chCLL-1/IL–2 was confirmed by supporting the growth of an IL-2–dependent T-cell line. Antibody-dependent cellular cytotoxicity against ARH-77 target cells demonstrated that both fusion proteins mediate enhanced tumor cell lysis by human mononuclear cells. Finally, biodistribution and imaging studies in nude mice bearing ARH-77 xenografts indicated that the fusion proteins specifically target the tumors. These in vitro and in vivo data suggest that chCLL-1/GM–CSF and chCLL-1/IL–2 have potential as immunotherapeutic reagents for the treatment of B-cell malignancies.

Description: Frommeyer, Epstein and Taylor: Refractoriness in hemophilia to coagulation-promoting agents: Whole blood and plasma derivatives. Blood 5: 401-420 (May), 1950. The authors wish to note that the following changes should have appeared in the article: Page 402, first line: "Lawrence and Johnson, 1941,8 reported" (instead of "Lawrence and Johnston, in 1946,8 reported"). Page 402, second sentence of same paragraph: "In this instance the patient had received whole blood and plasma in therapeutic management prior to the appearance of resistance to therapy." (instead of ". . . had received chemically prepared plasma fractions in the form of Fraction I of Cohn in addition to whole blood and plasma in therapeutic management prior to . . . "). Page 420, reference 8. To this reference should be added: "(Presented before the American Clinical and Climatological Society, October 1941.)".

Description: The molecular mechanisms by which multiple myeloma (MM) cells evade glucocorticoid-induced apoptosis have not been delineated. Using a human IgAκ MM cell line (ARP-1), we found that dexamethasone (Dex)-induced apoptosis is associated with decreased NF-κB DNA binding and κB-dependent transcription. Both nuclear p50:p50 and p50:p65 NF-κB complexes are detected in ARP-1 cells by supershift electrophoretic mobility shift assay (EMSA). Dex-mediated inhibition of NF-κB DNA binding precedes a notable increase in annexin V binding, thereby indicating that diminished NF-κB activity is an early event in Dex-induced apoptosis. Overexpression of bcl-2 in ARP-1 cells prevents Dex-mediated repression of NF-κB activity and apoptosis. Sustained NF-κB DNA binding is also observed in two previously characterized Dex-resistant MM cell lines (RPMI8226 and ARH-77) that express moderate levels of endogenous bcl-2 and IκB proteins. In addition, enforced bcl-2 expression in ARP-1 cells did not prevent the augmentation of IκB protein by Dex. We also noted a possible association between Dex-mediated downregulation of NF-κB in freshly obtained primary myeloma cells and the patients’ responsiveness to glucocorticoid-based chemotherapy. Collectively, our data suggest that the protective effects of bcl-2 in MM cells act upstream in the NF-κB activation–signaling pathway and the potential use of NF-κB as a biomarker in progressive MM.

Description: Between August 1990 and August 1995, 231 patients (median age 51, 53% Durie-Salmon stage III, median serum β-2-microglobulin 3.1 g/L, median C-reactive protein 4 g/L) with symptomatic multiple myeloma were enrolled in a program that used a series of induction regimens and two cycles of high-dose therapy (“Total Therapy”). Remission induction utilized non–cross-resistant regimens (vincristine-doxorubicin-dexamethasone [VAD], high-dose cyclophosphamide and granulocyte-macrophage colony-stimulating factor with peripheral blood stem cell collection, and etoposide-dexamethasone-cytarabine-cisplatin). The first high-dose treatment comprised melphalan 200 mg/m2 and was repeated if complete (CR) or partial (PR) remission was maintained after the first transplant; in case of less than PR, total body irradiation or cyclophosphamide was added. Interferon--2b maintenance was used after the second autotransplant. Fourteen patients with HLA-compatible donors underwent an allograft as their second high-dose therapy cycle. Eighty-eight percent completed induction therapy whereas first and second transplants were performed in 84% and 71% (the majority within 8 and 15 months, respectively). Eight patients (3%) died of toxicity during induction, and 2 (1%) and 6 (4%) during the two transplants. True CR and at least a PR (PR plus CR) were obtained in 5% (34%) after VAD, 15% (65%) at the end of induction, and 26% (75%) after the first and 41% (83%) after the second transplants (intent-to-treat). Median overall (OS) and event-free (EFS) survival durations were 68 and 43 months, respectively. Actuarial 5-year OS and EFS rates were 58% and 42%, respectively. The median time to disease progression or relapse was 52 months. Among the 94 patients achieving CR, the median CR duration was 50 months. On multivariate analysis, superior EFS and OS were observed in the absence of unfavorable karyotypes (11q breakpoint abnormalities, -13 or 13-q) and with low β-2-microglobulin at diagnosis. CR duration was significantly longer with early onset of CR and favorable karyotypes. Time-dependent covariate analysis suggested that timely application of a second transplant extended both EFS and OS significantly, independent of cytogenetics and β-2-microglobulin. Total Therapy represents a comprehensive treatment approach for newly diagnosed myeloma patients, using multi-regimen induction and tandem transplantation followed by interferon maintenance. As a result, the proportion of patients attaining CR increased progressively with continuing therapy. This observation is particularly important because CR is a sine qua non for long-term disease control and, eventually, cure.

Description: Twenty-two patients with classic hemophilia were studied from the point of view of refractoriness to the usual coagulation-promoting effect of whole blood, and of chemically prepared plasma fractions. Of these, 5 patients showed clinical and laboratory evidence of refractoriness to therapy. All had previously received by intravenous injection for therapeutic purposes during bleeding episodes large amounts of plasma fractions in addition to whole blood and plasma. Subsequently, each refractory patient exhibited a poor clinical response to therapy during hemorrhagic episodes. Thus, despite the administration of whole blood, plasma, or plasma derivatives in amounts which were usually effective, bleeding continued unabated and the blood coagulation time was not significantly reduced. An anticoagulant distinct from antithrombin, antithromboplastin, antiprothrombin and heparin was demonstrated in the plasma of each patient. Each had adequate amounts of fibrinogen and prothrombin in the circulating plasma. However, antibodies were demonstrated in the plasma of each patient by means of precipitin reactions with antigens possessing antihemophilic activity, including various plasma derivatives and Fraction I of Cohn. No such antibodies were demonstrable in a normal individual, in 2 anemic patients who had received many transfusions of whole blood, or in a patient with afibrinogenemia who had received large amounts of antihemophilic globulin because of its fibrinogen content. Prompt reduction in plasma anticoagulant activity, in precipitin titer, and coagulation time were effected only by the administration of large quantities of fresh whole blood. A secondary rise of antibodies always followed such a procedure within several days. When therapy was withheld, the anticoagulant effect and precipitin titer decreased in one patient over a period of six months and in another, had essentially disappeared by the end of a year. From these studies the following conclusions may be drawn: 1. A refractory state in hemophilia may develop following the therapeutic use of whole blood, plasma or chemically prepared plasma fractions. 2. This state is characterized by defective clinical and laboratory responses to therapy with whole blood, plasma or plasma fractions. An anticoagulant is demonstrable in the plasma as well as precipitins to various plasma derivatives possessing antihemophilic activity. 3. The formation of antibodies is apparently an immunologic response to a substance closely associated with antihemophilic activity in normal blood and in plasma derivatives previously employed in therapy. 4. The refractory state can be promptly and temporarily abolished and good clinical and laboratory responses can be effected by transfusions of very large amounts of fresh whole blood. 5. Because of their demonstrated ability to act as antigens in certain hemophilic patients, whole blood, plasma and chemically prepared plasma fractions should be withheld unless their employment as an emergency therapeutic measure is necessary.

Description: Murine monoclonal antibody (MoAb) Lym-1 is an IgG2a able to bind HLA-DR variants on malignant B cells and suitable for serotherapeutic approaches in B-lymphoma patients. We have previously shown that Lym-1 can synergize with granulocyte-macrophage colony-stimulating factor (GM-CSF) to trigger neutrophil cytolysis towards Raji cells used as a model of B-lymphoma targets. Here we provide evidence for the intervention of certain neutrophil receptors or surface molecules in this model of cell-mediated lysis. The lysis was completely inhibited by the anti-FcγRII MoAb IV.3 and unaffected by the anti-FcγRIII MoAb 3G8. This suggests that neutrophil cytolysis involves FcγRII without cooperation of this receptor with FcγRIII. Moreover, the lysis was inhibited by an anti-CD18 MoAb (MEM48) and by a MoAb specific for carcinoembryonic antigen (CEA)-like and glycophosphatidyl inositol (GPI)-linked glycoproteins (CD66b). Using an immunofluorescence staining procedure, cross-linking of CD66b induced the redistribution of CD11b on neutrophils with distinct areas of CD11b clustering via a process susceptible of inhibition by D-mannose. This is consistent with the ability of CD11b-CD18 and CD66b to undergo lectin-like physical interactions on the neutrophil surface. Such a type of interaction is presumably instrumental for neutrophil cytolytic activity in that the lysis was inhibited by D-mannose and enhanced by the MoAb VIM-12, which mimics the cooperation between CD11b and GPI-anchored molecules by specifically interacting with CD11b lectin-like sites. Therefore, the present results prove the absolute requirement for FcγRII in neutrophil GM-CSF/Lym-1–mediated cytolysis and, on the other hand, define the crucial role of CD66b and CD11b/CD18 in the expression of the cell lytic potential.

Description: The mannose receptor (MR) is a transmembrane protein that functions primarily as a phagocytic receptor for a wide range of microorganisms. Its expression appears to be restricted to tissue macrophages and Langerhans cells. To gain an understanding of the regulation of the gene, we have isolated the 5′ flanking sequence of the murine MR gene and have analyzed a 536-bp sequence upstream of the ATG start site for transcriptional activity. This sequence lacks a TATA box but contains an initiator (Inr) consensus element overlapping the single transcriptional start site. Transcription factor binding sites contained within this sequence include PU.1, Sp1, ETS, GATA, and MYB motifs. Serial 100-bp deletions of this promoter fragment fused to a luciferase reporter gene showed various patterns of activity when transfected into different cell types. In myeloid cells, sequence elements upstream of bp −300 appeared to have a silencing effect on promoter activity. Of the four potential PU.1 binding sites contained within the fragment, one site (at −164) bound the PU.1 factor most strongly, whereas the adjacent PU.1 site (at −177 bp) bound PU.1 to a lesser degree. Mutations of these sites decreased transcriptional activity but did not abolish it. However, promoter activity was abrogated when both the −164 bp PU.1 site and the adjacent −177 bp PU.1 site were mutated. In addition, mutation of the Sp1 site also significantly reduced promoter activity. Cotransfection studies in Drosophila Schneider cells indicated that PU.1 and Sp1 may function synergistically in transactivating the murine MR. This study indicates that MR gene expression is regulated in part by the interaction between the ubiquitously expressed factor Sp1 and the lymphoid/myeloid factor PU.1 and provides a basis for studying the regulation of this gene.

Description: Progress in unraveling the biology of myeloma has suffered from lack of an in vitro or in vivo system for reproducible growth of myeloma cells and development of disease manifestations. The SCID-hu mouse harbors a human microenvironment in the form of human fetal bone. Myeloma cells from the bone marrow of 80% of patients readily grew in the human environment of SCID-hu mice. Engraftment of myeloma cells was followed by detectable human Ig levels in the murine blood. Myeloma-bearing mice had high levels of monotypic human Igs, high blood calcium levels, increased osteoclast activity, and severe resorption of the human bones. The human microenvironment was infiltrated with Epstein-Barr virus-negative monoclonal myeloma cells of the same clonality as the original myeloma cells. Active angiogenesis was apparent in areas of myeloma cell infiltration; the new endothelial cells were of human origin. We conclude that the SCID-hu mouse is a favorable host for studying the biology and therapy of myeloma and that a normal bone marrow environment can support the growth of myeloma cells. © 1998 by The American Society of Hematology.

Description: The low proliferative activity of myeloma plasma cells prompted the notion that the clonotypic B cells that exist in the blood and bone marrow of all myeloma patients contain the proliferative myeloma cells (stem cell). We have exploited our severe combined immunodeficiency (SCID)-hu host system for primary myeloma to investigate whether myeloma plasma cells are capable of sustained proliferation. Purified CD38++CD45− plasma cells consistently grew and produced myeloma and its manifestations in SCID-hu hosts (8 of 9 experiments). In contrast, the plasma cell-depleted bone marrow cells from 6 patients did not grow or produce myeloma in SCID-hu hosts. Similarly, whereas plasma-cell containing blood cells from 4 patients grew and produced myeloma in hosts, neither the PC-depleted blood cells from 3 of the patients nor a blood specimen that did not contain plasma cells grew in SCID-hu hosts, regardless of their CD19-expressing cell contents. Also, in hosts injected with blood cells, although the myeloma cells were able to disseminate through the murine host system, they were only able to grow in the human bones within a human microenvironment and were not detectable in the murine blood or other organs. Interestingly, the circulating plasma cells appear to grow more avidly in the SCID-hu hosts than their bone marrow counterparts, suggesting that they represent a subpopulation of the plasma cells in the bone marrow. Although our studies clearly demonstrate the proliferative potential of myeloma plasma cells, they are suggestive, not conclusive, as to the existence of a preplasmacytic myeloma progenitor cell.