Preliminary results of pinpoint plantar long-wavelength infrared light irradiation on blood glucose, insulin and stress hormones in patients with type 2 diabetes mellitus.

BACKGROUND AND AIMS: This study was aimed at the development of a novel noninvasive treatment system, "pinpoint plantar long-wavelength infrared light irradiation (PP-LILI)", which may be able to relieve mental stress and normalize blood glucose level via the reduction of stress hormones in type 2 (non-insulin-dependent) diabetes mellitus (DM) patients. MATERIALS (SUBJECTS) AND METHODS: Based on this hypothesis, the present study was undertaken to examine effects of PP-LILI on stress hormones (ACTH and cortisol), blood glucose, HbA1c, and insulin levels in 10 patients with type 2 DM. Each patient received PP-LILI of the foot for 15 minutes once weekly using a stress free apparatus (infrared wavelength, 9,000-12,000 nm/power 30 mW). RESULTS: In response to this therapy, ACTH (P<0.01) and cortisol (P<0.05) levels decreased significantly. Fasting blood glucose (P<0.05) and insulin (P<0.05) levels also decreased significantly along with a tendency for HbA1c to decrease. CONCLUSIONS: The present data raise the possibility that PP-LILI can normalize blood glucose levels by reducing stress hormones such as cortisol, which aggravate DM, and by improving insulin sensitivity, thereby contributing to prevention and treatment of DM.

Effect of pinpoint plantar long-wavelength infrared light irradiation on subcutaneous temperature and stress markers.

BACKGROUND AND AIMS: The current investigation was aimed at the development of a novel non-invasive treatment system, "pinpoint plantar long-wavelength infrared light irradiation (PP-LILI)", which may be able to relieve mental stress and reduce stress-related hormones. Materials (Subjects) and methods: We compared the subcutaneous temperature, blood pressure, the degree of secretion of stress hormones before and after pinpoint irradiations (wavelength: 8-11 µm; output: 30mW). The study enrolled 15 subjects (Japanese healthy adults; 8 males, 7 females; average age 47.8 ± 14.6 years). Two parts of the planter region were irradiated for 15 min respectively. The stress markers such as ACTH, salivary amylase and cortisol were measured. As well, core body temperature and blood pressure were analyzed before and after the irradiation. RESULTS: A series of experiments revealed increased body temperature, decreased levels of blood pressure and stress markers described above after the irradiation. CONCLUSIONS: These results clearly suggest that the PP-LILI system will be quite useful for relieving stress and improvement of homeostatic functions in the body.

Screening of novel malaria DNA vaccine candidates using full-length cDNA library.

No licensed malaria vaccine exists, in spite of intensive development efforts. We have been investigating development of a DNA vaccine to prevent malaria infection. To date, we have established a full-length cDNA expression library from the erythrocytic-stage murine malaria parasite, Plasmodium berghei. We found that immunization of mice with combined 2000 clones significantly prolonged survival after challenge infection and that splenocytes from the immunized mice showed parasite-specific cytokine production. We determined the 5'-end one-pass sequence of these clones and mapped a draft genomic sequence for P. berghei for use in screening vaccine candidates for efficacy. In this study, we annotated these cDNA clones by comparing them with the genomic sequence of Plasmodium falciparum. We then divided them into several subsets based on their characteristics and examined their protective effects against malaria infection. Consequently, we selected 104 clones that strongly induced specific IgG production and decreased the mortality rate in the early phase. Most of these 104 clones coded for unknown proteins. The results suggest that these clones represent potential novel malaria vaccine candidates.

N-acetylglucosaminyltransferase V-deficiency increases susceptibility to murine malaria.

It is considered that several glycoproteins on erythrocytes in mammalian species are involved in malaria parasite infection. To elucidate the role of N-glycans on malaria parasite infection, we induced experimental murine malaria infection (using Plasmodium berghei ANKA) in mice deficient in N-acetylglucosaminyltransferase V (Mgat5), which is one of the enzymes involved in ß1,6-GlcNAc N-glycan biosynthesis. After infection, Mgat5(-/-) mice showed severe body weight loss and parasitemia compared with wild-type mice. The Mgat5(-/-) mice, but not wild-type mice, also showed severe pathology accompanied by marked infiltration of plasma cells into the lungs and liver. These results suggest that ß1,6-GlcNAc N-glycans on/in host erythrocytes may interfere with invasion of the parasites and progression to severe malaria.

Alteration of immune responses by N-acetylglucosaminyltransferase V during allergic airway inflammation.

BACKGROUND: ß-1,6-N-acetylglucosaminyltransferase V (Mgat5 or GlcNac-TV), which is involved in the glycosylation of proteins, is known to be important for down-regulation of TCR-mediated T-cell activation and negatively regulates induction of contact dermatitis and experimental autoimmune encephalomyelitis. However, the role of Mgat5 in the induction of allergic airway inflammation remains unclear. METHODS: To elucidate the role of Mgat5 in the pathogenesis of allergic airway inflammation, ovalbumin (OVA)-induced airway inflammation was induced in Mgat5-deficient mice. The OVA-specific lymphocyte proliferation and cytokine production levels, OVA-specific IgG1, IgG2a and IgE levels in the serum, and the number of leukocytes and cytokine levels in the bronchoalveolar lavage (BAL) fluid were compared between wild-type and Mgat5-deficient mice. RESULTS: OVA-specific lymphocyte proliferation and production of IFN-γ and IL-10, but not IL-4, were increased in Mgat5-deficient mice, suggesting that Th2-type immune responses are seemed to be suppressed by increased IFN-γ and IL-10 production in these mice. However, Th2-type responses such as OVA-specific IgG1, but not IgE, and IL-5 levels in BAL fluids were increased in Mgat5-deficient mice. Meanwhile, the number of eosinophils was normal, but the numbers of neutrophils, macrophages and lymphocytes were reduced, in these mutant mice during OVA-induced airway inflammation. CONCLUSIONS: Mgat5-dependent glycosylation of proteins can modulate acquired immune responses, but it is not essential for the development of OVA-induced eosinophilic airway inflammation.

Epidermal gammadelta T cells sense precancerous cellular dysregulation and initiate immune responses.

Hyperplasia associated with a loss of tissue homeostasis can induce DNA replication stress, leading to precancerous dysregulation. Epidermal gammadelta T cells reside in the primary barrier that protects against diverse environmental insults; however, the functions of these T cells in tissue surveillance are not completely understood. In mice with inducible Notch1 inactivation in keratinocytes that causes epidermal hyperplasia, epidermal gammadelta T cells sensed stressed keratinocytes and migrated into the cutaneous draining lymph nodes. Simultaneous induction of beta-galactosidase (beta-Gal) as a putative antigen expressed in the process of precancerous dysregulation and Notch1 ablation in the epidermis resulted in elevated beta-Gal-specific IgG2a production. Epidermal gammadelta T cells were found to have the capacity to express chemokine (C-C motif) receptor 7 and migrate into the lymph nodes. Cutaneous draining lymph node cells in Notch1-inactivated mice expressed high levels of IFN-gamma upon anti-CD3 plus anti-CD28 stimulation. Furthermore, induced expression of beta-Gal in mice that lacked epidermal gammadelta T cells failed to induce anti-beta-Gal IgG. These results suggest that epidermal gammadelta T cells play an essential role in the initiation process of epidermal antigen-specific humoral immune responses and demonstrate the importance of epidermal gammadelta T cells in sensing precancerous dysregulation and activating adaptive immunity.

CD4(+) T cell response in early erythrocytic stage malaria: Plasmodium berghei infection in BALB/c and C57BL/6 mice.

Plasmodium berghei ANKA causes lethal malaria in mice. It is well established that C57BL/6 mice die early with fulminant symptoms including convulsion, whereas BALB/c mice survive this phase and die later of anemia and prostration. Early death in C57BL/6 mice has been considered to result from the adverse effects of inflammatory cytokines. To elucidate the CD4(+) T cell responses in early death due to severe malaria, the kinetics of CD4(+) T cells were compared by analyzing cell surface markers and the production of cytokines and transcription factors. The results revealed that cytokine production by CD4(+) T cells was induced as early as 5 days after infection and the maintenance of higher levels of IL-4 and IL-10 may be associated with the protection of BALB/c mice from early death. These results suggest that parasite control in the early phase of infection may be important for the development of an effective vaccine.

Reconstitution of Schwannian stroma in neuroblastomas using human bone marrow stromal cells.

The Schwannian stroma in neuroblastomas is related to patient prognosis. There is debate surrounding the origin of Schwannian stroma in neuroblastomas: one theory is that the Schwann cells are derived from neoplastic cells, and the other is that they arise from normal cells surrounding the neuroblastoma. We examined whether human bone marrow stromal cells (hBMSCs) or human mesenchymal stem cells (hMSCs) could differentiate into Schwann cells in neuroblastomas. hBMSCs or hMSCs along with enhanced green fluorescent protein (EGFP) were injected into xenotransplanted neuroblastomas in nonobese diabetic mice with severe combined immunodeficiency and the resulting tumors were analyzed using immunohistochemistry. HBMSCs and hMSCs were co-cultured with neuroblastoma cells, and the induction of Schwann cell-specific molecules, S100beta and Egr-2, was monitored. S100beta-positive Schwannian stroma was observed only in neuroblastomas containing either hBMSCs or hMSCs, but not in neuroblastomas lacking these cells. Double staining with anti-S100 and anti-EGFP antibodies showed that S100-positive cells in neuroblastomas were also EGFP-positive. By contrast, hBMSCs did not develop into Schwann cells in Ewing's sarcoma, demonstrating that differentiation of transplanted hBMSCs or hMSCs into Schwann cells occurs specifically in neuroblastomas. Both S100beta and Egr-2 were expressed in hBMSCs or hMSCs co-cultured with neuroblastoma cells. HBMSCs or hMSCs may contribute to the formation of human tumor stroma. The Schwannian stroma of neuroblastomas appears to be derived from nonneoplastic stromal cells rather than neuroblastoma cells, further clarifying its developmental origins.

Two of four alternatively spliced isoforms of RUNX2 control osteocalcin gene expression in human osteoblast cells.

Runx2 is a Runt domain transcription factor that transcriptionally regulates osteoblast differentiation and bone formation. In this study, we show that human chondro- and osteosarcoma cell lines, human mesenchymal stem cells (hMSC) and a human primary chondrocytes (HC), osteoblst cells (HOb) express an intact isoform (RUNX2wt) and 3 alternatively spliced isoforms (RUNX2Delta5, Delta7, and Delta5Delta7) that are generated by skipping exon 5 and/or exon 7. Two of the truncated forms of RUNX2 (RUNX2Delta5 and RUNX2Delta5Delta7) did not localize in the nucleus and had lost their DNA binding activity. In cotransfection experiments with an osteocalcin (OC) promoter construct, we confirmed that only RUNX2wt and RUNX2Delta7 could upregulate the OC promoter activity in the osteosarcoma cell line. In addition, the coactivator CBP/p300 enhanced the transcriptional activity of the OC promoter when coexpressed with RUNX2wt or RUNX2Delta7, but not when coexpressed with RUNX2Delta5 or RUNX2Delta5Delta7. In contrast, the corepressor HDAC3 only repressed the activation from the OC promoter when coexpressed with RUNX2wt. These results support the hypothesis that RUNX2 both up- and downregulates its target gene promoters, as exemplified by the OC gene, using various isoforms and context-dependent formation of transcriptional complexes.

NF-AT-mediated expression of TGF-beta1 in tolerant T cells.

During T cell development in the thymus, a certain population of self-reactive thymocytes differentiates into regulatory T cells that suppress otherwise harmful self-reactive T cells. In transgenic mice expressing both TCR that specifically recognizes moth cytochrome c and the moth cytochrome c ligand, a large proportion of CD4+ T cells expresses CD25 and secretes TGF-beta1 upon Ag stimulation. Because TGF-beta1 expression by these T cells can be decreased by cyclosporin A, a NF-AT inhibitor, NF-AT-mediated TGF-beta1 expression in T cells was addressed by characterizing a NF-AT response element in the TGF-beta1 promoter. Analysis of the mouse TGF-beta1 promoter (-1799 to +793) in transfection experiments in T cell 68-41 hybridoma cells detected NF-AT binding sites at positions +268 and +288 in the proximal promoter region. Binding of NF-AT to this region was detected only in tolerant CD4+ T cells, but not in fully activated CD4+ T cells by chromatin immunoprecipitation assays. Activation of these NF-AT sites was sufficient to induce TGF-beta1 promoter activity; however, additional signaling due to full Ag stimulation blocked NF-AT-mediated TGF-beta1 expression. This suppression of the TGF-beta1 promoter is mediated by the -1079 to -406 region, in which deletion of a GATA-binding motif at position -821 abrogates NF-AT-mediated activation of the TGF-beta1 promoter. Therefore, TGF-beta1 expression in T cells is controlled by multiple regulatory factors that have distinct functions in response to partial or full TCR activation.

Involvement of Notch signaling in initiation of prechondrogenic condensation and nodule formation in limb bud micromass cultures.

Notch signaling is an evolutionarily conserved mechanism that plays a critical role in the determination of multiple cellular differentiation pathways and morphogenesis during embryogenesis. The limb bud high-density culture is an established model that recapitulates mesenchymal condensation and chondrocyte differentiation. Reverse transcription-polymerase chain reaction (RT-PCR) showed that Notch and its related genes were expressed in such cultures on day 1 and reached a peak between day 3 and day 5, when cell condensation and nodule formation were initiated. Immunohistochemical experiments revealed that the expression of Notch1 was initially localized within the nodules and shifted to their peripheral region as the cell differentiation progressed. We disrupted Notch signaling by using a gamma-secretase inhibitor, N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT), to analyze the function of Notch signaling in the culture system. The blocking of Notch signaling by DAPT apparently promoted the initiation of prechondrogenic condensation and fusion of the nodules, and such an effect was reversed by exogenous expression of the Notch cytoplasmic domain. DAPT treatment also induced chondrogenic markers and bone morphogenetic protein (BMP)-related molecules, including type II collagen, Sox9, GDF5, and Id1. These observations imply that the Notch signal may have an important role in chondrogenic differentiation by negatively regulating the initiation of prechondrogenic condensation and nodule formation.

A novel avian homologue of CD72, chB1r, down modulates BCR-mediated activation signals.

The avian B cell differentiation antigen chB1 is a C-type lectin membrane protein most homologous to mammalian CD72. Here, we report a new chB1-related gene, chB1r, that is located 18 kb away the chB1 gene. The cytoplasmic domain of chB1r protein contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs: ITIM1 and 2), which are identical to those found in CD72, whereas chB1 lacks the second ITIM2. Although chB1 expression is restricted to the bursa and an immature B cell line, chB1r is highly expressed in the bursa, spleen and both immature and mature B cell lines, a pattern that parallels CD72 expression. SHP-1 and Grb2 interact with phosphorylated tyrosine residues within chB1r ITIM1 and ITIM2, respectively. By contrast, ITIM1 of chB1 does not interact with SHP-1. Functional characterization using chB1r/chB1 double-deficient DT40 B cells demonstrated that ITIM1 in chB1r transduces a negative signal for BCR-mediated nuclear factor of activated T cells (NF-AT) activation and that ITIM2 attenuates this negative signal. This study has established chB1r as the genuine avian homologue of mammalian CD72, and revealed an opposing role for the two ITIMs through binding with SHP-1 and Grb2 for regulation of BCR-mediated NF-AT activation.

Use of the SST-REX method for the identification of genes expressed at the condensation stage of chondrogenic cell line ATDC5.

In endochondral ossification, chondrogenesis precedes bone development. Cartilage differentiation is initiated by the formation of chondrogenic cell condensates. Thus, it is essential to investigate genes expressed at the condensation stage for a better understanding of chondrogenesis and ossification. To this end, we constructed a cDNA library from the mRNA fraction derived from a chondrogenic cell line (ATDC5) at the cell condensation stage by using the signal sequence trap by retrovirus-mediated expression (SST-REX) method. We obtained 486 factor (IL-3)-independent clones by screening 5.7 x 10(3) clones. DNA sequencing analysis of the clones identified genes encoding 157 known proteins and 4 novel proteins. These 4 genes encoding novel proteins were expressed not only in chondrogenic ATDC5 cells but also in osteogenic MC3T3-E1 and myogenic C2C12 cells. The mRNA expression level of 1 of the 4 clones increased at the calcification stage of the differentiation of MC3T3-E1 cells. The results demonstrate that the SST-REX method is a useful experimental system to identify genes involved in the complicated mechanisms of bone formation.

1'-acetoxychavicol acetate is a novel nuclear factor kappaB inhibitor with significant activity against multiple myeloma in vitro and in vivo.

1'-Acetoxychavicol acetate (ACA) is a component of a traditional Asian condiment obtained from the rhizomes of the commonly used ethno-medicinal plant Languas galanga. Here, we show for the first time that ACA dramatically inhibits the cellular growth of human myeloma cells via the inhibition of nuclear factor kappaB (NF-kappaB) activity. In myeloma cells, cultivation with ACA induced G0-G1 phase cell cycle arrest, followed by apoptosis. Treatment with ACA induced caspase 3, 9, and 8 activities, suggesting that ACA-induced apoptosis in myeloma cells mediates both mitochondrial- and Fas-dependent pathways. Furthermore, we showed that ACA significantly inhibits the serine phosphorylation and degradation of IkappaBalpha. ACA rapidly decreased the nuclear expression of NF-kappaB, but increased the accumulation of cytosol NF-kappaB in RPMI8226 cells, indicating that ACA inhibits the translocation of NF-kappaB from the cytosol to the nucleus. To evaluate the effects of ACA in vivo, RPMI8226-transplanted NOD/SCID mice were treated with ACA. Tumor weight significantly decreased in the ACA-treated mice compared with the control mice. In conclusion, ACA has an inhibitory effect on NF-kappaB, and induces the apoptosis of myeloma cells in vitro and in vivo. ACA, therefore, provides a new biologically based therapy for the treatment of multiple myeloma patients as a novel NF-kappaB inhibitor.

Dual regulation of BCR-mediated growth inhibition signaling by CD72.

CD72 has been reported to regulate BCR-mediated signals both positively and negatively. SHP-1 and Grb2 bind, respectively, to ITIM1 and ITIM2 of CD72. We generated transformed B cell lines with an immature phenotype following J2 virus infection of splenocytes from CD72(-/-) and wild-type (Wt) mice. The transformed lines were infected with retroviral vectors carrying Tyr (Y) to Phe (F) substitutions in the ITIM sequences (ITIM1 mutated: Y7/F; ITIM2 mutated: Y39/F; and both ITIM mutated: Y7,39/F). Cross-linking of the BCR induced growth inhibition in transfectants expressing Wt CD72, but this response was less sensitive in transfectants with Y7,39/F. The Y7/F transfectants demonstrated the least sensitive response. We were not able to obtain transfectants with Y39/F, suggesting that CD72 associated with SHP-1, but not with Grb2, delivers a strong negative signal. Pre-ligation of CD72, which induces dephosphorylation of the molecule, partially rescued the Wt transfectants from growth inhibition, leading to a growth response profile similar to that of Y7,39/F transfectants. These results suggest that ITIM1/SHP-1 delivers a very strong negative signal that is down-modulated by signals through ITIM2/Grb2, leading to delivery of an attenuated negative signal. Thus, pre-ligation of CD72 results in the manifestation of an ostensible positive signal.

Catechin, a green tea component, rapidly induces apoptosis of myeloid leukemic cells via modulation of reactive oxygen species production in vitro and inhibits tumor growth in vivo.

BACKGROUND AND OBJECTIVES: The aim of this study was to investigate the possibility of green tea polyphenol, (-)-epigallocatechin-3-gallate (EGCG) as a novel therapeutic agent for the patients with myeloid leukemia. DESIGN AND METHODS: We investigated the effects of EGCG on the induction of apoptosis in leukemic cells in vitro and in vivo. We further examined the molecular mechanisms of EGCG-induced apoptosis in myeloid leukemic cells. RESULTS: EGCG rapidly induced apoptotic cell death in retinoic acid (RA)-resistant acute promyelocytic leukemia (APL), UF-1 cells within 3 h. EGCG induced apoptosis in UF-1 cells was in association with the loss of mitochondrial transmembrane potentials (Deltapsim) and activation of caspase-3 and -9. Elevation of intracellular reactive oxygen species (ROS) production was also demonstrated during EGCG-induced apoptosis of UF-1 as well as fresh myeloid leukemic cells. In NOD/SCID mice transplanted with UF-1 cells, EGCG effectively inhibited tumor growth in vivo, and the number of mitoses among the cells significantly decreased in comparison to that of control mouse cells. INTERPRETATION AND CONCLUSIONS: In summary, EGCG has potential as a novel therapeutic agent for myeloid leukemia via induction of apoptosis mediated by modification of the redox system.

Ca2+ signaling down-regulates TGF-beta1 gene expression in CD4+ T cells.

In the immune system, TGF-beta1 exerts two major functions, anti-inflammatory and immuno-suppressive effects. This work aims to investigate the molecular mechanisms involved in the regulation of the TGF-beta1 gene expression in CD4(+) T cells. The TGF-beta1 gene expresses three transcripts of 2.5, 1.9, and 1.4kb. The 1.9kb mRNA which has the highest translation activity was the major transcript. The relationship between T cell receptor (TCR) stimulation and the expression of the gene was investigated. TCR stimulation with a low dose of antigen peptide enhanced the gene expression, whereas a higher dose suppressed the expression. TCR stimulation activates PKC/MAPK and Ca(2+) signaling pathways. PMA increased the gene expression, whereas ionomycin decreased the gene expression, markedly. The results indicate that Ca(2+) signaling down-regulates TGF-beta1 gene expression. The molecular regulation of TGF-beta1 gene expression is unique when comparing to other cytokine genes which are generally activated by Ca(2+) signaling.

Hepatitis C virus infection in human liver tissue engrafted in mice with an infectious molecular clone.

BACKGROUND/AIMS: Recent advances in molecular cloning of hepatitis C virus (HCV) have enabled us to apply some available HCV molecular clones to experimental studies. However, these investigations have been restricted to chimpanzee models or 'isolated hepatocytes' from tree shrews. In this study, we engrafted 'human liver tissue' into immunodeficient mice and investigated HCV infection using an infectious molecular clone. METHODS: Human liver tissues from normal (non-HCV-infected) liver were transplanted into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. We then inoculated the mice with sera from HCV-infected patients or an infectious HCV molecular clone. HCV RNA was assessed using nested reverse-transcription polymerase chain reaction (PCR), real-time detection PCR and in situ PCR. RESULTS: Without any growth support, normal human liver tissues survived in NOD/SCID mice while maintaining the original viable hepatic architecture. HCV RNA was detected in the mice serum until the fourth week after the inoculation. In situ PCR and immunohistochemistry clearly demonstrated positive signals for HCV in the cytoplasm of infected hepatocytes, while the engrafted human liver tissues showed no apparent morphological changes indicative of infection. CONCLUSION: Engraftment of human liver tissues into NOD/SCID mice and infection with HCV molecular clones could offer a reverse genetic strategy for HCV infection.

Tumor angiogenesis in the bone marrow of multiple myeloma patients and its alteration by thalidomide treatment.

Angiogenesis in solid tumors is important to tumor growth, invasion and metastasis. Recently, it has been suggested that angiogenesis plays a certain role in the development of hematopoietic malignancies, including leukemia and multiple myeloma. We evaluated tumor angiogenesis in the bone marrow (BM) of multiple myeloma (MM) patients by calculating microvessel density (MVD) in needle-biopsy specimens obtained from 51 cases of untreated MM or monoclonal gammopathy of undetermined significance (MGUS). The MVD in the BM of donors for transplantation and patients with non-hematological diseases was calculated as a control. There was an obvious increase in MVD in the BM of MM patients, and the MVD correlated with the grade of myeloma cell invasion of the BM in the untreated MM cases. It was recently reported that thalidomide might be effective for the treatment of MM. We assessed the effect of thalidomide on angiogenesis in BM treatment of 11 patients with refractory MM. The concentration of M-protein in the serum or urine of seven of the 11 patients was reduced by at least 30% after thalidomide treatment, and MVD in the BM decreased in three of these seven cases in response to thalidomide. Increased plasma concentrations of basic fibroblast growth factor (FGF-2) and vascular endothelial growth factor (VEGF) were observed in all 11 cases before thalidomide administration and both levels were reduced after treatment with thalidomide. Augmented angiogenesis in the bone marrow of MM patients was confirmed in the present study. It seems that thalidomide is effective in the treatment of MM through the impairment of angiogenesis by decreasing FGF-2 and VEGF production. This is the first report on pathological evidence in the bone marrow of MM before and after thalidomide treatment, in Japan.