Functional and Biochemical Characteristics of a Soluble B Lymphocyte Proliferation-Inhibiting Activity Produced by Bone Marrow Cells from Multiple Myeloma Patients

Radioautographic DNA labeling and rosetting techniques were combined to study the development of surface IgM, Fc, and complement receptors (FcR, CR) on small lymphocyte populations in mouse bone marrow. [3H]thymidine was either infused continuously to label newly formed cells for periods up to 4 days, or injected daily, 21--35 days before use, to label a sample of long-lived cells. Bone marrow cells were incubated with sensitized sheep erythrocytes to detect surface IgM, FcR, and CR, respectively, and examined radioautographically after cytocentrifugation. During [3H]thymidine infusion, marrow small lymphocytes lacking surface markers were the first to show [3H]thymidine labeling. Most of these cells became labeled by 4 days (IgM--ve, 89%; FcR--ve, 92%; Cr--ve, 88%). Labeling of small lymphocytes bearing surface IgM, FcR, and Cr began after an initial lag and increased to high values by 4 days (IgM + ve, 73%; FcR + ve, 82%; CR + ve, 83%). Labeled IgM + ve small lymphocytes formed progressively larger rosettes as cell age increased. Some proliferating large lymphoid cells formed rosettes for IgM, FcR, and CR. Labeled long-lived small lymphocytes expressed surface IgM, FcR, and CR, the incidence of each receptor being uniformly high (38--43%) and the rosettes tending to be larger than those formed by newly formed lymphocytes. In double-surface marker studies, FcR and CR rosettes were formed by some IgM--ve small lymphocytes as well as IgM + ve cells in the marrow. After transfusion of marrow cells from donor mice infused with [3H]thymidine for 24 h, many labeled newly formed lymphocytes homed into the splenic red pulp of unlabeled syngeneic recipients. Subsequently, these cells showed a rapid increase in the incidence of rosettes for surface IgM, FcR, and CR, together with a progressive enlargement of each type of rosette. Although all the labeled small lymphocytes recovered from the spleen developed both surface IgM and FcR by 3 days, only approximately one-half developed CR. The results demonstrate that most of the small lymphocytes bearing FcR, CR, and surface IgM in mouse bone marrow are newly formed indigenous cells. Each receptor becomes detectable by rosetting soon after the small lymphocytes are first produced. The newly formed, marrow-derived small lymphocytes are able to continue their development of surface IgM, FcR, and CR after migrating into the spleen, consistent with a maturation of primary B lymphocytes. In addition, the data indicate the genesis in mouse marrow of a non-B lineage of lymphocytes (notably, IgM--ve FcR + ve cells.). A minority of small lymphocytes bearing IgM, FcR, and CR in mouse marrow are long-lived cells, presumptive recirculating immigrants, differing in receptor status from the newly formed cells. The results are discussed with regard to the heterogeneity of marrow lymphocytes and proposed models of primary B lymphocyte and null lymphocyte production.

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C-met Receptor as a Potential Target for the Treatment of Patients with Multiple Myeloma.

Blood ◽

10.1182/blood.v106.11.3395.3395 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3395-3395

Author(s):

Marcin Majka ◽

Artur Jurczyszyn ◽

Anna Zebzda ◽

Wojciech Czogala ◽

Ewa Lesko ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Cell Lines ◽

Protein Level ◽

Bone Marrow Cells ◽

Response To Therapy ◽

Poor Responders ◽

Met Receptor ◽

Potential Target ◽

Marrow Cells

Abstract Despite progress in the treatment of Multiple Myeloma (MM), it is still an incurable disease with average survival of 3–4 years. Because MM is often resistant to conventional therapies, new treatment strategies are necessary. The presence of elevated HGF (Hepatocytic Grow Factor) expression has been well documented in multiple myeloma. The c-met oncogene has been shown to be present in MM cell lines at the mRNA and protein level. Some data suggested that this axis could be responsible for proliferation and inhibition of apoptosis in MM cells. In this study we have analyzed c-met expression in 15 patients with (MM) before and after treatment. Seven of these pts responded well and eight pts responded poorly to the employed therapy. All 15 pts were c-met positive before therapy. Bone marrow cellularity of patients who responded well was 76% before (range: 10% – 100%) and 46% after treatment (range: 40% – 60%). In this group plasmocyte infiltration of bone marrow consisted of 59% before (range: 10% – 80%) and 9% after chemotherapy (range: 0% – 20%). Five of them had undetectable c-met positive cells among bone marrow cells after treatment. In the group of poor responders cellularity of bone marrow was 40% (range: 20% – 70%) before treatment and 46% (range: 20% – 70%) after therapy. Plasmocytes consisted of 20% (range: 10% – 50%) of bone marrow cells before and 44% (range: 10% – 90%) after treatment. All patients in this group had cells positive for c-met receptor after therapeutic regiment. This results suggested that c-met-HGF axis might be a good target for alternative therapy in MM. We looked for potential therapeutics that interferes with this axis and we found that geldanamycin (GA) has been shown to decrease expression of c-met at the protein level in several different cell types. Using inhibitors that belongs to geldanamycin family (GA, 17AAG and 17DMAG) we treated MM cell lines and primary sample. We found that these molecules strongly inhibited expression of c-met in both MM cell lines and patients sample as assessed by western blot analysis. We also tested the influence of these inhibitors on proliferation of MM cells. We found that 100nM dose of GA and 17DMAG inhibited growth of MM cell lines by 80% and 100nM dose of 17AAG inhibited growth of these cells by 20%. Primary cells were more resistant to treatment but we still obtained 30% inhibition with GA and 17DMAG. 17AAG was ineffective and proliferation decreased by less than 10%. Grow inhibition was probably not only due to c-met-HGF axis blockade because these molecules also inhibit other proteins (AKT, RAF). In our experiments we have shown that the level of c-met expression correlates with response to therapy. Patients who respond well had substantially decreased number of c-met positive plasmocytes after chemotherapy in comparison to poor responders. We have also showed that drugs that block c-met-HGF axis could be used in treatment of MM. These drugs could potentially inhibit cells proliferation, increase apoptosis and disrupt MM cells interaction with bone marrow environment. Based on these data we postulate that the c-met receptor is a potential target for MM therapy especially in patients who do not respond to the first line of treatment.

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Neutralization Of Vascular Endothelial Growth Factor-A Induced CD138-Expression In Bone Marrow Cells From Multiple Myeloma Patients

Blood ◽

10.1182/blood.v122.21.5331.5331 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5331-5331

Author(s):

Ryosuke Shirasaki ◽

Takuji Matsuo ◽

Yoko Oka ◽

Jun Ooi ◽

Naoki Shirafuji

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Vascular Endothelial Growth Factor ◽

Growth Factor ◽

Poor Prognosis ◽

Bone Marrow Cells ◽

Vascular Endothelial ◽

Normal Individuals ◽

Endothelial Growth Factor ◽

Marrow Cells

Abstract Background We previously reported that when adult human dermal fibroblasts were cultured with interleukin (IL)-1-b, vascular endothelial growth factor (VEGF)-A was produced significantly (54th ASH). And, when antihuman VEGF-A neutralizing antibody (VEGF-A Ab) was added to the cultures, CD138 (Syndecan-1) expressed significantly. CD138 is a member of cell-surface transmembrane haparan sulfate proteoglycans, and expresses in plasma cells from multiple myeloma (MM) cases. Membrane-anchoring CD138 shows a better prognosis in an immunodeficiency murine transplantation model in vivo; however, when extra-domain of CD138 is digested by heparanase to be shed from the cell-surface, MM cells invade to various kinds of tissues, and the patients show poor prognosis. Aims To validate a biological implication of inhibition of VEGF-A-signaling in MM cells, we observed effects of VEGF-A Ab to bone marrow cells from MM patients. Cell-proliferations as well as morphological changes were also observed time-dependently. Materials and Methods Institutional ethical committee approved our study, and bone marrow cells were obtained from the informed MM patients as well as normal individuals. Cells were separated with gravity-sedimentation method, and the prepared mononuclear cells were cultured with or without VEGF-A Ab, and the expression of specific genes was analyzed. Results Twenty MM patients were eligible, in which three showed significant poor prognosis, and worsened after underwent intensive chemotherapy or allogeneic hematopoietic stem cells transplantation. Thirteen out of twenty expressed CD138, and when cells were cultured with VEGF-A Ab for four days, CD138-expression increased significantly in all cases. Four did not express CD138; however, CD138-expression was observed after 4 day’s culture with VEGF-A Ab. In three progressed cases CD138-expression decreased in accordance with the disease-progression; however, when VEGF-A Ab was added to the cell-cultures, CD138 was induced to express. Heparanase-expression was observed in 10 cases out of 20, which were down-regulated when VEGF-A Ab was added to the cultures. In contrast, in bone marrow cells from seven normal individuals CD138-expression was very low, which was down-regulated with the addition of VEGF-A Ab. Heparanase-expression was not observed in these normal cells, and were induced to be observed in four out of seven when VEGF-A Ab was added to the cultures. Discussion Expression of CD138 is induced in fibroblast by the addition of fibroblast growth factor-2, and in keratinocytes by epidermal growth factor and keratinocyte growth factor; however, an induction of CD138 by the VEGF-A Ab has not been reported. Several cytokines including VEGF-A influence plasma cell-proliferation; however, little is reported on cytokine-suppression therapy. Inhibition of the signaling of VEGF receptors by the chemicals including solafenib is not specific for VEGF-A. Currently we validate the efficacy of the inhibition of VEGF-A-signaling to MM cells and their environmental cells using RNA interference. Disclosures: No relevant conflicts of interest to declare.

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Monoclonal origin of B lymphocyte colony-forming cells in spleen colonies formed by multipotential hemopoietic stem cells

Journal of Experimental Medicine ◽

10.1084/jem.148.6.1468 ◽

1978 ◽

Vol 148 (6) ◽

pp. 1468-1477 ◽

Cited By ~ 27

Author(s):

PK Lala ◽

GR Johnson

Keyword(s):

Bone Marrow ◽

B Lymphocyte ◽

Bone Marrow Cells ◽

Fetal Liver ◽

Absolute Number ◽

Hemopoietic Stem Cell ◽

Adult Bone Marrow ◽

Clonal Origin ◽

Adult Bone ◽

Marrow Cells

Spleen colonies produced by transplanting lethally irradiated mice with either 12 day fetal liver or adult bone marrow cells were found to contain B- lymphocyte colony-forming cells (BL-CFC) . The proportion of BL-CFC positive spleen colonies did not increase substantially between 8 and 14 days after transplantation, the range being 18-45 percent. However, the absolute number of BL-CFC per spleen colony varied considerably (between 1 and 10,318), although the majority of colonies contained less than 200 BL-CFC. Irrespective of the time after transplantation, smaller spleen colonies were found to have a higher frequency of BL-CFC than larger spleen colonies. To determine the possible clonal origin of BL-CFC from spleen colony- forming unit (CFU-S), CBA mice were injected with equal numbers of CBA and CBA T(6)/T(6) fetal liver or adult bone marrow cells. Analysis of 7-15-day spleen colonies demonstrated that 90 percent were either exclusively T(6) positive or T(6) negative and approximately equal numbers ofboth colony types were observed. B-lymphocyte colonies were grown and successfully karyotyped from 19 spleen colonies. When compared with the original spleen colony karyotype the B-lymphocyte colony cells karyotype was identical in all 19 cases. In 3 of the 19 colonies analyzed a mixture of T(6) positive and T(6) negative karyotypes was present and identical proportions of the karyotypes were present in the pooled B-lymphocyte colony cells and spleen colony cells. The data indicate that the B-lymphocyte colony-forming cells detected in spleen colonies are genuine members of the hemopoietic clone derived from the initiating hemopoietic stem cell (CFU-S).

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3200 Effect of chemotherapy drugs in vitro on the death of bone marrow cells in patients with multiple myeloma

European Journal of Cancer ◽

10.1016/s0959-8049(15)30061-7 ◽

2015 ◽

Vol 51 ◽

pp. S649

Author(s):

I. Lysenko ◽

E. Zlatnik ◽

I. Novikova ◽

N. Nikolaeva ◽

O. Shatokhina ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Bone Marrow Cells ◽

Chemotherapy Drugs ◽

Marrow Cells

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Antibody-Inhibiting Activity of Bone Marrow Cells in vitro

International Archives of Allergy and Immunology ◽

10.1159/000230910 ◽

1972 ◽

Vol 43 (6) ◽

pp. 934-951 ◽

Cited By ~ 13

Author(s):

S.K. Singhal ◽

S. King ◽

P.J. Drury

Keyword(s):

Bone Marrow ◽

Bone Marrow Cells ◽

Inhibiting Activity ◽

Marrow Cells

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Evidence for the clonal abortion theory of B-lymphocyte tolerance.

Journal of Experimental Medicine ◽

10.1084/jem.141.4.904 ◽

1975 ◽

Vol 141 (4) ◽

pp. 904-917 ◽

Cited By ~ 215

Author(s):

G J Nossal ◽

B L Pike

Keyword(s):

Bone Marrow ◽

Tissue Culture ◽

B Lymphocytes ◽

B Lymphocyte ◽

Cultured Cells ◽

Bone Marrow Cells ◽

Mouse Bone Marrow ◽

Immune Competence ◽

Self Recognition ◽

Marrow Cells

This paper deals with the behavior of adult mouse bone marrow cells placed in tissue culture with or without antigen, and subsequently assessed for immune competence after adoptive transfer into lethally X-irradiated, syngeneic hosts. Attention was focussed on B lymphocytes through using hapten human gamma globulin (HGG) preparations as putative tolerogens in tissue culture, the T-cell-independent antigens DNP-POL and NIP-POL as challenge injections in adoptive hosts, and numbers of hapten-specific PFC in host spleens for the quantitation of immune competence. It was found that the capacity of bone marrow cells to mount an adoptive immune response rose by a factor of about fivefold over 3 days in tissue culture. This rise was completely abolished by the presence in the culture of hapten-HGG conjugates with about one mole of hapten per carrier molecule. The prevention of the emergence of immune competence amongst maturing B cells was termed clonal abortion tolerogenesis. Dose-response studies showed the lowest effective antigen concentration to be between 2.5 times 10- minus 10 and 2.5 times 10- minus 9 M, and a standard concentration of 2.5 times 10- minus 8 M was chosen as producing near maximal effects. The tolerance was antigen-specific and time-dependent, being maximal only when antigen was present continuously as the cultured cells was maturing. It did not depend on the presence of T lymphocytes in marrow, and was not of an "infectious" type. In contrast to tolerogenesis of mature B lymphocytes by high antigen concentrations, it could not be abolished by lipopolysaccharide. We speculate that clonal abortion may be a tolerance mechanism of great physiological significance for self-recognition, and discuss the results in the framework of other recent tolerance models, including those involving receptor blockade and suppressor T cells.

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G-banding analysis of complex aneuploidy in multiple myeloma bone marrow cells

Blood ◽

10.1182/blood.v47.1.69.69 ◽

1976 ◽

Vol 47 (1) ◽

pp. 69-77 ◽

Cited By ~ 41

Author(s):

P Philip ◽

A Drivsholm

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Chromosomal Abnormalities ◽

Bone Marrow Cells ◽

Clonal Evolution ◽

Banding Technique ◽

Chromosome Loss ◽

Banding Analysis ◽

Myeloproliferative Diseases ◽

Marrow Cells

Abstract Chromosome studies with the banding technique have been performed in a considerable number of cases of myeloproliferative diseases, but technical difficulties have so far prevented detailed studies of chromosomal abnormalities in multiple myeloma. The karyotypes of bone marrow cells from two patients with multiple myeloma have been analyzed by a trypsin-Giemsa banding technique. Evidence is given for clonal evolution which in one patient has probably occurred by cell fusion and subsequent chromosome loss. Eight different marker chromosomes are characterized. Nonrandom chromosomal participation in the translocations and the existence of specific vulnerable points on chromosomes 1, 3, and 16 are suggested.

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