Biomedical subjects

Kimihiro Yamashita

Publications and source records attributed to Kimihiro Yamashita.

At least 19 recordsLinked to original sources

PubMed2026

Translational Gap in Biomarker Discovery: Tumor Surface Markers Rarely Mirror Circulating Levels.

BACKGROUND: Tumor-associated cell surface proteins are frequently proposed as circulating biomarkers for colorectal cancer (CRC) based on their high tumor expression. However, many candidates identified through tissue-based analyses fail to translate into clinically useful biomarkers. We investigated the translational gap between tissue-level expression and circulating detectability in CRC, focusing on molecular subtypes defined by caudal-type homeobox 2 (CDX2) expression. METHODS: Transcriptomic data from The Cancer Genome Atlas (TCGA) were analyzed to identify cell surface markers differentially expressed between CDX2-Low and CDX2-High CRCs. A clinical cohort of right-sided CRC patients was evaluated using paired tumor tissue and preoperative plasma samples. CDX2 expression was assessed by immunohistochemistry, and circulating concentrations of selected cell surface proteins were quantified using a multiplex ELISA platform. RESULTS: Several tumor-associated cell surface markers exhibited marked CDX2-dependent differences in tissue expression. However, for most markers, circulating plasma levels did not mirror tissue-level patterns. CEACAM1 was the sole marker demonstrating concordant CDX2-dependent differences in both tumor tissue and plasma, with significantly lower levels in CDX2-Low CRCs. In contrast, CEACAM5 showed a dissociation between tissue expression and circulating levels, despite analytical validation against serum carcinoembryonic antigen (CEA). CONCLUSIONS: Our findings demonstrate that tumor overexpression of cell surface markers does not necessarily translate into detectable circulating biomarkers. This translational disconnect underscores limitations of biomarker selection strategies based solely on tissue expression and highlights the importance of integrating systemic biology into biomarker development. While some tumor-associated proteins may lack utility as circulating biomarkers, they may still represent viable therapeutic targets in CRC.

CDX2↗

PubMed2007

Hydrothermal crystallization of carbonate-containing hydroxyapatite coatings prepared by radiofrequency-magnetron sputtering method.

Carbonate-containing hydroxyapatite (HA) films were prepared by low-temperature hydrothermal annealing from carbonate-containing calcium phosphate amorphous coatings on titanium substrates. The biocompatibility of the carbonate-containing HA layers was estimated by in vitro tests using simulated body fluid (SBF). Precursory amorphous coatings were deposited with rf-magnetron sputtering apparatus, using calcium phosphate glass target in Ar/CO2 atmosphere. The carbonate-containing HA coatings were successfully formed by the annealing at above 130 degrees C for 20 h. On the basis of SEM observation, about 2-microm thickness films coated rigidly were durable enough for the hydrothermal treatment. The coating layer was revealed to consist of single phase of PO4(-) and OH- partially carbonated HA by XRD and IR analyses. Overgrowing of bone-like apatite layers on the carbonate-containing HA surfaces in the SBF implied that the obtained films acquired a sufficient osteoconductivity, while it was still unclear that activity was enhanced, compared to pure HA coatings. The low-temperature hydrothermal annealing method was effective for preparation of rigid HA coatings on titanium as well as modification of their chemical compositions.

Body Fluids↗

PubMed2007

Interaction of a blood coagulation factor on electrically polarized hydroxyapatite surfaces.

Although the polarization treatment of hydroxyapatite (HA) remarkably enhances the osteoconductivity, the mechanisms have not yet been completely understood. The interaction of proteins in blood and tissue fluids with biomaterials are reportedly triggers for later cellular responses and played a major role in osteoconductive processes. Considering this, we disclosed the interaction of polarized HA surface with a coagulation factor, fibrin stabilizing factor XIII (FXIII). The HA activated FXIII even in Ca2+ free buffer, based on the SDS-PAGE detections of alpha-polymer and gamma-dimer bands assigned to stabilized fibrin. The Ca2+ ions, possibly released from the HA surfaces, were examined whether they initiate the activation of the FXIII. It was experimentally proved by ICP analysis that the induced large negative charges on the electrically polarized HA significantly increased the released Ca2+ concentration for the short pre-incubation time of 3 min. The more Ca2+ ions released from the negatively charged HA (N-HA) surfaces were more effective in the activation of the FXIII, resulting in the rapider disappearance of the gamma-chain bands in fibrin. The slightly lower Ca2+ concentration in the positively charged HA, compared to the nonpolarized HA activated the FXIII at an almost equal rate. The accelerated activation contributed to the stabilization of fibrin scaffold. Therefore, the polarity difference of the induced charges of the polarized HA surface altered the rate of the FXIII activation. The early stage interaction of the HA surfaces with blood proteins was considered to be an essential process of the accelerated new bone formation near implanted N-HA surface.

Bone Substitutes↗

PubMed2006

Role of blood coagulation components as intermediators of high osteoconductivity of electrically polarized hydroxyapatite.

The immediate interactions of an implanted hydroxyapatite (HA) surface and blood coagulation components were detected in vivo, and the mechanism of the enhanced osteoconductivity caused by electrical polarization was discussed. Fibrin was presented as being a key protein in the early stages of osteoconduction. Scanning electron microscope observation and immunohistochemical detection indicated that fibrin adsorption is accelerated on negatively charged surfaces (N-surfaces) and on positively charged surfaces (P-surfaces) of implanted polarized HA. The acceleration of fibrin adsorption is caused by the ionic and pH changes near the N- and P-surfaces by the attraction of calcium ions. The adsorbed fibrin formed a network scaffold for subsequent cell components, such as platelets and osseous cells. Near the N-surface, the higher concentration of calcium ions than that on the conventional nonpolarized surface (O-surface) and P-surface contributed to the adhesion of the platelets to the fibrin through integrin alpha(IIb)beta(3) and platelet activation. The activated platelets release a variety of growth factors that stimulate the osseous cells. These continuous reactions from the action of the fibrin adsorption as a trigger induced the early osteoconduction near the N-surface. The coagulation components played an important role at an early stage of the osteoconductive mechanism.

Animals↗

PubMed2006

Enhanced bone ingrowth into hydroxyapatite with interconnected pores by Electrical Polarization.

Hydroxyapatite (HA) ceramics are used as implants to repair damaged/removed bone, and negative or positive electrical polarization enhances osteoblast and decreases osteoclast activity, respectively, in vivo. We compared the ability of electrically polarized and non-polarized HA with interconnected pores (IPHA) implants to promote bone growth. Polarized or non-treated IPHAs were implanted into the right or left femoral condyle of rabbits (N = 10 in each group), and we performed histological examination, including enzymatic staining for osteoblasts and osteoclasts, 3 and 6 weeks after implantation. We observed improved bone ingrowth and increased osteoblast activity in polarized implants with complete bone penetration into polarized implants occurring as early as 3 weeks after surgery. In contrast, non-polarized implants were not fully ossified at 6 weeks after surgery. Furthermore, positively charged implant regions had decreased osteoclast activity compared to negatively charged or uncharged regions. We propose two different models to explain these observations.

Animals↗

PubMed2006

Expression of MCP-1 in the hippocampus of SHRSP with ischemia-related delayed neuronal death.

1. The expression of monocyte chemoattractant protein-1 (MCP-1) was examined in stroke-prone spontaneously hypertensive rats with transient global ischemia in order to study the involvement of the infiltration of blood monocytes in the mechanism of ischemia-related neuronal death. 2. The brains of the animals with occlusion of the bilateral carotid arteries for 10 min were removed at 8 h, 1, 2, 4 and 7 days after reperfusion. Frozen sections were used for in situ hybridization and tissue specimens from the hippocampus and the cerebral cortex were used to measure the concentration of MCP-1 by ELISA. 3. No MCP-1 mRNA was detected in the hippocampus of the sham group animals. One day after ischemia-reperfusion, MCP-1 mRNA was clearly expressed in the CA4 subfield and the molecular layer of the dentate gyrus, while it was slightly expressed in the lacnosum moleculare of the CA1 subfield. A dramatic expression was demonstrated in the entire CA1 subfield at 2 days after the operation. Most of the cells expressing MCP-1 were astrocytes. At 4 and 7 days after reperfusion, no MCP-1 mRNA was detected in the hippocampus. The concentration of MCP-1 protein dramatically increased in the hippocampus at 2 days after reperfusion. 4. Taken together with the findings of our previous study showing an increased permeability of the blood-brain barrier in the hippocampus from 12 h after ischemia-reperfusion, the astrocytes expressing MCP-1 might therefore induce the migration of monocytes into the brain parenchyma. As a result, such astrocytes expressing MCP-1 may therefore be related to the pathological events of delayed neuronal death in the pyramidal neurons.

Animals↗

PubMed2006

Chromosomal numerical abnormality profiles of gastrointestinal stromal tumors.

BACKGROUND: Biological variations in and the heterogeneity of gastrointestinal stromal tumors (GISTs) are well known, but chromosomal numerical abnormality (CNA) has not been fully examined especially in this context. The aim of this study is to test CNA as a possible biological predictor of biological behavior of GISTs. METHOD: We applied microwave-assisted FISH protocol to pathological archives of GIST tumors displaying different clinical features to characterize the CNA profile of these tumors. A panel of 18 centromere enumeration probes (CEP) and 24 bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC) probes containing genes like Aurora kinases (AURKs) and other candidate genes involved in human carcinogenesis were used. CNA profiles, histopathological risk categorization and Ki-67 labeling indexes of 23 primary and/or metastatic GIST tumors of 12 subjects (both primary and metastatic in 7 subjects) were compared between primary GIST with and without metastases, and between metastatic and primary portions in 7 individuals. RESULTS: CNA in the primary sites was more extensive in the GISTs with recurrence and metastasis than in those without, especially as to the loss of chromosome 20 and genomic imbalance of AURKA-containing BAC probe on 20q in the cases with metastasis. The consistent loss of one allele of chromosome 14q was also noted. Interestingly, both primary and metastatic tumors in identical individuals had similar CNA profiles. CONCLUSION: The extent of CNA differed between GISTS with and without recurrence or metastasis; thus, FISH analysis of specimens from the primary sites may predict the biological behavior of this tumor.

Adult↗

PubMed2006

Bcl6 controls granzyme B expression in effector CD8+ T cells.

Bcl6, a sequence-specific transcriptional repressor, is important for generation and maintenance of memory CD8(+) T cells. Although memory CD8(+) T cells are generated from effector CD8(+) T cells, a role for Bcl6 in effector CD8(+) T cells is largely unknown. We show here that Bcl6 expression was transiently induced in activated CD8(+) T cells and continuously up-regulated in effector CD8(+) T cells. The amount of granzyme B mRNA among effector molecules produced by effector CD8(+) T cells inversely correlated with the amount of Bcl6 mRNA in CD8(+) T cells. Overexpression of Bcl6 in CD8(+) T cells resulted in lower killing activity at their effector phase, supporting the reduction of granzyme B expression in effector CD8(+) T cells by Bcl6. We identified a putative Bcl6-binding DNA sequence in the promoter region of the granzyme B gene. Binding of Bcl6 to the Bcl6-binding sequence was detected in naive CD8(+) T cells but not in activated CD8(+) T cells by chromatin immunoprecipitation assay. Furthermore, the Bcl6-binding sequence was required for Bcl6 to repress the luciferase reporter gene expression controlled by the granzyme B promoter. Thus, the granzyme B gene is a molecular target of Bcl6 in effector CD8(+) T cells.

Animals↗

PubMed2006

Specific response of osteoblast-like cells on hydroxyapatite layer containing serum protein.

Accelerations of bone-like apatite deposition and cell growth on an electrically polarized ceramic hydroxyapatite have been reported. A relationship between these phenomena was investigated in a previous report, and then it was suggested that osteoblast-like cell's (MC3T3-E1) growth had relevance to the mineral growth. The effect of the formed apatite layer especially appeared to be on the cell adhesion. The acceleration of cell proliferation on the polarized HAp has been shown using fibroblastic cell (L929) and nerve cell (SK-N-SH) lines, therefore the effect of the layer on L929 and SK-N-SH was investigated to support the mechanism of acceleration of cell proliferation by polarization of HAp. In this study, the effect of the bone-like apatite layer was not confirmed on L929 cell's growth. On the other hand, the acceleration of nerve cell's proliferation was confirmed on the formed apatite layer. However, the remarkable improvement of the cell adhesion of SK-N-SH was not confirmed on the apatite layer. Consequently, it was considered that the bone-like apatite containing serum protein obtained by the coprecipitation of bone-like apatite and serum protein has a pronounced role only in the activity of osteoblast-like cells.

3T3 Cells↗

PubMed2006

Immunohistochemical and genetic features of gastric and metastatic liver gastrointestinal stromal tumors: sequential analyses.

Metastatic gastrointestinal stromal tumors (GIST) have an extremely poor prognosis; however, their immunohistochemical and genetic features have not been assessed satisfactorily and the mechanisms responsible for their high malignant potential remain unclear. We examined the immunohistochemical differences between gastric GIST and metastatic lesions in the liver of four patients who had undergone a postgastrectomy hepatectomy for metachronous liver metastases. We also carried out genetic analysis of the tumors in three of the four cases. In all cases, the immunoreactivity profiles, including KIT (CD117), CD34, smooth muscle actin (SMA), desmin, S-100 and vimentin, were similar between the gastric and metastatic tumors, but the Ki67 labeling index in the metastatic GIST was higher than that of the primary GIST. Interestingly, in the case who had received neoadjuvant imatinib therapy before gastrectomy, its therapeutic effect was observed in most of the primary lesion, with the exception of a specific small area with high cellularity. Genetic analysis revealed no acquired mutations in the c-kit or PDGFRA genes in the metastatic lesions in any of the patients, but loss of heterozygosity (LOH) of the c-kit gene was observed mainly in the metastatic tumors in two of the three cases. Furthermore, in the case of neoadjuvant imatinib therapy, LOH of the c-kit gene was shown in the high cellularity area in the primary lesion and metastatic liver GIST. It is suggested that LOH of the c-kit gene is an important event that leads to imatinib resistance and metastatic progression of GIST. In conclusion, both gastric and metastatic GIST had almost the same immunohistochemical features, except for their proliferative activity, and LOH of the c-kit gene played an important role in the process of liver metastasis.

Aged↗

PubMed2006

Enhanced bone regeneration by electrical polarization of hydroxyapatite.

We examined osteogenic cell responses and new bone formation on electrically polarized hydroxyapatite (HAp) plates implanted into calvarial bone defects in rats. In the first group of rats, test pieces were placed with their positively charged surfaces face down on the dura mater. In a second group, test pieces were placed with their negatively charged surfaces facedown on the dura mater. A third group received noncharged test pieces. Histological examination was carried out to characterize the newly formed bone as well as quantification. Enzyme histochemistry involving the detection of alkaline phosphatase and tartrate-resistant acid phosphatase was performed to quantify osteogenic cell activity. Bone growth was enhanced in the groups that received polarized HAp plates, both at the negatively and positively charged surfaces. In addition to the electrostatic force that attracts Ca2+ to the negatively charged surfaces, the suppressive effects on osteoclasts proliferation on the positively charged surfaces may enhance bone formation.

Animals↗

PubMed2005

Electrical polarization of plasma-spray-hydroxyapatite coatings for improvement of osteoconduction of implants.

We have established the electrical polarization method of hydroxyapatite (HA) coatings (HAC) for clinical use, such as dental and orthopedic implants. The HAC examined in the current study was prepared in titanium substrates by plasma spraying of beta-tricalcium phosphate (TCP) powders followed by hydrothermal treatment. The prepared HAC consisted of a single phase of HA, because the starting TCP phase was completely transformed to the HA phase during the posthydrothermal treatment. Polarization was carried out at the elevated temperature of 400 degrees C under a d.c. field of 1 kV . cm(-1). The electrical measurements showed that the stored charges of the polarized HAC were greater than the reported value of the sintered ceramic HA. The enhanced bioactivity of the polarized HAC was demonstrated using 1.5 simulated body fluid (SBF). The crystal growth from the SBF solution was accelerated on the negatively charged surface in comparison with the untreated HAC. Similar to the polarized ceramic HA, the current results confirmed that the bioactivity of HAC would be effective for improving the initial fixation by polarization.

Calcium Phosphates↗

PubMed2005

A role for c-fos/activator protein 1 in B lymphocyte terminal differentiation.

Expression of B lymphocyte-induced maturation protein 1 (Blimp-1) transcription factor is essential for promoting B cell differentiation into plasma cells. However, a critical transcription factor for Blimp-1 expression in activated B cells is unclear. When splenic B cells were stimulated with CD40 ligand (CD40L) and IL-4, terminal differentiation was induced in the B cells from c-fos transgenic (H2-c-fos) mice but barely in those from control littermates and from c-fos-deficient mice. AP-1 family and Blimp-1 mRNAs were transiently induced in the control B cells, and overexpression of c-Fos induced a sufficient amount of Blimp-1 for terminal differentiation in the H2-c-fos B cells. When normal and c-fos-deficient B cells were stimulated with LPS, a sufficient amount of Blimp-1 for terminal differentiation was induced in those B cells. However, expression of c-fos/AP-1 family mRNAs in LPS-stimulated normal B cells was similar to that of normal B cells stimulated with CD40L and IL-4. EMSA and chromatin immunoprecipitation assays using the AP-1-binding DNA sequence in the murine Blimp-1 promoter region demonstrated that AP-1-binding activity in nuclear protein of LPS-stimulated normal B cells was prolonged more than that in normal B cells stimulated with CD40L and IL-4. Furthermore, the percentage of CD138(+) B cells within germinal center B cells in the spleen and the number of Ab-forming cells in the bone marrow of H2-c-fos mice was larger than that of control mice 12 days after immunization. Thus, although c-Fos is not essential for Blimp-1 expression, c-Fos/AP-1 positively regulates Blimp-1 expression and terminal differentiation of activated B cells.

Adjuvants, Immunologic↗

PubMed2005

Targeted disruption of TGF-beta-Smad3 signaling leads to enhanced neointimal hyperplasia with diminished matrix deposition in response to vascular injury.

The role of transforming growth factor (TGF)-beta and its signal in atherogenesis is not fully understood. Here, we examined mice lacking Smad3, a major downstream mediator of TGF-beta, to clarify the precise role of Smad3-dependent signaling in vascular response to injury. Femoral arteries were injured in wild-type and Smad3-null (null) male mice on C57Bl/6 background. Histopathological evaluation of the arteries 1 to 3 weeks after the injury revealed significant enhancement of neointimal hyperplasia in null compared with wild-type mice. Transplantation of null bone marrow to wild-type mice did not enhance neointimal thickening, suggesting that vascular cells in situ play a major role in the response. Null intima contained more proliferating smooth muscle cells (SMC) with less amount of collagen compared with wild-type intima. TGF-beta caused significant inhibition of cellular proliferation in wild-type aortic SMC, whereas the growth of null SMC was only weakly inhibited by TGF-beta in vitro, indicating a crucial role of Smad3 in the growth inhibitory function. On the other hand, Smad3-deficiency did not attenuate chemotaxis of SMC toward TGF-beta. TGF-beta increased transcript level of alpha2 type I collagen and tissue inhibitor of metalloproteinases-1, and suppressed expression and activity of matrix metalloproteinases in wild-type SMC. However, these effects of TGF-beta were diminished in null SMC. Our findings altogether show that the loss of Smad3 pathway causes enhanced neointimal hyperplasia on injury through modulation of growth and matrix regulation in vascular SMC. These results indicate a vasculoprotective role of endogenous Smad3 in response to injury.

Animals↗

PubMed2005

c-fos overexpression in splenic B cells augments development of marginal zone B cells.

Marginal zone (MZ)-B cells participate in very early immune responses and play a pivotal role in the first-line of defense against blood-borne Ags including bacterial LPS. Since splenic B cells from c-fos transgenic (H2-c-fos) mice are hyper-sensitive to LPS stimulation, we examined LPS-sensitivity of MZ-B cells in the spleen of H2-c-fos mice. Here, we show that proliferation of MZ-B cells from H2-c-fos mice stimulated with LPS was larger than that from control mice. Proliferation and IgM production of the H2-c-fos MZ-B cells were also larger than those of splenic follicular (FO)-B cells from the H2-c-fos mice, suggesting that c-fos overexpression augments LPS-sensitivity of MZ-B cells more than that of FO-B cells. Furthermore, the number of MZ-B cells but not that of FO-B cells in the spleen of H2-c-fos mice was two- to three-fold larger than that in control littermates. The number of transitional type II (T2)-B cells in H2-c-fos mice was also larger than that of control littermates. However, the number of transitional type I (T1)-B cells in the spleen of H2-c-fos mice was not larger than that of control mice. Moreover, this c-fos effect on differentiation of MZ-B cells was intrinsic in B cells by the competitive repopulation assay with hematopoietic stem cells of H2-c-fos and control mice. These results suggest that c-fos overexpression in B cells augments differentiation and accumulation of MZ-B cells.

Animals↗

PubMed2005

Simultaneous imaging of membrane antigen and the corresponding chromosomal locus in pathology archives.

A new procedure for the simultaneous staining of membranous antigens, such as tyrosine kinase-type cell surface receptor HER2 (c-erbB2), and the corresponding chromosome (chromosome 17 for c-erbB2) in the same cell for use in examining pathology archives is presented. A multistep procedure involving microwave-assisted fluorescence in situ hybridization and immunofluorescence yielded cell images having c-erbB2 on the membrane and genomic signals from the chromosome 17 centromere and the c-erbB2 locus. Furthermore, a combination of microwave-assisted chromogenic in situ hybridization and immunohistochemistry found colorized signals from both chromosome 17 centromere in the nuclei and c-erbB2 on the membranes of individual cells. Quantitative image analysis further confirmed the presence of a significantly stronger c-erbB2 immunoreactivity on cells containing three or more signals from chromosome 17 than from those with less than three signals. It was possible to extend the constellation of cell surface markers and corresponding chromosomes or locus-specific makers to several other genes including CDH1. In this case, the disappearances of CDH1 expression, a CDH1 locus signal, and a centromere enumeration probe (CEP) 16 signal were simultaneously demonstrated in the less-adhesive tumor cells. Thus, it is believed that this procedure might pave the way for exploiting pathology archives for the genotype-phenotype analysis of individual cells.

Antigens, Surface↗

PubMed2004

Effect of c-fos overexpression on development and proliferation of peritoneal B cells.

We examined effects of c-fos overexpression on the development and property of peritoneal B-1 cells using transgenic (H2-c-fos) mice carrying the c-fos gene under the control of the constitutive H-2Kb promoter. The number of B-1b cells in the peritoneal cavity of H2-c-fos mice was 4-fold larger than that in control littermates. Although the numbers of total peritoneal B cells and B-1a cells were similar between them, the peritoneal B-2 cell number in H2-c-fos mice was reduced to 50% of control littermates, suggesting the effect of c-fos overexpression on a balance of B-1b and B-2 cells in a peritoneal cavity. Adoptive transfer experiments with hematopoietic stem cells of H2-c-fos and control mice into irradiated H2-c-fos mice demonstrated that the augmentation of B-1b cells is due to the c-fos effect in B cells and the effect on environment of the peritoneal cavity of H2-c-fos mice. When peritoneal B cells were cultured with LPS in the presence or absence of IL-4, cell proliferation of B-1b cells was the highest among these peritoneal B cell subsets, and the proliferation of H2-c-fos B-1b cells was 3-fold higher than that of control B-1b cells. This augmentation is due to the c-fos effect in B cells. IgG1 production of B-1b cells in these cultures was slightly higher than those of B-1a and peritoneal B-2 cells. Thus, the c-fos overexpression augments development of B-1b cells in a peritoneal cavity and proliferation of peritoneal B-1b cells to LPS.

Adoptive Transfer↗

PubMed2004

Numerical osteobonding evaluation of electrically polarized hydroxyapatite ceramics.

Enhanced osteobonding abilities of electrical polarized hydroxyapatite (HA) ceramics were statistically evaluated by a bone morphometric technique. HA ceramics with an average charge of 3.9 microCcm(-2) were implanted in tibial and femoral diaphyses of New Zealand white rabbits. Estimation of the affinity index, defined as the ratio of the direct bonding length to the HA ceramic length, significantly showed more active osteobonding ability with a negatively charged surface than with a positively charged surface and a nonpolarized HA 1 week after administration. The enhanced osteobonding ability attributed to the stimulated osteoids in the vicinity of the positively charged surface ranked above the nonpolarized surface. Although amounts of the newly formed bone on the HA surfaces increased with implantation period in all of the observation areas, the negatively charged surface was almost covered with bone and had the best index value of 94.0% at 4 weeks. The superior osteobonding activity of the negatively charged surfaces was statistically proven.

Animals↗