Quantitation of monoclonal plasma cells in bone marrow biopsies in plasma cell dyscrasia.

Direct measurement of monoclonal plasma cell mass in bone marrow biopsies may be a useful parameter to establish in plasma cell dyscrasia. In this study monoclonal plasma cells/mm in light chain immunoglobulin immunostained archival bone marrow sections from 22 patients in whom a diagnosis of multiple myeloma (MM) had been excluded but who had monoclonal proteins were counted by two observers at light microscopic level. There was good correlation between the counts of the two observers. The levels of monoclonal plasma cells/mm in biopsies were not related to the % counts in the aspirates taken at the same time as the biopsies. Three of seven patients with biopsy levels in excess of the polyclonal levels in patients without plasma cell dyscrasia developed progressive MM within the observation time. Monoclonal plasma cell levels/mm of bone marrow biopsies can be measured and they provide a useful parameter for the assessment of patients with low volume plasma cell dyscrasia.     

Enhancement of bone formation by genetically-engineered bone marrow stromal cells expressing BMP-2, VEGF and angiopoietin-1

To explore the potential of combined delivery of osteogenic and angiogenic factors to bone marrow stromal cells (BMSCs) for repair of critical-size bone defects, we followed the formation of bone and vessels in tissue-engineered constructs in nude mice and rabbit bone defects upon introducing different combinations of BMP-2, vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) to BMSCs with adenoviral vectors. Better osteogenesis and angiogenesis were found in co-delivery group of BMP-2, VEGF and angiopoietin-1 than any other combination of these factors in both animal models, indicating combined gene delivery of angiopoietin-1 and VEGF165 into a tissue-engineered construct produces an additive effect on BMP-2-induced osteogenesis.     

The bone marrow plasma cell labeling index by flow cytometry

The bone marrow plasma cell labeling index is the most important prognostic indicator for patients with multiple myeloma. Traditionally, this test has been performed as a two color immunofluorescent microscope technique which is time consuming and requires a degree of subjectivity in its interpretation. We have assessed various adaptations of this method to flow cytometry. A bromodeoxyuridine method has been compared with a propidium iodide DNA method to detect cells in S phase and CD38-FITC has been compared with CD38-FITC + CD138-FITC and CD38-biotin + streptavidin FITC to identify plasma cells. The mean channel fluorescent intensity of the plasma cell peaks for each of these markers was 12. 7, 17.4 and 35.3 respectively demonstrating the superiority of CD38-biotin + streptavidin FITC. Analysis after propidium iodide staining provided a good correlation with the slide technique (r = 0. 71; P < 0.0001) but the bromodeoxyuridine method did not correlate with the slide method (r = 0.09; P = NS). The labeling index values obtained from either of the flow methods were greater than the microscopic method. Thus a labeling index of >4% will replace the traditional >1% threshold for identifying patients with a significantly increased labeling index. The advantages of the new method are that it takes less time to perform, is more objective and provides additional data on ploidy and cell cycle status.     

Unique biomechanical interactions between myeloma cells and bone marrow stroma cells

We observed that BMSCs (bone marrow stromal cells) from myeloma patients (myeloma BMSCs) were significantly stiffer than control BMSCs using a cytocompression device. The stiffness of myeloma BMSCs and control BMSCs was further increased upon priming by myeloma cells. Additionally, myeloma cells became stiffer when primed by myeloma BMSCs. The focal adhesion kinase activity of myeloma cells was increased when cells were on stiffer collagen gels and on myeloma BMSCs. This change in myeloma stiffness is associated with increased colony formation of myeloma cells and FAK activation when co-cultured with stiffer myeloma BMSCs or stiffer collagen. Additionally, stem cells of RPMI8226 cells became stiffer after priming by myeloma BMSCs, with concomitant increases of stem cell colony formation. These results suggest the presence of a mechanotransduction loop between myeloma cells and myeloma BMSCs to increase the stiffness of both types of cells via FAK activation. The increase of stiffness may in turn support the growth of myeloma cells and myeloma stem cells.     

Influence of a fibroblastoid cell line derived from murine bone marrow (H-1 cells) on stem cell proliferation

A murine fibroblastoid cell line (H-1) with properties similar to those of adventitial reticular cells can support granulopoiesis and the development of mononuclear phagocytes in vitro. In the current study the effect of these cells on stem cell maintenance in vitro was assessed. The H-1 cells were unable to support CFUs replication in liquid culture, while treatment of some stem cells with H-1 conditioned medium appeared to inhibit their proliferation.     

Multilineage hematopoietic disorders induced by transplantation of bone marrow cells expressing the v-fms oncogene

Mouse bone marrow cells infected with a helper-free retrovirus containing v-fms were engrafted into lethally irradiated mice. Dominant provirus-positive clones emerged in the spleens of some recipients within 1 month. When spleen cells were transplanted into lethally irradiated secondary recipients, clonal erythroleukemias or B cell lymphomas expressing the v-fms-coded glycoprotein developed. Other secondary recipients repopulated by “unmarked” progenitor cells or by cryptic provirus-positive precursors present in the spleens of the same donor mice did not develop disease; thus cells expressing v-fms did not invariably have a proliferative advantage after transplantation. Several primary engrafted recipients developed myeloproliferative disorders that were provirus-positive without evidence of clonality. Although expression of the c-fms product (CSF-1 receptor) is normally restricted to cells of the mononuclear phagocyte series, the v-fms-coded glycoprotein can contribute to proliferative abnormalities of multiple hematopoietic lineages.     

Differential expression of MUC1 on transfected cell lines influences its recognition by MUC1 specific T cells

In adenocarcinomas of the breast and pancreas, underglycosylation of the glycoprotein MUC1, also expressed by normal breast and pancreatic ductal epithelial cells, results in new protein epitopes to which the immune system mounts a cytotoxic T cell response. This cytotoxic immune response is directed primarily against epitopes on the tandem repeat domain of MUC1, and is unconventional in that it is major histocompatibility complex (MHC)-unrestricted. It is therefore necessary to investigate the molecular basis of this immune response in order to enhance and optimize it for immune therapy purposes. In the present study, we characterize new MUC1 transfected human lymphoblastoid cell lines C1R and T2, and a pig kidney epithelial line LLC-PK₁, that express MUC1 with either two repeats (MUC1–2R) or 22 repeats (MUC1–22R), and use them as stimulators and targets for cytotoxic T cells (CTL)in vitro. We show that MUC1–2R is processed and glycosylated similarly to MUC1–22R. In contrast to MUC1–22R, MUC1–2R is not recognized by CTL on T2 and C1R cells known for no or low MHC class I expression. It is however recognized when expressed at high density on xenogeneic LLC-PK₁ cells. We propose that in MHC-unrestricted recognition, a large number of MUC1 epitopes is necessary to effectively engage the T cell receptor, and that in the presence of a low number of epitopes, engagement of the CD8 co-receptor by MHC class I molecules may be required for completing the signal through the T cell receptor.     

T-cell depleted bone marrow transplantation for plasma cell myeloma. Report of a case and review of the results of BMT for myeloma

A 37-year-old male patient with advanced refractory plasma cell myeloma underwent T-cell depleted bone marrow transplantation (BMT) after 7 years of active disease previously treated with combination chemotherapy and irradiation. After the BMT there was marked clinical improvement and the patient is currently in good clinical condition two years after the BMT was performed. However, residual myeloma cells are still seen in the marrow and stable levels of paraprotein are still present in the serum. No GVHD was encountered after BMT. The problems of BMT in myeloma are discussed with a review of the current pertinent literature.     

T cell regulation of interferon-alpha/beta (IFN-alpha/beta) production by alloantigen-stimulated bone marrow cells

We have previously reported that mouse bone marrow cells produce high levels of interferon-alpha/beta (IFN-alpha/beta) after 5 to 6 days of in vitro culture with irradiated allogenic spleen cells. The current study was initiated to determine whether or not T cells are important for alloantigen-induced IFN-alpha/beta production by mouse bone marrow cells. Bone marrow cells and spleen cells were obtained from C57BL/6 mice. These cells were treated with different monoclonal antisera and complement, and then were cultured 5 to 6 days with irradiated DBA spleen cells. The results from these experiments indicated that optimal IFN-alpha/beta production by alloantigen-stimulated bone marrow cells required Lyt-1+2+ T cells. In addition, when bone marrow cells obtained from nu/nu B10 mice were cultured with alloantigen, only low levels of IFN were produced when compared with IFN production by bone marrow cells obtained from normal littermate B10 mice. The addition of nylon wool-enriched splenic T cells to cultures containing bone marrow cells and alloantigen resulted in an augmentation of IFN-alpha/beta production by three-fold to fivefold. Furthermore, bone marrow cells obtained from alloantigen-immunized mice produced much higher levels of IFN-alpha/beta and in a shorter period of time (2 to 3 days) when compared with bone marrow cells obtained from control or non-immunized mice. Cyclosporin A (CsA) has been shown to inhibit predominantly T cell-dependent responses. The effect of CsA on IFN production by alloantigen-stimulated bone marrow and spleen cells was investigated. The addition of CsA at concentrations as low as 0.1 micrograms/ml inhibited not only IFN-gamma production by alloantigen-stimulated spleen cells, but also IFN-alpha/beta production by alloantigen-stimulated bone marrow cells. In contrast, IFN-alpha/beta production by Newcastle disease virus-infected spleen cells, bone marrow cells, or L cells was not inhibited by the addition of CsA (1 microgram/ml). Thus, the ability of bone marrow cells to produce high levels of IFN-alpha/beta after in vitro culture with alloantigen is dependent upon T cells resident in the bone marrow. IFN-alpha/beta production by alloantigen-stimulated bone marrow cells may play a major role in the pathogenesis associated with graft-vs-host disease and in T cell regulation of hematopoiesis.     

Molecular cytogenetic aberrations in Tunisian patients with multiple myeloma identified by cIg-FISH in fixed bone marrow cells

Cytogenetic studies in multiple myeloma (MM) are hampered by the hypo-proliferative nature of plasma cells. In order to circumvent this problem, we have used a combination of immunolabeling of cytoplasmic Ig light chains (λ or κ) and FISH (cIg-FISH), which allowed a comprehensive detection of the most common and/or recurrent molecular cytogenetic aberrations on fixed bone marrow cells of 70 Tunisian patients. Translocations involving the chromosome 14q32 region were observed in 32 cases (45.7%), including 18 cases with a t(11;14), 8 cases with a t(4;14), and 2 cases with a t(14;16). Deletions of the 13q14 region (D13S319/RB1) were detected in 18.6%, and deletions of the 17p13 region (TP53) in 5.7% of the cases, respectively. Of all patients with a D13S319/RB1 deletion, 61.5% also carried a 14q32 translocation, whereas TP53 deletions were associated with a t(11;14) in 2 cases (50%) and a D13S319 deletion in 1 case (25%). Our results suggest that there is a correlation between the presence of 14q32 translocations and chromosome 13q14 deletions in MM patients and that cIg-FISH is more sensitive as compared to conventional karyotyping in detecting molecular cytogenetic abnormalities in this disease.     

Bone marrow stromal cell interaction reduces Syndecan-1 expression and induces kinomic changes in myeloma cells

CD138 (Syndecan 1) is a heparan sulfate proteoglycan that concentrates heparan sulfate-binding growth factors on the surface of normal and malignant plasma cells (multiple myeloma, MMC). Recent studies have shown the presence of a CD138-negative fraction of MMC within myelomatous bone marrow (BM). We employed kinome array technology to characterize this fraction at a molecular level, using a myeloma cell line model. Compared to CD138-positive cells, CD138-negative MMC showed (i) a reduced activity of kinases involved in cell cycle progression, in agreement with a decreased labeling index and (ii) reduced Rho signaling to F-actin. Interestingly, CD138 mRNA and protein expression was reduced upon interaction of MM cells with stromal cell lines and primary mesenchymal cultures, which was accompanied by the acquisition of an increased Bcl6/Blimp1 ratio. Co-culture induced an increased activity of kinases involved in adhesion and a decreased S-phase transition in both CD138-positive and -negative fractions. In addition, CD138-negative MMC demonstrated an increased STAT3 and ERK1/2 activation compared to CD138+ MMC, in agreement with a lower sensitivity to compound exposure. The presence of a less mature, more resistant CD138-negative myeloma cell fraction within bone marrow microniches might contribute to high incidence of relapse of Myeloma patients.     

Separation of osteoblast-like cells from bone marrow by fluorescence-activated cell sorting

The purification of the osteoblast-like cells (2-3%) among the bone marrow cells (BMC) of C57BL/6 mice using a specific anti-osteoblast serum and a fluorescence-activated cell sorter is described. The antiserum was raised against osteoblast cells isolated from calvaria from neonatal mice. The majority of the cells of the osteoblast-enriched fraction from bone marrow showed a parathormone-induced increase in cyclic adenine monophosphate but no response to calcitonin. This is similar to the response of osteoblast cells obtained from the calvaria. Electron microscopic studies of the extracellular matrix of cultured osteoblast-like cells purified from bone marrow showed the deposition of apatite crystals within and in close apposition to the vesicles. These findings suggest that the isolated cell population was enriched in osteoblasts. Such a cell system from bone marrow might provide an experimental system for investigating the mechanism of bone formation.     

Serglycin constitutively secreted by myeloma plasma cells is a potent inhibitor of bone mineralization in vitro

Although the biological significance of proteoglycans (PGs) has previously been highlighted in multiple myeloma (MM), little is known about serglycin, which is a hematopoietic cell granule PG. In this study, we describe the expression and highly constitutive secretion of serglycin in several MM cell lines. Serglycin messenger RNA was detected in six MM cell lines. PGs were purified from conditioned medium of four MM cell lines, and serglycin substituted with 4-sulfated chondroitin sulfate was identified as the predominant PG. Flow cytometry and confocal microscopy showed that serglycin was also present intracellularly and on the cell surface, and attachment to the cell surface was at least in part dependent on intact glycosaminoglycan side chains. Immunohistochemical staining of bone marrow biopsies showed the presence of serglycin both in benign and malignant plasma cells. Immunoblotting in bone marrow aspirates from a limited number of patients with newly diagnosed MM revealed highly increased levels of serglycin in 30% of the cases. Serglycin isolated from myeloma plasma cells was found to influence the bone mineralization process through inhibition of the crystal growth rate of hydroxyapatite. This rate reduction was attributed to adsorption and further blocking of the active growth sites on the crystal surface. The apparent order of the crystallization reaction was found to be n=2, suggesting a surface diffusion-controlled spiral growth mechanism. Our findings suggest that serglycin release is a constitutive process, which may be of fundamental biological importance in the study of MM.     

Enhanced differentiation of splenic plasma cells but diminished long-lived high-affinity bone marrow plasma cells in aged mice

In the present work, we have dissected the mechanisms responsible for the impaired humoral responses in aging. We found that there was a substantially higher level of Ab-forming cells in the spleens of aged mice than that of young controls. However, the number of high-affinity, class-switched Ab-forming cells was severely decreased in the spleen of aged mice. The accumulation of low-affinity IgM Ab-forming cells in the spleens of aged animals was not due to a deficiency in isotype switching because the number of total IgG1 splenic plasma cells was not significantly reduced. Remarkably, plasma cells of both low and high affinity were significantly diminished in the bone marrow of aged mice compared with that of young mice. The results from reconstitution experiments showed that aged bone marrow was less supportive for plasma cells derived from young splenic B cells. These findings suggest that humoral immune deficiency in aging results from at least two mechanisms: the inability to generate sufficient numbers of high-affinity Ab-forming cells, which is a result of diminished germinal center reaction, and the defective bone marrow environment that has diminished ability to support the selection and survival of long-term Ab-forming cells.     

Detection of murine adult bone marrow stroma-initiating cells in Lin(-)c-fms(+)c-kit(low)VCAM-1(+) cells

We attempted to characterize the phenotype of cells which initiate fibroblastic stromal cell formation (stroma-initiating cells: SICs), precursor cells for fibroblastic stromal cells, based on the expression of cell surface antigens. First, we stained adult murine bone marrow cells with several monoclonal antibodies and separated them by magnetic cell sorting. SICs were abundant in the c-kit(+), Sca-1(+), CD34(+), VCAM-1(+), c-fms(+), and Mac-1(-) populations. SICs were recovered in the lineage-negative (Lin(-)) cells but not the Lin(+) cells. When macrophage colony-stimulating factor (M-CSF) was absent from the culture medium, no stromal colony appeared among the populations enriched in SICs. Based on these findings, the cells negative for lineage markers and positive for c-fms (M-CSF receptor) were further divided on the basis of the expression of c-kit, VCAM-1, Sca-1 or CD34 with a fluorescence-activated cell sorter. SICs were found to be enriched in the Lin(-)c-fms(+)c-kit(low) cells and Lin(-)c-fms(+)VCAM-1(+) cells but not in Lin(-)c-fms(+)Sca-1(+) cells and Lin(-)c-fms(+)CD34(low) cells. As a result, the SICs were found to be present at highest frequency in Lin(-)c-fms(+)c-kit(low)VCAM-1(+) cells: a mean of 64% of the SICs in the Lin(-) cells were recovered in the population. In morphology and several characteristics, the stromal cells derived from Lin(-)c-fms(+)c-kit(low)VCAM-1(+) cells resembled fibroblastic cells. The number of Lin(-)c-fms(+)c-kit(low)VCAM-1(+) cells in bone marrow of mice injected with M-CSF was higher than that in control mice. In this study, we identified SICs as Lin(-)c-fms(+)c-kit(low)VCAM-1(+) cells and demonstrated that M-CSF had the ability to increase the cell population in vivo.