Search

Objective.To assess the utility of dose frequency adjustment of tocilizumab (TCZ) in rheumatoid arthritis (RA).Methods.Patients who received TCZ at 3-week (n = 24) or 5-week (n = 61) interval were evaluated.Results.Disease Activity Score at 28 joints based on erythrocyte sedimentation rate in the 3-week group significantly improved after 3 administrations at 3-week intervals (from 4.2 to 2.7, p = 0.001). Forty-five of the patients in the 5-week group (74%) successfully continued 5-week interval administration without disease exacerbation. Lower C-reactive protein level at TCZ initiation and shorter duration to remission achievement were key to successful dose frequency reduction.Conclusion.Adjusting the dose frequency of intravenous TCZ is a useful strategy.

The development of gefitinib (‘Iressa’, ZD1839) by targeting the EGFR tyrosine kinase is a recent therapeutic highlight. We have reported that gefitinib is antiangiogenic in vitro, as well as in vivo.In this study, we asked if the anti‐angiogenic action of gefitinib is due to a direct effect on activation of vascular endothelial cells by EGF. EGF, as well as VEGF, caused pronounced angiogene‐sis in an avascular area of the mouse cornea, and i.p. administration of gefitinib almost completely blocked the response to EGF, but not to VEGF. Immunohistochemical analysis demonstrated phosphorylation of EGFR by EGF in the neovasculature, and gefitinib markedly reduced this effect. Gefitinib also inhibited downstream activation of ERK 1/2 via EGFR in cultured microvascular endothelial (HMVE) cells. These findings suggest that the anti‐angiogenic effect of gefitinib in the vascular endothelial cells of neo‐vasculature is partly attributable to direct inhibition of EGFR activation, and that endothelial cells in malignant tumors play a critical role in the cancer therapeutic efficacy of gefitinib.

Circulating CD14+monocytes are precursors of phagocytes, such as macrophages and dendritic cells. Here we report primitive cells with a fibroblast‐like morphology derived from human peripheral blood CD14+monocytes that can differentiate into several distinct mesenchymal cell lineages. We named this cell population monocyte‐derived mesenchymal progenitor (MOMP). MOMPs were obtained in vitro from human peripheral blood mononuclear cells cultured on fibronectin in the presence of fetal bovine serum alone as a source of growth factors. MOMPs had a unique molecular phenotype–CD14+CD45+CD34+type I collagen+–and showed mixed morphologic and molecular features of monocytes and endothelial and mesenchymal cells. MOMPs were found to be derived from a subset of circulating CD14+monocytes, and their differentiation required that they bind fibronectin and be exposed to one or more soluble factors derived from peripheral blood CD14−cells. MOMPs could be expanded in culture without losing their original phenotype for up to five passages. The induction of MOMPs to differentiate along multiple limb‐bud mesodermal lineages resulted in the expression of genes and proteins specific for osteoblasts, skeletal myoblasts, chondrocytes, and adipocytes. Our findings represent the first evidence that human circulating CD14+monocytes are a source of progenitors that exhibit mesenchymal cell differentiation.

To examine whether Long‐Evans Cinnamon (LEC) rats, a mutant rat model of Wilson's disease, have a susceptibility gene(s) to hepatocarcinogenesis in addition to the causative gene, Atp7b, we established a new congenic strain, WKAH.C‐Atp7brats, in which the Atp7bgene of the LEG rats is inserted into the normal Wistar‐King Aptekman Hokkaido (WKAH) background. Hepatocellular tumors developed spontaneously in both sexes of WKAH.C‐Atp7brats, their incidence being slightly lower than that in LEG rats. Incidences of spontaneous liver tumors in LEG, WKAH.C‐Atp7band WKAH rats correlated with hepatic copper and iron concentrations. Medium‐term liver bioassay showed that LEG rats were more susceptible to the induction of glutathione S‐transferase placental form‐positive preneoplastic foci than WKAH.C‐Atp7brats, and WKAH.C‐Atp7brats were more susceptible than WKAH rats. In an N‐diethylnitrosamine (DEN)‐induced long‐term carcinogenicity study, 1) LEC rats were similarly or rather less susceptible to hepatocellular tumors than WKAH.C‐Atp7band WKAH rats, indicating that the progression of the preneoplastic foci to liver cancer in LEG rats was worse than that in WKAH.C‐Atp7band WKAH rats, 2) the incidences of kidney tumors in LEG and WKAH.C‐Atp7brats were higher than that in WKAH rats and high copper concentrations in the kidneys were observed in LEG and WKAH.C‐Atp7brats, 3) LEC rats were resistant to lung carcinogenesis. These data indicate that the susceptibility of LEG rats to liver and kidney carcinogenesis could be explained by Atp7bgene mutation and that the susceptibility to lung carcinogenesis is controlled by gene(s) other than Atp7b.

We studied the susceptibilities to N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine (BBN)‐induced urinary bladder carcinogenesis of male Long‐Evans Cinnamon (LEC), F344 and Long‐Evans Agouti (LEA) rats. Male rats (n=21) were given 0.1% BBN in their drinking water from week 6, 8 and 10 for one week, and killed in week 56. The incidences of transitional cell tumors (papillomas plus carcinomas) in BBN‐treated LEC and F344 rats were 12% and 76%, respectively (P< 0.001, experiment 1), and those in LEC and LEA rats were 11% and 95%, respectively (P< 0.001, experiment 2). When male LEC and F344 rats were given 0.1% BBN in their drinking water for 7 days, the intake of BBN and the urinary concentration of its active metabolite, N‐butyl‐N‐(3‐carboxypropyl)nitrosamine (BCPN), were higher in the LEC rats (P< 0.01). The urinary pHs of untreated LEC and F344 rats were similar between week 6 and 30. The urinary copper concentration was lower in LEC rats before jaundice than in F344 rats, but its concentrations in 28‐ and 50‐week‐old LEC rats were 1.7 and 2.3 times those in F344 rats. In a two‐stage carcinogenesis study using F344 rats, i.p. injections of cupric nitrilotriacetate increased urinary copper excretion, and inhibited BBN induced bladder carcinogenesis. In a two‐stage carcinogenesis study using LEC rats, oral administration of D‐penicillamine decreased urinary copper excretion, and increased BBN‐induced bladder cancer, although the difference was not significant. These data show that LEC rats are resistant to bladder carcinogenesis and suggest that urinary copper has a significant role in their resistance.

Spontaneous renal cell tumors in totals of 223 male and female Long‐Evans Cinnamon (LEC) rats of 51–120 weeks old, 157 male F344 rats of 51–120 weeks old, and 14 male Long‐Evans Agouti (LEA) rats of 51–70 weeks old were examined histologically. The incidences of renal cell tumors increased with age in male and female LEC rats, but no tumors developed in F344 or LEA rats. Dilated atypical tubules of the kidneys were observed at high incidence in aged LEC rats. Copper staining of LEC rat kidneys showed a positive reaction in proximal tubules of the cortex and the outer stripe of the medulla. The renal copper concentration of LEC rats reached a peak in the period of necrotizing hepatitis with renal tubular necrosis, and was higher than that in F344 rats for up to 106 weeks. In contrast, the renal iron concentration of LEC rats was lower than that in F344 rats except in the period of necrotizing hepatitis. Long‐term treatment of LEC rats with d‐penicillamine, a copper‐chelating agent, inhibited accumulation of copper, but not iron, in the kidneys, and inhibited the development of karyomegaly of proximal tubules and dilated atypical tubules. These results suggest that persistent copper accumulation after toxic necrosis of tubules is the major cause of spontaneous renal carcinogenesis in LEC rats.

To examine the effect of nongenotoxic chemicals on hepatocarcinogenesis in Long‐Evans Cinnamon (LEC) rats, we gave 6‐week‐old male and female LEC rats (n=18) weekly subcutaneous injections of d‐galactosamine hydrochloride (GalN, 300 mg/kg) in 0.9% NaCl or only 0.9% NaCl for 50 weeks, and killed them in week 62. GalN‐treated male rats unexpectedly showed no lethal necrotizing hepatitis. GalN treatment increased the incidence of cholangiofibrosis in males and its severity in females, but did not cause significant increases of hepatocellular tumors in either sex. GalN treatment increased the 5‐bromo‐2′‐deoxyuridine (BrdU)‐labeling index of hepatocytes and plasma hepatocyte growth factor, and accelerated megalocytic alterations without reduction of the hepatic copper concentration. Next, male and female LEC rats were subjected to two‐thirds partial hepatectomy (PH) or sham hepatectomy in week 8 (n=12) or in week 14 (n=9), and killed in week 62. PH in week 14 inhibited lethal hepatitis, but PH in week 8 was less effective. PH reduced the hepatic copper concentration to half that of controls. The present data suggest that induction of hepatocyte regeneration by repeated injections of GalN, or by PH just before the onset of jaundice has a significant effect in prevention of hepatic injury of LEC rats, but not enhancement of spontaneous hepatocarcinogenesis.

We reported that LEC rats are genetically deficient in the development of thymic CD4+8−cells and that this defect is caused by bone marrow (BM)‐derived stem cells. To determine which BM‐derived cells are responsible for the arrest of T‐cell development in LEC rats, fetal thymuses of LEC rats, or LEA rats which bear the same major histocompatibility complex (MHC) as LEC rats but are immunologically normal, were engrafted under the kidney capsule of severe combined immunodeficiency (SCID) mice (LEC‐TG and LEA‐TG mice, respectively). We then examined the differentiation of T cells and their immunological functions in the SCID mice. A large number of rat‐derived CD4+T cells appeared in the peripheral blood, lymph nodes (LN) and spleens in LEC‐TG mice. Furthermore, the peripheral LN cells in LEC‐TG mice appeared to be functional. These cells produced IL‐2 upon Con A stimulation, whereas LN cells from LEC rats produced no IL‐2 in the same conditions. Thymopoiesis was observed at 3 weeks in LEC‐TG as well as LEA‐TG mice. The distribution of thymocyte subsets with respect to CD4 and CD8 expression in LEC‐TG mice closely resembled that of LEA rat thymus and that in LEA‐TG mice, suggesting that normal T‐cell differentiation occurred in LEC‐TG mice. The results indicated that BM‐derived progenitor T cells of LEC rats could differentiate to functional CD4+T cells.

We have recently reported the presence of a novel perchloric acid soluble protein in rat liver (PSP1) that inhibits cell-free protein synthesis in a rabbit reticulocyte system. While studying the perchloric acid soluble proteins from different tissues of rats, we found that the kidney protein cross-reacted with antibody against the PSP1. In this investigation, we have purified a perchloric acid soluble protein from the rat kidney and studied its characterization and expression. The protein extracted from the postmitochondrial supernatant fraction with 5% perchloric acid was purified by ammonium sulfate fractionation and CM-Sephadex chromatography. By immunoscreening with the rabbit antisera against the PSP1, we detected a cDNA that contained an open reading frame of 411 bp, encoding a 137 amino-acid protein with a molecular mass of 14,149 daltons. The deduced amino acid sequence was completely identical with that of PSP1 from rat liver. The perchloric acid soluble protein from rat kidney (K-PSP1) also inhibited cell-free protein synthesis in the rabbit reticulocyte lysate system in a different manner than RNase A. Immunohistochemistry showed that the expression of K-PSP1 increased from fetal 17th day to postnatal 4th week, and it remained almost the same until the 7th week of postnatal age. Furthermore, the expression of K-PSP1 in the kidney of the nephrotic rat model was shown to be differentiation dependent. On the other hand, the expression of K-PSP1 in renal tumor cells was downregulated as compared with intact tissue. These results suggest that the expression of K-PSP1 is regulated in a differentiation-dependent manner in the kidney.

A case of primary endometrial squamous cell carcinoma (ESCC) in a 75-year-old woman is reported. In her endometrium, mature stratified squamous epithelium was associated with mucinous glands in which epithelium and glands were identical to those of the uterine cervix, but which were not connected with the cervix. Thus, the endometrial squamous epithelium with mucinous glands was diagnosed as heterotopic cervical tissue. This heterotopia was surrounded by ESCC and connected with it. The present case suggests that some ESCCs may arise in heterotopic cervical epithelium in the endometrium.

The carcinogenicity of 1,2-dimethylhydrazine dihydrochloride (DMH) by oral, intragastric and subcutaneous administration was examined in 339 BALB/c mice. Subcutaneous injection of DMH induced intestinal tumors in the lower colon of all mice. After oral administration it induced a high incidence of vascular tumors in the liver and soft tissues, but colon tumors were found in only 2 mice when given at a high dosage. On intragastric administration, it induced a fairly high incidence both of colon and vascular tumors. The sites and incidences of vascular tumors and squamous cell carcinomas of the perianal glands were also described.

A mutant strain of Long-Evans cinnamon (LEC) rats was found to have a novel defect in T-cell maturation, i.e., arrest of differentiation from CD4⁺8⁺ to CD4⁺8⁻, but not to CD4⁻8⁺ thymocytes. Despite the maturational arrest of CD4⁺8⁻ thymocytes, some CD4⁺ T cells occurred in peripheral lymphoid organs in LEC rats. In the present study, expression of adhesion molecules was compared between normal and LEC rat T cells. Three-color flow cytometry analysis showed that the expression pattern of LFA-1 and LECAM-1 of LEC rat CD4⁺ T cells was different from that of normal rat CD4⁺ T cells; F344 rat CD4⁺ T cells were LFA-1⁺, LECAM-1⁺⁺, whereas LEC rat CD4⁺ cells were LFA-1⁺⁺, LECAM-1⁻. However, the expression pattern of these adhesion molecules on LEC rat CD8⁺ T cells was the same as that on F344 rat CD8⁺ T cells, suggesting that LEC rat T cells do not have an intrinsic defect in the expression of these molecules. To address the question of whether the abnormal expression of adhesion molecules is correlated with the thid mutation, we generated WKAH.C-thid congenic rats and analyzed these expression patterns. The expression of adhesion molecules on WKAH.C-thid congenic rat CD4⁺ T cells was the same as that of WKAH rat CD4⁺ T cells, suggesting that abnormal expression of adhesion molecules of LEC rat CD4⁺ T cells is independent of the thid mutation. These results indicate that LEC rat CD4⁺ T cells show a unique expression of adhesion molecules, and these phenotypes are independent of the thid mutation that causes maturational arrest of CD4⁺8⁻ thymocytes. J. Trace Elem. Exp. Med. 10:81–88, 1997. © 1997 Wiley-Liss, Inc.

We generated transgenic mice homozygous for the tyrosine kinase-deficient human insulin receptor (hIRK1030M(+/+)) under control of the insulin receptor promoter. Similar growth patterns and results of glucose tolerance tests were observed among normal, heterozygous, and homozygous mice. Insulin tolerance test indicated no significant difference in the hypoglycemic response to insulin among the three genotypes. However, the serum insulin levels of the homozygous mice before and after glucose loading (201.42 ± 58.15 pg/ml to 578.57 ± 49.03 pg/ml) were significantly higher than in the control mice (100.92 ± 19.55 pg/ml to 356.36 ± 55.08 pg/ml; p<0.01 and p<0.01, respectively) and heterozygous mice (74.46 ± 18.55 pg/ml to 352.33 ± 52.43 pg/ml; p<0.005 and p<0.01, respectively). Immunohistological evidence of pancreatic islets showed no significant difference among the three genotypes. Taken together, these results suggest that the tyrosine kinase-deficient insulin receptor causes hyperinsulinemia but not diabetes in these homozygous transgenic mice.

Peanut agglutinin was used in a lectin-anti-lectin immunoperoxidase technique to assess the status of T antigen-like substance in histological sections of benign hyperplasia and adenocarcinoma of the prostate. Of 30 benign lesions 26 (86.7 per cent) had negative staining in the glandular epithelia and 4 showed uneven staining of the epithelial cells in a small proportion of the glands. Additionally, 15 of 30 benign lesions (50 per cent) had positive staining restricted to the basal myoepithelial cells in a variable proportion of the glands. All benign lesions demonstrated the presence of normal cryptic T antigen after neuraminidase digestion. Of 25 adenocarcinomas 14 (56 per cent) were positive and 7 (25 per cent) were negative for T antigen but demonstrated the presence of normal cryptic T antigen after neuraminidase digestion, and 4 (16 per cent) were negative for T and cryptic T antigens. Of 8 grade 1, 9 grade 2 and 8 grade 3 tumors, and 9 tumors from patients with bone metastases 3 (37.5 per cent), 8 (88.9 per cent), 7 (87.5 per cent) and 7 (77.8 per cent), respectively, were positive for T antigen or negative for T and cryptic T antigens.

A number of cancer genes have been identified by the study of hereditary human cancers and shown to be involved in sporadic genesis of the same tumors. We have identified a germline mutation in the rat homologue of the human tuberous sclerosis (TSC2) predisposing gene in the Eker rat model. In this study, we searched for mutations of the Tsc2 gene in chemically induced non-Eker rat renal cell carcinomas (RCs). N-ethyl-N-hydroxyethylnitrosamine (EHEN)- and diethylnitrosamine (DEN)-induced non-Eker rat primary RCs were subjected to polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis using specific primers covering all exons of the Tsc2 gene (41 coding exons and 1 non-coding exon). We simultaneously searched for mutations in the Vhl gene, a rat homologue of von Hippel-Lindau disease (VHL) gene, as well as the Tsc2 gene. Mutations in the Vhl gene were not detected in any rat RCs (0/8). In contrast, Tsc2 gene mutations were detected at a high frequency in EHEN-induced RCs (2/3) and DEN-induced RCs (3/5) (total 5/8) (p &lt; 0.05). By a direct cloning approach utilizing PCR analysis in 2 applicable cases, we could demonstrate the presence of intragenic somatic mutations in both alleles of the Tsc2 gene. Our results suggest that Tsc2 gene inactivation plays an important role in EHEN- and DEN-induced RCs as well as in Eker rat RCs. Int. J. Cancer 77:895–900, 1998.© 1998 Wiley-Liss, Inc.

Abstract.: Genetic maps are the primary resources for genetic study. Genetic map construction was quite difficult in the past decade for lack of polymorphic markers. This situation has been changed since the development of microsatellite markers or simple sequence length polymorphisms (SSLPs) because they are abundant and more polymorphic. Here we report the construction of an integrated genetic map of the rat derived from two F2 intercrosses. A map of 376 markers from 160 (OLETF × F344)F2 progenies and a map of 333 markers from 71 (F344 × LEC)F2 animals are integrated by use of common set of 120 anchor markers chosen to be spaced at an average of 15 cM in the genome. The resulting integrated map with 194 newly developed rat markers from WIBR/MIT CGR, 269 Mit/Mgh markers, 94 Wox markers, and 5 markers of various origins covers the majority of 21 chromosomes of the rat with a total genetic distance of 1797 cM and an average marker spacing of 3.2 cM. The current map provides detailed information for markers from different sources and, therefore, should be helpful to the research community.

Immunoperoxidase localization of carcinoembryonic antigen was done on tissue sections of benign hyperplasia and adenocarcinoma of the prostate using murine monoclonal antibody against carcinoembryonic antigen. The study was done to determine whether carcinoembryonic antigen would be a tissue marker of prostatic malignancy. Of 30 benign lesions 25 (83 per cent) were negative and 5 had focally weak staining, while of 36 adenocarcinomas 31 (86 per cent) had diffusely positive staining and 5 were negative. Diffusely moderate or strong staining was noted in 5 of 10 grade I (50 per cent), 11 of 12 (92 per cent) grade II and 13 of 14 (93 per cent) grade III tumors, as well as 14 of 15 (93 per cent) tumors from patients with bone metastasis.

Solitonic solution-generating methods are powerful tools to construct nontrivial black hole solutions of the higher-dimensional Einstein equations systematically. In five dimensions particularly, the solitonic methods can be successfully applied to the construction of asymptotically Minkowski spacetimes with multiple horizons. We review the solitonic methods applicable to higher-dimensional vacuum spacetimes and present some five-dimensional examples derived from the methods.

We revisit the problem of the helicity-0 ghost mode of a massive graviton in the de Sitter background. In general, the presence of a ghost particle, which has negative energy, drives the vacuum to be unstable through the pair production of ghost particles and ordinary particles. In the case that the vacuum state preserves the de Sitter invariance, the number density created by the pair production inevitably diverges owing to unsuppressed ultraviolet (UV) contributions. In such cases, one can immediately conclude that the model is not viable. However, in the massive gravity theory, we cannot construct a vacuum state that respects the de Sitter invariance. Therefore, the presence of a ghost does not immediately mean the breakdown of the model. Explicitly estimating the number density and the energy density of particles created by the pair production of two conformal scalar particles and one helicity-0 ghost graviton, we find that these densities both diverge. However, since models with a helicity-0 ghost graviton have no de Sitter invariant vacuum state, it is rather natural to consider a UV cutoff scale in the three-dimensional momentum space. Then, even if we take a cutoff scale as large as the Planck scale, the created number density and energy density are well suppressed. In many models, the cutoff scale is smaller than the Planck scale. In such models, the created number density and energy density are negligible as long as only the physics below the cutoff scale is concerned.

In this paper, we point out that the massive gravity theory with a graviton ghost mode in the de Sitter background cannot possess a de Sitter invariant vacuum state. In order to avoid a negative norm state, we must associate the creation operator of the ghost mode with a negative-energy mode function instead of a positive-energy one as the mode function. Namely, we have to adopt a different procedure of quantization for a ghost. When a theory has a symmetry mixing a ghost mode with ordinary nonghost modes, the choice of a ghost mode is not unique. However, the quantization of a ghost is impossible without specifying a choice of ghost mode, which breaks the symmetry. For this reason, the vacuum state cannot respect the symmetry. In the massive gravity theory with a graviton ghost mode in the de Sitter background, the ghost is the helicity-0 mode of the graviton. This ghost mode is mixed with the other helicity graviton modes under the action of de Sitter symmetry. Therefore, there is no de Sitter invariant vacuum in such models. This leads to an interesting possibility that the non-covariant cutoff of the low-energy effective theory may naturally arise. As a result, the instability due to the pair production of a ghost and normal non-ghost particles gets much milder and that the model may escape from being rejected.

We construct a five dimensional exact solution of the orthogonal black di-ring which has two black rings whose S1-rotating planes are orthogonal. This solution has four free parameters which represent radii of and speeds of S1-rotation of the black rings. We use the inverse scattering method. This method needs the seed metric. We also present a systematic method how to construct a seed metric. Using this method, we can probably construct other solutions having many black rings on the two orthogonal planes with or without a black hole at the center.

Recently, we have identified a primitive cell population termed monocyte-derived multipotential cells (MOMCs), which have a fibroblast-like morphology and a unique phenotype positive for CD14, CD45, CD34, and type I collagen. MOMCs are derived from in vitroculture of circulating CD14+monocytes, and contain progenitors capable of differentiating into a variety of mesenchymal cells, neuron, and endothelium. Thus, human MOMCs are attractive candidates for an autologous transplantable cell source for therapeutic tissue regeneration. In vitroinduction of MOMCs from circulating CD14+monocytes requires their binding to matrix proteins such as fibronectin, and exposure to soluble factors derived from peripheral blood CD14−cells, but detailed processes remain unknown. Here, we investigated molecular factors involved in MOMC induction using human peripheral blood monocyte cultures. Induction of MOMCs was defined as having all of features, including fibroblastic morphology, CD34+phenotype, and potentials of differentiating into multiple cell lineages. First, we set up MOMC cultures on fibronectin, type I collagen, laminin, or poly-L-lysine, and found that MOMCs were efficiently obtained exclusively in the presence of fibronectin. Since fibronectin is a ligand for β1 integrins, we examined effects of blockade of these interactions on MOMC generation. Induction of MOMCs was completely inhibited by anti-α5-integrin monoclonal antibody, but not by anti-α4-integrin antibody. In addition, a synthetic peptide that competed with the RGD domain of fibronectin suppressed generation of MOMC, but a CS-1 domain peptide had no effect, indicating that binding of α5β1integrin to the RGD domain of fibronectin is required for monocytes to acquire a multipotential property. Next, highly enriched monocytes were cultured on fibronectin with various peripheral blood cell subsets to identify cells producing soluble factors required for MOMC generation. As a result, MOMCs were obtained exclusively in the presence of platelets. MOMC induction was also observed when conditioned medium prepared by stimulating platelets with various agonists, such as thrombin and ADP, was used instead of whole platelets. When platelet-derived condition medium was fractionated based on the molecular weight (MW), MOMC induction activity was retained in the fraction of MW < 30,000. Then, we selected a list of 16 candidate factors that are released from activated platelets and < 30,000 in MW, and screened them using two different strategies: evaluation of MOMC induction efficiency in monocyte cultures by adding individual candidate factors; and FACS sorting of circulating monocytes into cells with high and low expressions of receptor for individual candidate factors, followed by evaluation of MOMC induction. As a result, SDF-1was selected as a sole factor with capacity to promote generation of MOMCs. Moreover, MOMC generation was significantly more prominent in cultures with monocytes with high expression of CXCR4, a receptor for SDF-1, than in cultures of monocytes with low CXCR4 expression. In summary, circulating monocytes are able to transdifferentiate into multipotential cells through the fibronectin-α5β1integrin and SDF-1-CXCR4 interactions. This information is helpful in establishing an optimal MOMC culture condition used in cell transplantation for tissue regeneration.

Recently, we have identified a primitive cell population termed monocyte-derived multipotential cells (MOMCs), which have a fibroblast-like morphology and a unique phenotype positive for CD14, CD45, CD34, and type I collagen. MOMCs are derived from in vitro culture of circulating CD14+ monocytes, and contain progenitors capable of differentiating into a variety of mesenchymal cells, neuron, and endothelium. Thus, human MOMCs are attractive candidates for an autologous transplantable cell source for therapeutic tissue regeneration. In vitro induction of MOMCs from circulating CD14+ monocytes requires their binding to matrix proteins such as fibronectin, and exposure to soluble factors derived from peripheral blood CD14− cells, but detailed processes remain unknown. Here, we investigated molecular factors involved in MOMC induction using human peripheral blood monocyte cultures. Induction of MOMCs was defined as having all of features, including fibroblastic morphology, CD34+ phenotype, and potentials of differentiating into multiple cell lineages. First, we set up MOMC cultures on fibronectin, type I collagen, laminin, or poly-L-lysine, and found that MOMCs were efficiently obtained exclusively in the presence of fibronectin. Since fibronectin is a ligand for β1 integrins, we examined effects of blockade of these interactions on MOMC generation. Induction of MOMCs was completely inhibited by anti-α5-integrin monoclonal antibody, but not by anti-α4-integrin antibody. In addition, a synthetic peptide that competed with the RGD domain of fibronectin suppressed generation of MOMC, but a CS-1 domain peptide had no effect, indicating that binding of α5β1 integrin to the RGD domain of fibronectin is required for monocytes to acquire a multipotential property. Next, highly enriched monocytes were cultured on fibronectin with various peripheral blood cell subsets to identify cells producing soluble factors required for MOMC generation. As a result, MOMCs were obtained exclusively in the presence of platelets. MOMC induction was also observed when conditioned medium prepared by stimulating platelets with various agonists, such as thrombin and ADP, was used instead of whole platelets. When platelet-derived condition medium was fractionated based on the molecular weight (MW), MOMC induction activity was retained in the fraction of MW < 30,000. Then, we selected a list of 16 candidate factors that are released from activated platelets and < 30,000 in MW, and screened them using two different strategies: evaluation of MOMC induction efficiency in monocyte cultures by adding individual candidate factors; and FACS sorting of circulating monocytes into cells with high and low expressions of receptor for individual candidate factors, followed by evaluation of MOMC induction. As a result, SDF-1was selected as a sole factor with capacity to promote generation of MOMCs. Moreover, MOMC generation was significantly more prominent in cultures with monocytes with high expression of CXCR4, a receptor for SDF-1, than in cultures of monocytes with low CXCR4 expression. In summary, circulating monocytes are able to transdifferentiate into multipotential cells through the fibronectin-α5β1 integrin and SDF-1-CXCR4 interactions. This information is helpful in establishing an optimal MOMC culture condition used in cell transplantation for tissue regeneration.

Infrared-laser-assisted reactions of O2with a graphite surface were studied with laser-ionization mass spectroscopy. CO was observed as a reaction product of O2and graphite. A time profile of CO was measured for the first time, and the mean translational temperature of CO was measured to be 790±80 K. The translational temperatures of carbon clusters of C, C2and C3evaporated from the surface were also measured.