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Biomedical subjects

Yoshinosuke Fukuchi

Publications and source records attributed to Yoshinosuke Fukuchi.

At least 55 records · Page 3Linked to original sources

Cutting edge: a potent adjuvant effect of ligand to receptor activator of NF-kappa B gene for inducing antigen-specific CD8+ T cell response by DNA and viral vector vaccination.

The ligand to receptor activator of NF-kappaB (RANK-L)/RANK interaction has been implicated in CD40 ligand/CD40-independent T cell priming by dendritic cells. In this report, we show that the coadministration of the RANK-L gene with a Trypanosoma cruzi gene markedly enhances the induction of Trypanosoma Ag-specific CD8(+) T cells and improves the DNA vaccine efficacy. A similarly potent adjuvant effect of the RANK-L gene on the induction of Ag-specific CD8(+) T cells was also observed when recombinant influenza virus expressing murine malaria Ag was used as an immunogen. In contrast, the coadministration of the CD40L gene was not effective in these systems. Our results demonstrated, for the first time, the potent immunostimulatory effect of the RANK-L gene to improve the CD8(+) T cell-mediated immunity against infectious agents.

Adjuvants, Immunologic↗

Osteopontin overproduced by tumor cells acts as a potent angiogenic factor contributing to tumor growth.

Angiogenesis, which is essential for tumor growth, is regulated by various angiogenic factors. Osteopontin (OPN) is expressed in various human tumors and is postulated to be involved in tumor progression. We have recently reported that culture medium with murine neuroblastoma C1300 cells transfected with OPN gene significantly stimulates human umbilical vein endothelial cell migration and induces neovascularization in mice by dorsal air sac assay. However, the effect of OPN on tumorigenesis as an angiogenic factor remains to be clarified. In this study, we injected the OPN-transfected C1300 cells and control cells into the nude mice subcutaneously. OPN-overexpressing C1300 cells significantly formed rapidly growing tumor as compared to the control cells in mice, although in vitro and in vivo cell growth rates were similar. In vivo tumorigenecity of these cells correlated with the amount of secreted OPN protein. In addition, neovascularization of OPN-transfected tumor was significantly increased in comparison with those of control cells by immunohistochemistry for CD31. In vitro chemoinvasiveness and gene expression of proteases including uPA, MMP2, 9, MT1-MMP, and cathepsin B, D, L, were not different between OPN-transfected and control cells determined with matrigel invasion assay and cDNA expression macroarray, respectively. Conclusively, these results strongly imply that OPN plays an important role in tumor growth through the enhancement of angiogenesis in vivo.

Angiogenesis Inducing Agents↗

Quantification of elastin cross-linking amino acids, desmosine and isodesmosine, in hydrolysates of rat lung by ion-pair liquid chromatography-mass spectrometry.

The elastin cross-linking amino acids, desmosine (DES) and isodesmosine (IDE), in hydrolysates of rat lungs were quantified by ion-pair liquid chromatography-electrospray mass spectrometry. The column was a 2.0 mm i.d. x 150 mm Develosil UG3 (ODS) with a mobile phase A of 7 mM pentafluoropropionic anhydride (PFPA) using ultrapure water as the ion-pair reagent, and a mobile phase B of 7 mM PFPA in 80% methanol. The retention times of IDE and DES were 25.5 and 26.6 min, respectively. The mean concentrations of IDE and DES in the lung were 191.6+/-54.5 nmol/g lung (dry tissue) (+/-SD) and 184.0+/-39.3 nmol/g lung, respectively, and the IDE/DES ratio was 1.04, in Wistar Kyoto rats. Our results indicate that ion-pair liquid chromatography-mass spectrometry is a useful procedure for quantitation of DES and IDE in hydrolysates of rat lung.

Animals↗

Alternative splicing of myeloid IgA Fc receptor (Fc alpha R, CD89) transcripts in inflammatory responses.

More than 10 splice variants of the Fc receptor for IgA (Fc alpha R, CD89) have been identified in human myeloid cells. In this study, we quantified Fc alpha R splice transcripts Delta EC2 and Delta 66 EC2, which lack the entire and a part of the homologous immunoglobulin-like extracellular domain 2 (EC2), respectively. Tumor necrosis factor-alpha was found to specifically increase the ratio of Delta EC2 to the wild type CD89 in neutrophils and conversely decrease the Delta EC2 ratio in monocytes. We also observed a significant decrease in the neutrophil Delta EC2/CD89 ratio in pneumonia patients. These results suggest that Delta EC2 is differentially regulated and could be involved in immunoregulation of IgA-mediated host defense.

Alternative Splicing↗

Restoration of CD44S in non-small cell lung cancer cells enhanced their susceptibility to the macrophage cytotoxicity.

CD44 is a cell surface receptor for osteopontin (OPN) and hyaluronate. Transformation of normal tissue to a variety of cancers has been demonstrated to be associated with alterations of CD44 isoform expression. However, few reports have paid attention on differences in CD44S expression between non-small cell lung cancer (NSCLC) and adjacent normal lung tissue. In this study, we demonstrate that CD44S expression is down-regulated in NSCLC tissue when compared with paired normal lung tissue. To investigate the role of CD44S down-regulation in NSCLC cells, we reintroduced the CD44S back into the NSCLC cell line, H322 cells, which originally lack CD44S expression. The cytotoxicity by activated macrophage (RAW264.7 cells stimulated with lipopolysaccharide and interferon-gamma) against the H322 cells transfected with the CD44S gene (H322DeltaS) is more prominent than that against the H322 control transfectants (H322Deltaneo). The enhanced susceptibility of H322DeltaS cells to the activated macrophage cytotoxicity appears to be mediated by the interaction between CD44S expression on H322DeltaS cells and OPN produced by activated macrophages since it is completely blocked by either anti-OPN or anti-CD44 antibody. Moreover, H322DeltaS cells are attracted toward OPN produced by activated RAW264.7 cells to a much greater extent than H322Deltaneo cells. These findings suggest that CD44S down-regulation in NSCLC cells may confer a protective advantage of allowing escape from tumoricidal effector cells including activated macrophages of the host.

Blotting, Western↗

Acid exposure potentiates intercellular adhesion molecule-1 and e-cadherin expression on A549 alveolar lining epithelial cells.

Recruitment of neutrophils into the alveoli plays a major role in the pathogenesis of acid-induced pneumonitis. Preliminary data suggest that alteration in the expression of cellular adhesion molecules on the airway epithelial cells may play an important role in the recruitment of neutrophils following acid-induced lung injury. The aim of this study was to evaluate the change in the surface expression of intercellular adhesion molecule-1 (ICAM-1), E-cadherin, and vascular cell adhesion molecule -1 (VCAM-1) on acid-exposed A549 alveolar lining epithelial cells by flow cytometry and confocal laser microscopy. Acid exposure changed cell morphology, increased cell adhesion after trypsin-EDTA treatment, and up-regulated the expression of ICAM-1 and E-cadherin but not of VCAM-1. The up-regulation of ICAM-1 expression will induce the dysfunction of epithelial cells with or without accumulation of neutrophils in air spaces. Because the distribution of E-cadherin in acid-exposed A549 cells was at the sites where the cells attached to culture dish but not at the intercellular junctions between adjoining cells, up-regulated expression of E-cadherin will rather result in alterations of epithelial morphology and function of epithelial barrier. In addition, pentoxifylline suppressed the up-regulation of ICAM-1 and E-cadherin expression and may therefore attenuated the airway inflammation in acid-induced pneumonitis.

Cadherins↗

Smad7 suppresses the inhibitory effect of TGF-beta2 on corneal endothelial cell proliferation and accelerates corneal endothelial wound closure in vitro.

PURPOSE: The inhibitory activity of transforming growth factor-beta2 (TGF-beta2) on corneal endothelial cell proliferation is thought to be a cause of the limited regenerative capacity of corneal endothelial cells that may be related to impaired corneal transparency when many corneal endothelial cells are lost due to various stresses. We determined whether Smad7, an intracellular antagonist of TGF-beta signaling, regulated the inhibitory activity of TGF-beta2 or aqueous humor on corneal endothelial cell proliferation. METHODS: The effect of Smad7 on TGF-beta2- or aqueous humor-mediated inhibition of corneal endothelial cell proliferation was evaluated using thymidine uptake assay with cultured rabbit corneal endothelial cells infected with adenovirus carrying Smad7. Expression of Smad or cell cycle-related proteins was detected by immunoblotting. In addition, a small scrape wound was made across a monolayer of Smad7-expressing cultured rabbit corneal endothelial cells to examine the effect of Smad7 on the wound-healing process in vitro. RESULTS: Overexpression of Smad7 abolished the inhibitory effect of TGF-beta2 or aqueous humor on the proliferation of cultured rabbit corneal endothelial cells associated with the inhibition of phosphorylation of Smad2 and downregulation of p27kip1. Smad7-overexpressing cultured rabbit corneal endothelial cells exhibited shorter wound closure time in the presence of aqueous humor than LacZ-expressing cells. CONCLUSION: Overexpression of Smad7 suppressed the inhibitory effect of TGF-beta2 or aqueous humor on corneal endothelial cell proliferation and accelerated corneal endothelial wound closure in vitro. Modification of Smad7 expression in corneal endothelial cells may thus have applicability in the treatment of wounded corneal endothelium.

Adenoviridae↗

Activation of natural killer T cells by alpha-galactosylceramide impairs DNA vaccine-induced protective immunity against Trypanosoma cruzi.

Innate immunity as a first defense is indispensable for host survival against infectious agents. We examined the roles of natural killer (NK) T cells in defense against Trypanosoma cruzi infection. The T. cruzi parasitemia and survival of CD1d-deficient mice exhibited no differences compared to wild-type littermates. NK T-cell activation induced by administering alpha-galactosylceramide (alpha-GalCer) to T. cruzi-infected mice significantly changed the parasitemia only in the late phase of infection and slightly improved survival when mice were infected intraperitoneally. The combined usage of alpha-GalCer and benznidazole, a commercially available drug for Chagas' disease, did not enhance the therapeutic efficacy of benznidazole. These results suggest that NK T cells do not play a pivotal role in resistance to T. cruzi infection. In addition, we found that the coadministration of alpha-GalCer with DNA vaccine impaired the induction of epitope-specific CD8(+) T cells and undermined the DNA vaccine-induced protective immunity against T. cruzi. Our results, in contrast to previous reports demonstrating the protective roles of NK T cells against other infectious agents, suggest that these cells might even exhibit adverse effects on vaccine-mediated protective immunity.

Animals↗

Critical contribution of CD28-CD80/CD86 costimulatory pathway to protection from Trypanosoma cruzi infection.

The CD28-CD80/CD86-mediated T-cell costimulatory pathway has been variably implicated in infectious immunity. In this study, we investigated the role of this costimulatory pathway in resistance to Trypanosoma cruzi infection by using CD28-deficient mice and blocking antibodies against CD80 and CD86. CD28-deficient mice exhibited markedly exacerbated T. cruzi infection, as evidenced by unrelenting parasitemia and 100% mortality after infection with doses that are nonlethal in wild-type mice. The blockade of both CD80 and CD86 by administering specific monoclonal antibodies also exacerbated T. cruzi infection in wild-type mice. Splenocytes from T. cruzi-infected, CD28-deficient mice exhibited greatly impaired gamma interferon production in response to T. cruzi antigen stimulation in vitro compared to those from infected wild-type mice. The induction of T. cruzi antigen-specific CD8(+) T cells was also impaired in T. cruzi-infected, CD28-deficient mice. In addition to these defects in natural protection against T. cruzi infection, CD28-deficient mice were also defective in the induction of CD8(+)-T-cell-mediated protective immunity against T. cruzi infection by DNA vaccination. These results demonstrate, for the first time, a critical contribution of the CD28-CD80/CD86 costimulatory pathway not only to natural protection against primary T. cruzi infection but also to DNA vaccine-induced protective immunity to Chagas' disease.

Animals↗

[Proteases involved in airway inflammation of COPD].

Chronic obstructive pulmonary disease(COPD) is one of major cause of morbidity and mortality worldwide. Cigarette smoking is the main risk factor for the development of COPD and causes airway inflammation and parenchymal destruction. In the pathogenesis of COPD, protease-antiprotease imbalance is considered to be one of the important processes through the study of alpha 1-antitrypsin deficiency. At present, not only neutrophil elastase but also cathepsin and matrix metalloproteinases produced by inflammatory cells are demonstrated to be important in the pathogenesis of COPD, In this review, the roles of various kinds of proteases in the airway inflammation are summarized.

Animals↗

Pro-inflammatory effect of TWEAK/Fn14 interaction on human umbilical vein endothelial cells.

TWEAK, a member of the TNF family, induces cell death in some tumor cell lines, but also induces proliferation of endothelial cells and angiogenesis. Recently, fibroblast growth factor-inducible 14 (Fn14) has been identified to be a TWEAK receptor, which may be responsible for the proliferation of endothelial cells and angiogenesis. In this study, we investigated the pro-inflammatory effect of TWEAK on human umbilical vein endothelial cells (HUVEC). We demonstrated that TWEAK could not only induce the proliferation and migration but also upregulate the cell surface expression of adhesion molecules such as ICAM-1 and E-selectin, and induce the secretion of chemokines such as IL-8 and MCP-1 in HUVEC. Moreover, by using an anti-Fn14 mAb that blocks the TWEAK/Fn14 interaction, we demonstrated that Fn14 was constitutively expressed on HUVEC and totally mediated the biological effects of TWEAK on HUVEC. These results indicated that TWEAK could induce pro-inflammatory reactions via Fn14 on HUVEC.

Animals↗

Osteopontin induces angiogenesis of murine neuroblastoma cells in mice.

Angiogenesis is an essential process for tumor growth and is regulated by tumor-derived angiogenic cytokines. Osteopontin (OPN) is one of the cytokines produced by various tumor cells and is suggested to be involved in angiogenesis by upregulating endothelial cell migration in cooperation with vascular endothelial cell growth factor (VEGF). To provide evidence of OPN involvement in a causal role in tumor angiogenesis, we generated a stable transfectant from murine neuroblastoma C1300 cells to constitutively secrete high levels of murine OPN. The OPN mRNA expression and protein secretion were confirmed by RT-PCR and ELISA, respectively. The biological activity of secreted OPN was determined with migration assay by using human umbilical vein endothelial cells (HUVEC). Transfection with OPN gene did not increase VEGF production and did not affect gene expression of other angiogenic factors confirmed by complementary DNA macroarray system. To demonstrate the effect of OPN on tumor-induced angiogenesis in vivo, millipore chambers containing OPN-transfected or control cells were implanted to the dorsal air sac of mice. The OPN-transfected cells significantly induced neovascularization in comparison to the control cells in mice. Conclusively, these results provide direct evidence of OPN involvement in the role of tumor angiogenesis.

Animals↗

Mutation analysis of the TSC1 and TSC2 genes in Japanese patients with pulmonary lymphangioleiomyomatosis.

Pulmonary lymphangioleiomyomatosis (LAM) is a destructive lung disease characterized by a diffuse hamartomatous proliferation of smooth muscle cells (LAM cells) in the lungs. Pulmonary LAM can occur as an isolated form (sporadic LAM) or in association with tuberous sclerosis complex (TSC) (TSC-LAM), a genetic disorder with autosomal dominant inheritance with various expressivity resulting from mutations of either the TSC1 or TSC2 gene. We examined mutations of both TSC genes in 6 Japanese patients with TSC-LAM and 22 patients with sporadic LAM and identified six unique and novel mutations. TSC2 germline mutations were detected in 2 (33.3%) of 6 patients with TSC-LAM and TSC1 germline mutation in 1 (4.5%) of 22 sporadic LAM patients. In accordance with the tumor-suppressor model, loss of heterozygosity (LOH) was detected in LAM cells from 3 of 4 patients with TSC-LAM and from 4 of 8 patients with sporadic LAM. Furthermore, an identical LOH or two identical somatic mutations were demonstrated in LAM cells microdissected from several tissues, suggesting LAM cells can spread from one lesion to another. Our results from Japanese patients with LAM confirmed the current concept of pathogenesis of LAM: TSC-LAM has a germline mutation but sporadic LAM does not; sporadic LAM is a TSC2 disease with two somatic mutations; and a variety of TSC mutations causes LAM. However, our study indicates that a fraction of sporadic LAM can be a TSC1 disease; therefore, both TSC genes should be examined, even for patients with sporadic LAM.

Chromosomes, Human, Pair 16↗

Clinical significance of nocturnal oximeter monitoring for detection of sleep apnea syndrome in the elderly.

OBJECTIVES: The sensitivity and specificity of overnight monitoring of arterial oxygen saturation (SaO(2)) using an oximeter were evaluated in elderly subjects who are being investigated for possible sleep apnea syndrome (SAS). METHODS: Seventy-five consecutive elderly subjects with habitual snoring (47 men, 28 women; mean (+/-SE) age 75.5+/-0.9 years (range 65-94 years)) were studied. The SaO(2) was measured with an oximeter and a chart recorder during the night immediately before detailed polysomnographic studies. The SaO(2) recordings were classified by two observers as positive or negative using a number of significant oxyhemoglobin desaturation (SDS) of more than 2, 4, and 6%. The sensitivity of the oximeter alone for the recognition of the SAS was calculated as the number of true positive SaO(2) records divided by the total number of positive definitive (polysomnographic) records. The specificity was defined as the number of true negative SaO(2) records divided by the total number of negative definitive records. RESULTS: Of the 75 subjects, 24 had moderate SAS (apnea index (AI)>15) and 55 had mild to moderate SAS (AI>5). The sensitivity and specificity of the dosimeter as a screening test were determined with the two diagnostic thresholds of the AI. For AI exceeding 5 or 15, the respective sensitivity by using the criterion of SDS of more than 4% was 85.5 or 91.7%, with corresponding specificity of 85.0 or 92.2%. CONCLUSIONS: The nocturnal oximeter monitoring allows recognition of elderly subjects with a mild to moderate SAS, and 4% desaturation of SaO(2) is a candidate index to detect a significant number of apneas in elderly SAS patients with an oximeter.

Journal Article↗

Adenovirus vector-mediated transfer of 9 kDa granulysin induces DNA fragmentation in HuD antigen-expressing small cell lung cancer murine model cells.

OBJECTIVE: Granulysin is a tumoricidal molecule secreted by cytotoxic T cells (CTL) and natural killer (NK) cells, that induces apoptotic cell death in tumour cells. It has been demonstrated that small cell lung cancer (SCLC) cell lines are susceptible to NK cells and lymphokine activated killer (LAK) cells, and HuD antigen is assumed to be a target molecule on SCLC cells for host cellular immunity. METHODOLOGY: In order to understand the mechanism of sensitivity of SCLC to cellular immunity, we evaluated granulysin-induced apoptosis using mouse adenocarcinoma Colon 26 (Colon 26/HuD) cells transfected with the 9 kDa active form of granulysin using an adenovirus vector as a murine model of SCLC cells. RESULTS: Adenovirus vector-mediated transfer of 9 kDa granulysin increased DNA fragmentation in Colon 26/HuD cells 2.5-fold and suppressed Colon 26/HuD proliferation by 21% on day 3 (P < 0.05 for each value) compared with the control adenovirus vector transfer. In contrast, adenovirus vector-mediated transfer of 9 kDa granulysin did not increase DNA fragmentation nor suppress the proliferation of Colon 26 parent cells. CONCLUSIONS: The sensitivity of HuD-expressing tumour cells to granulysin is likely to partially explain the susceptibility of SCLC to cell-mediated immunity.

Adenocarcinoma↗

Increased circulating CD16+ CD14dim monocytes in a patient with pulmonary alveolar proteinosis.

Pulmonary alveolar proteinosis (PAP) is characterized by filling of the alveoli with a periodic acid-Schiff-positive proteinaceous material. Although the pathogenesis of primary or idiopathic PAP remains unknown, it has been proposed that a deficiency or loss of responsiveness of the monocyte/macrophage lineage to granulocyte-macrophage colony stimulating factor (GM-CSF) is involved in PAP. Secondary PAP is associated with haematological malignancies, especially in myeloid disorders. Herein, we report on an adult with PAP associated with myelodysplastic syndrome (MDS). The CD16+ CD14dim monocytes comprise 5-10% of circulating monocytes in healthy volunteers. Flow cytometric analysis of the patient in the present study revealed increased CD16+ CD14dim monocytes in the peripheral blood. It has been demonstrated that the expression of CD16 and CD14 is regulated by macrophage colony stimulating factor (M-CSF) and GM-CSF. Hence, serum cytokines were analysed in our patient and the concentration of serum GM-CSF was found to be less than the lower limit of the assay. In addition, serum M-CSF and granulocyte colony stimulating factor levels were only slightly increased above the normal range. These results suggest that the increase in the CD16+ CD14dim subpopulation in the circulation of our patient indicates another pathogenetic mechanism for secondary PAP, such as hyperresponsiveness of the monocyte/macrophage lineage to these cytokines.

Aged↗