Electromyographic analysis of deltoid muscle fatigue during abduction on scapular and frontal planes.

The aim of the study was to compare the fatigue rates of the deltoid muscle portions in the scapular and frontal planes. Ten healthy men without shoulder muscle impairment took part in the study. They performed isometric arm abduction for 30 seconds against a resistance of load cell while the electromyographic data were collected. The electromyographic data were transformed by the Fast Fourier Transform, to obtain the median power frequency (MDPF). The changes in MDPF of the three deltoid portions in the scapular and frontal planes were compared. The acromialis and spinalis portions fatigued during the exercises. The clavicularis portion presented no fatigue. A statistically significant difference occurred between the clavicularis and the other two portions (P < 0.05). No differences were found when the planes were compared. It represents to practice no preferential order during rehabilitation. Moreover, the acromialis and spinalis portions fatigue, although the clavicularis portion did not fatigue. The actions of other muscles of the shoulder girdle can explain this fact. Moreover, these two portions need more attention to avoid fatigue during exercises. In general, therapeutic strategies for injured patients should not only be directed towards increased force, but also towards fatigue control during shoulder exercises.

Increased susceptibility to apoptosis in CD45(+) myeloma cells accompanied by the increased expression of VDAC1.

Expression of CD45 is quite variable in human myeloma cells and cell lines, such as U266, and CD45(+) U266 proliferates in response to a growth factor, interleukin-6. Here, we show that CD45(+) myeloma cell lines were more sensitive to various apoptotic stimuli, such as oxidative stress and endoplasmic reticulum (ER)-stress, than CD45(-) cells. Reactive oxygen species and calcium ion seemed to be involved in the susceptibility to apoptosis of CD45(+) U266. The activation of the src family kinases associated with CD45 phosphatase played an important role in the augmented apoptosis in CD45(+) U266 by oxidative stress. These results indicate that the CD45-expression renders myeloma cells competent for not only mitogenic but also apoptotic stimuli, resulting in either proliferation or apoptosis of CD45(+) myeloma cells dependently upon the circumstantial stimuli. Furthermore, voltage-dependent anion channel (VDAC) 1 was identified as a gene highly expressed in CD45(+) U266 by cDNA subtraction. The increased expression of VDAC1 seemed to augment the sensitivity to the ER-stress because the VDAC1-transfected U266 was more susceptible to the thapsigargin-induced apoptosis. Thus, CD45 expression accompanied by the increased VDAC1 expression sensitizes myeloma cells to the various extracellular stimuli that trigger apoptosis via the mitochondrial pathways.

Biological significance of heterogeneity in human myeloma cells.

B cells differentiate into plasma cells which produce antibodies in the bone marrow (BM). Multiple myeloma (MM) is a hematologic malignancy in human plasma cells, and myeloma cells grow mainly in BM. According to phenotypic differences, such as expression of adhesion molecules, human myeloma cells as well as normal plasma cells can be classified into several differentiation stages. We have found that cells strongly expressing CD38 antigens (CD38++(+)) in BM are all plasma cells, and that there also are no plasma cells in either CD38- cell fraction or fraction of cells weakly expressing CD38 antigens (CD38+). Myeloma cells in BM consist of CD38++(+)MPC-1-CD49e (VLA-5)-immature and CD38++(+)MPC-1+CD49e+ mature myeloma cells. Immature myeloma cells proliferate markedly in vitro and respond to interleukin-6 (IL-6), a growth factor for myeloma cells, whereas mature myeloma cells show very low proliferative activities and show no response to IL-6. Immature myeloma cells expressing CD21 molecules on their surface seem to attach to stromal cells in BM through binding to CD23 molecules. Thus, there is a heterogeneity in human myeloma cells, and immature myeloma cells appear to proliferate in response to IL-6.

A new phenotypic classification of bone marrow plasmacytosis.

Here, we propose a new phenotypic classification of bone marrow plasmacytosis. By 2-color phenotypic analysis with FITC anti-CD38 and PE anti-CD19, -CD56, -VLA-5 or MPC-1 antibody, plasma cells are easily identified on the histogram, even though no more than 1% of plasma cells are found in the bone marrow. Hence, plasma cells are phenotypically classified into polyclonal (reactive) (CD19+CD56-) or monoclonal (neoplastic) plasma cells (mostly CD19-CD56+), and furthermore immature (VLA-5-MPC-1-), intermediate (VLA-5-MPC-1+) and mature plasma cells (VLA-5+MPC-1+). According to these findings, plasmacytosis in the bone marrow can be classified into polyclonal marrow plasmacytosis (POMP) and monoclonal marrow plasmacytosis (MOMP) states. The MOMP state is further subclassified into MOMP-1 and MOMP-2, MOMP-3 and MOMP-4; MOMP-1 is defined by co-existence of monoclonal plasma cells and polyclonal plasma cells, and MOMP-2 to MOMP-4 are dependent on increased proportions of VLA-5-MPC-1- immature myeloma (plasma) cells. We found that the cases of benign monoclonal gammopathy (BMG) according to the conventional classification were in the MOMP-1 state, and myelomas could be classified into the MOMP-2 to MOMP-4 state. Subclassification of the MOMP state may be useful in determining the prognosis of myelomas, where an increase in immature myeloma cells is reported to correlate well with their aggravation during the clinical courses. Therefore, this new phenotypic classification of bone marrow plasmacytosis (POMP and MOMP-1 to MOMP-4) will contribute to differential diagnosis and understanding of therapeutic responses and prognosis in myelomas.

A possible mechanism of inability of immunoglobulin heavy-chain production in Bence-Jones type myeloma cells.

To clarify the mechanism of the inability of immunoglobulin heavy (IgH) chain production in Bence-Jones (BJ) type myeloma cells, we examined the rearrangement of the IgH gene, the expression of IgH mRNA and the presence of functional nuclear factors that bind to the IgH gene enhancer in myeloma cells from 6 cases of BJ-type myelomas, and three myeloma cell lines and one Epstein-Barr virus-transformed B cell line having no IgH chain production. All BJ-type myeloma cells we examined showed rearranged bands by Southern blotting using the JH probe, and the rearrangement of VDJ segments in these cells was confirmed by PCR amplification of VDJ segments with consensus VH and JH primers. However, the expressions of IgH mRNA were not detected by Northern blotting in any of the BJ-type myeloma cells or the three non-producer myeloma cell lines. These findings suggested that the transcription of the IgH gene ceased in these cells, and alteration of this transcriptional apparatus may explain this inability. The functional nuclear factors that bind to IgH gene enhancers (HE2 and HE3) were absent in the myeloma cells from one case of BJ-type myelomas among these BJ-type myeloma cells and myeloma cell lines. This suggests that alteration of the enhancer binding factors may be one of the reasons why BJ-type myeloma cells are unable to produce the IgH chain.

Proliferation of immature myeloma cells by interleukin-6 is associated with CD45 expression in human multiple myeloma.

Multiple myeloma (MM) is a hematologic malignancy of human plasma cells, and myeloma cells can be classified into several subpopulations according to phenotypic differences, such as CD38 MPC-1- CD49e- immature, CD38 MPC-1+ CD49e- intermediate and CD38 MPC-1+ CD49e+ mature myeloma cells. The expression of the CD45 molecule on myeloma cells is quite variable, and the physiological consequence of CD45 on myeloma cells is still unknown. Recently, we have found that a few MPC-1- immature myeloma cells express CD45 antigens while most myeloma cells do not express the CD45. MPC-1- CD45+ CD49e- but not MPC-1- CD45- CD49e- immature cells contain proliferating cells in response to interleukin-6 (IL-6). IL-6 can also induce expression of CD45 on the MPC-1- CD45- subpopulation of immature myeloma cells. In addition, myeloma cell lines responding to IL-6 express CD45, whereas cell lines proliferating independent of IL-6 do not express CD45. In the U266 cell line, IL-6 leads to the induction of CD45 expression and cell proliferation, indicating that IL-6-induced effects are closely linked to CD45 expression. Thus, there is a heterogeneity in human myeloma cells, and among these subpopulations immature myeloma cells expressing the CD45 molecules appear to proliferate in response to IL-6. In this review we propose the involvement of CD45 in MM pathogenesis, and the possible implications of CD45 as both a phenotypic marker and a functional molecule is discussed.

Involvement of CD8+ RT1.B+ and CD4+ RT1.B+ cells of cervical lymph nodes in the immune response after corneal transplantation in the rat.

Graft rejection reactions have been observed with concomitant lymphocyte infiltrations after allogenic corneal transplantation, although the cornea is considered to be relatively protected from the systemic immune response. In order to characterize the lymphocytes that accumulate in cervical lymph nodes following transplantation, we used a model of orthotopic penetrating keratoplasty in inbred rats. After grafting, the time course of the pathological scoring was monitored, and subpopulations of CD4+ RT1.5+ and CD8+ RT1.B+ cells were analyzed in the cells harvested from the cervical lymph nodes. The number of CD8+RT1.B+ cells increased 1 week after grafting, reaching the maximum at 3 weeks; whereas CD4+ RT1.B+ cells were induced 1 week after the grafting and remained constant during the next 3 weeks. There were four times as many CD4+ RT1.B+ cells as CD8+ RT1.B+ cells 1 week after grafting when there was no rejection. Therefore, it appears that CD8+RT1.B+ and CD4+RT1.B+ cells in the cervical lymph nodes do participate in ocular immunologic responses.

[Myeloma precursor cells].

Heterogenous biological character of myeloma cells was associated with different expression of adhesion molecules. Myeloma cells could be phenotypically divided into two subpopulations: CD38++/VLA5+/MPC-1+(VLA-5+) cells and CD38++/VLA5-/MPC-1-(VLA-5-) cells. VLA-5- myeloma cells were morphologically immature and proliferated markedly with response to IL-6 in vitro, while VLA-5+ cells showed very low uptakes of 3H-TdR but secreted higher amounts of M-protein in vitro. These results suggest VLA-5- cells are proliferative precursor in myeloma. With respect to VLA-5 and MPC-1 expression, myeloma precursor cells (CD38++/VLA-5-/MPC-1-/CD10-/CD24-) showed similar phenotype to germinal center B cells (CD38+/VLA-5-/MPC-1-/CD10+/CD24-), rather than that of pre-B cells in the bone marrow (CD38+/VLA-5+/MPC-1-/CD10+/CD24+). Identification of precursor cells and characterization of their growth is important for the understanding of pathophysiology of myeloma and the therapeutic strategy.

[Myeloma precursor cells and their differentiation].

Recently, differentiation pathway of plasma cells from germinal center B cells has been clarified in detail. Most of bone marrow plasma cells are considered to be derived from germinal center B cells. Early plasma cells are detected in the peripheral blood, and in the bone marrow, immature, intermediate and mature plasma cells are identified by expression of adhesion molecules such as VLA-5 and MPC-1. Myeloma cells are also subclassified into immature, intermediate and mature myeloma cells. Immature myeloma cells can respond to interleukin 6 (IL-6) to proliferate, and circulate in the peripheral blood and markedly expand in relapse. Therefore, immature myeloma cells are considered to be clonogenic cells for myeloma, so-called myeloma precursor cells.

[Phenotypic analysis of myeloma cells].

Recent 2-color phenotypic analysis using anti-CD38 antibody reveals that plasma cells alone locate at CD38strong positive (CD38++) fraction and expression of adhesion molecules such as VLA-5 and MPC-1 can define VLA-5-MPC-1- immature, VLA-5-MPC-1+ intermediate and VLA-5+ MPC-1+ mature plasma (myeloma) cells. Furthermore, phenotypic analysis of plasma cells with anti-CD19 and -CD56 antibodies can distinguish normal (polyclonal) plasma cells from malignant (monoclonal) plasma cells; normal plasma cells from various tissues are all CD19+ CD56-, while malignant plasma cells are mostly CD19- CD56+. Therefore, this 2-color phenotypic analysis is very useful for differential diagnosis of bone marrow plasmacytosis, that is, myeloma, benign monoclonal gammopathy or polyclonal gammopathy, and furthermore contributes to understanding of differentiated stages of myeloma cells (immature, intermediate or mature myeloma cells).

Cloning and analysis of the human Pax-5 gene promoter.

The expression of Pax-5 gene is altered in human myeloma cells (malignant plasma cells). This altered expression is considered to be closely involved in oncogenesis of human myeloma. To investigate the possible mechanism(s) underlying this alteration, we first cloned the 1,050 bp fragment in the 5' upstream region of human Pax-5 gene by PCR-mediated gene walking method. The cloned fragment has predicted regulatory motifs for Lyf-1(Ik-1), IK-2, bHLH, E-47, Sox-5, Oct-1, GATA-1,-2, and -3, but it lacks a TATA box. By constructing deletion mutants of this fragment, its basal promoter activity was analyzed by transfecting these mutants to Cos 7 cells. The maximal promoter activity was recovered by the fragment that extends between -70 to -820 upstream of the transcription start site. Also, three DNA fragments from this cloned sequence were used as templates in gel shift assay; these fragments covered most of the predicted regulatory sites. Specific binding activities were found in each DNA fragment. Therefore, we could clone the functionally active fragment of 5' upstream region of human Pax-5 gene.

Cyclin D1 and p16INK4A are preferentially expressed in immature and mature myeloma cells, respectively.

Myeloma cells consist of immature, intermediate and mature cells with respect to expression of VLA-5 (CD49e) and MPC-1 adhesion molecules. VLA-5(-)MPC-1(-) immature myeloma cells respond to interleukin 6 (IL-6) to proliferate in vitro. but VLA-5+MPC-1+ mature myeloma cells have almost no proliferative activity with higher secretory activity of M-protein in vitro. In order to further clarify the biological differences between these immature and mature myeloma cells, we examined survival of these cells with or without IL-6 in vitro, and investigated the underlying mechanism of the proliferative or non-proliferative character of these cells by examining expression of cell cycle regulators such as cyclin D1 and inhibitors for cyclin-dependent kinase (Cdk), p16INK4A, p21CIP1 and p27KIP1 by RT-PCR and immunohistochemistry. In vitro survival of these myeloma cells was examined by flow cytometric quantification of fluorescein diacetate (FDA) and propidium iodide (PI) staining. Immature myeloma cells rapidly entered apoptosis without IL-6, but mature myeloma cells could survive without IL-6 as well as normal mature plasma cells. Immature myeloma cells as well as myeloma cell lines expressed cyclin D1 mRNA and protein, but not any Cdk inhibitors. On the other hand, mature myeloma cells did not express cyclin D1 but expressed p16, not p21 or p27, as well as normal mature plasma cells. Therefore these results show that immature myeloma cells constitutively express cyclin D1 and can proliferate, and mature myeloma cells as well as normal mature plasma cells preferentially express p16 and can survive for a long time without proliferation.

Differentiation of early plasma cells on bone marrow stromal cells requires interleukin-6 for escaping from apoptosis.

The bone marrow (BM) is well known to be the major site of Ig production in secondary immune responses; thus, the microenvironment of BM is considered to be essential for final differentiation of plasma cells. We identified in the peripheral blood (PB) early plasma cells (CD38++CD19+VLA-5-) committed to entering the BM. The sorted early plasma cells rapidly entered apoptosis in vitro, but these cells could survive and further differentiate into mature plasma cells (CD38 CD19+) just as BM plasma cells in the presence of a BM-derived stromal cell line (KM-102). Culture supernatants of KM-102 cell lines could also support survival of these cells, and antibody to interleukin-6 (IL-6) completely blocked the effect of these supernatants. Furthermore, recombinant IL-6, but not IL-1 or IL-3, could support their survival and their differentiation into mature plasma cells (CD38 CD19+VLA-5+) with expression of VLA-5 mRNA. Therefore, here is direct evidence that early plasma cells found in the PB differentiated into mature plasma cells with stromal cell-derived IL-6 in vitro; thus, BM stromal cells control the final checkpoint of plasma cell differentiation with secretion of IL-6 in the BM.

Induction of CD45 expression and proliferation in U-266 myeloma cell line by interleukin-6.

Recently, there has been an increasing interest in the expression pattern and biological significance of the CD45 molecule in myeloma cells. In this study, we have further defined the phenotypic pattern of CD45 expression on myeloma cells. Using a panel of myeloma cell lines, we showed that CD45 showed a remarkably heterogeneous pattern of expression. Whereas some cell lines were CD45(+) and others were CD45(-), the U-266 cell line, although predominantly CD45(-), still had a considerable subpopulation of CD45(+) cells. Among the myeloma cell lines examined, there was a direct correlation between interleukin-6 (IL-6) dependency and CD45 positivity. Moreover, we showed that IL-6 stimulation led to the induction of expression of CD45 and cellular proliferation. Using independent experimental approaches, we could show that the IL-6-induced effects were closely linked to CD45 expression. First, sorting out CD45(+) and CD45(-) subsets of U-266 cell line followed by IL-6 stimulation, only the CD45(+) cells showed a proliferative advantage after IL-6 stimulation. Second, IL-6 stimulation of sorted CD45(-) cells was gradually followed by phenotypic conversion to CD45(+) cells that started after 2 days as judged by the detection of CD45 mRNA by reverse transcription polymerase chain reaction (RT-PCR) and immunophenotypic analysis by flow cytometry. Withdrawal of IL-6 from the medium led to gradual loss of CD45 expression in CD45(+) flow-sorted U-266 cells. Third, the use of vanadate, a potent inhibitor of protein tyrosine phosphatase (PTP), abrogated the IL-6-induced proliferation in the CD45(+) myeloma cells. On the other hand, cellular proliferation induced by IL-6 was not affected by the serine-threonine phosphatase inhibitor okadaic acid. Our data show that the expression pattern of CD45 in myeloma cell lines is heterogeneous and show for the first time that CD45 expression can be induced by IL-6 stimulation. Finally, these data shed some light on the biological role of CD45 in myeloma by determining the proliferative population among myeloma cells.

MPC-1-CD49e- immature myeloma cells include CD45+ subpopulations that can proliferate in response to IL-6 in human myelomas.

Using three-colour phenotypic analysis, we detected five subpopulations of myeloma cells (CD38++) in the bone marrow mononuclear cells of human myeloma patients: MPC-1-CD45-CD49e-, MPC-1-CD45+CD49e-, MPC-1+CD45-CD49e-, MPC-1+CD45+CD49e- and MPC-1+CD45+CD49e+. Most of the myeloma cells did not express CD45 but a few MPC-1- immature myeloma cells and some MPC-1+ myeloma cells expressed CD45 and CD45RO but not CD45RA, whereas all of normal early plasma cells in the peripheral blood, lymph node plasma cells and bone marrow plasma cells expressed CD45 and CD45RA, CD45RB but not CD45RO. In order to clarify the biological character of these myeloma subpopulations, we examined the expression of Ki-67 antigen. Proliferating myeloma cells (Ki-67+) were found in the MPC-1- fractions and the MPC-1-CD45+ fractions rather than MPC-1-CD45- fractions. Next, in order to further clarify the biological difference of two immature subpopulations (MPC-1-CD45-CD49e- and MPC-1- CD45+CD49e-), determined cell viability and phenotypic change after culturing with interleukin 6 (IL-6) in vitro. In the presence of IL-6, MPC-1-CD45+ cells kept their viability more than MPC-1-CD45- cells and some MPC-1-CD45- cells could be converted to MPC-1-CD45+ cells. In conclusion, these data suggest that human myeloma cells are phenotypically subdivided into five subpopulations, and among these subpopulations MPC-1-CD45+CD49e- but not MPC-1-CD45-CD49e- immature cells contain proliferating cells in response to IL-6, and IL-6 can also induce expression of CD45 on MPC-1-CD45- subpopulation of immature myeloma cells.

Plasma cells induce apoptosis of pre-B cells by interacting with bone marrow stromal cells.

By using two-color phenotypic analysis with fluorescein isothiocyanate-anti-CD38 and phycoerythrin-anti-CD19 antibodies, we found that pre-B cells (CD38+CD19+) signifcantly decreased depending on the number of plasma cells (CD38++CD19+) in the bone marrow (BM) in the cases with BM plasmacytosis, such as myelomas and even polyclonal gammopathy. To clarify how plasma cells suppress survival of pre-B cells, we examined the effect of plasma cells on the survival of pre-B cells with or without BM-derived stromal cells in vitro. Pre-B cells alone rapidly entered apoptosis, but interleukin-7 (IL-7), a BM stromal cell line (KM-102), or culture supernatants of KM-102 cells could support pre-B cell survival. On the other hand, inhibitory factors such as transforming growth factor-beta1 (TGF-beta1) and macrophage inflammatory protein-1beta (MIP-1beta) could suppress survival of pre-B cells even in the presence of IL-7. Plasma cells alone could not suppress survival of pre-B cells in the presence of IL-7, but coculture of plasma cells with KM-102 cells or primary BM stromal cells induced apoptosis of pre-B cells. Supernatants of coculture with KM-102 and myeloma cell lines (KMS-5) also could suppress survival of pre-B cells. Furthermore, we examined the expression of IL-7, TGF-beta1, and MIP-1beta mRNA in KM-102 cells and primary stromal cells cocultured with myeloma cell lines (KMS-5). In these cells, IL-7 mRNA was downregulated, but the expression of TGF-beta1 and MIP-1beta mRNA was augmented. Therefore, these results suggest that BM-derived stromal cells attached to plasma (myeloma) cells were modulated to secrete lesser levels of supporting factor (IL-7) and higher levels of inhibitory factors (TGF-beta1 and MIP-1beta) for pre-B cell survival, which could explain why the increased number of plasma (myeloma) cells induced suppression of pre-B cells in the BM. This phenomenon may represent a feedback loop between pre-B cells and plasma cells via BM stromal cells in the BM.

Enforced CD19 expression leads to growth inhibition and reduced tumorigenicity.

In multiple myeloma (MM), the cell surface protein, CD19, is specifically lost while it continues to be expressed on normal plasma cells. To examine the biological significance of loss of CD19 in human myeloma, we have generated CD19 transfectants of a tumorigenic human myeloma cell line (KMS-5). The CD19 transfectants showed slower growth rate in vitro than that of control transfectants. They also showed a lower capability for colony formation as evaluated by anchorage-independent growth in soft agar assay. The CD19 transfectants also had reduced tumorigenicity in vivo when subcutaneously implanted into severe combined immunodeficiency (SCID)-human interleukin-6 (hIL-6) transgenic mice. The growth-inhibitory effect was CD19-specific and probably due to CD19 signaling because this effect was not observed in cells transfected with a truncated form of CD19 that lacks the cytoplasmic signaling domain. The in vitro growth-inhibitory effect was confirmed in a nontumorigenic human myeloma cell line (U-266). However, introduction of the CD19 gene into a human erythroleukemia cell line (K-562) also induced growth inhibition, suggesting that this effect is CD19-specific, but not restricted to myeloma cells. These data suggest that the specific and generalized loss of CD19 in human myeloma cells could be an important factor contributing to the proliferation of the malignant plasma cell clones in this disease.

Identification of immature and mature myeloma cells in the bone marrow of human myelomas.

With regard to the expression of adhesion molecules, human myeloma cells freshly isolated from bone marrow were heterogeneous. By two-color analysis with anti-VLA-5 antibody (PE staining) and FITC-labeled anti-CD38 antibody, we found all myeloma cells located at CD38-strong positive (CD38++) fraction and identified two subpopulations among these myeloma cells: CD38++ VLA-5-(VLA-5-) myeloma cells and CD38++ VLA-5+ (VLA-5+) myeloma cells. To clarify the biologic character of these two subpopulations, the morphology, in vitro proliferative activity and in vitro M-protein secretion were examined in each fraction isolated by the purification procedure or a cell sorter. Morphologic examination showed that VLA-5- myeloma cells were mostly immature or plasmablastic and VLA-5+ cells were mature myeloma cells. Furthermore, VLA-5- myeloma cells proliferated markedly in vitro and responded to interleukin 6 (IL-6), a growth factor for myeloma cells, while VLA-5+ myeloma cells showed very low uptakes of 3H-thymidine and no responses to IL-6 but secreted higher amounts of M-protein (immunoglobulin) in vitro significantly. Therefore, we could clarify here heterogeneity of human myeloma cells in the bone marrow with regard to the expression of VLA-5, one of integrin adhesion molecules; VLA-5- myeloma cells were proliferative immature cells and VLA-5+ cells were mature myeloma cells.