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H Tsujimoto

Publications and source records attributed to H Tsujimoto.

At least 37 records · Page 2Linked to original sources
PubMed2002

Intravenous immunoglobulin (IVIG) preparations induce apoptosis in TNF-alpha-stimulated endothelial cells via a mitochondria-dependent pathway.

Endothelial cells (ECs) are a target in inflammation, and the death of EC is regulated by various factors. Although intravenous immunoglobulin (IVIG) preparations are known to be beneficial therapeutic agents for the treatment of autoimmune diseases and systemic inflammatory disorders, their mechanism of action have not yet been completely elucidated. The aim of the present study is to investigate the possible role of IVIG in EC apoptosis. We demonstrate herein that IVIG induced the apoptosis of human umbilical vein ECs (HUVECs) prestimulated by TNF-alpha in vitro, but not in unstimulated HUVECs, in a dose- and time-dependent manner, using a proportion of cells with hypodiploid DNA, DNA ladder formation and morphological changes. Anti-Fas MoAbs had no effect on the IVIG-induced apoptosis in the TNF-alpha-stimulated HUVECs. IVIG decreased the intracellular expression of anti-apoptotic proteins of the Bcl-2 family (A1 and Bcl-XL) while IVIG increased the intracellular expression of pro-apoptotic proteins (Bax and Bcl-XS) in the TNF-alpha-stimulated HUVECs. Furthermore, IVIG increased the intracellular production of reactive oxygen species and decreased the mitochondrial membrane potential (Delta(psi)m). Caspase-inhibitors inhibited the IVIG-induced apoptosis of the TNF-alpha-stimulated HUVECs. The present results show a novel action in which IVIG can induce the apoptosis of TNF-alpha-stimulated HUVECs through a mitochondrial apoptotic signalling pathway. These observations suggest that the clinical use of IVIG preparations may thereby regulate the cell death of activated ECs in inflammation.

Apoptosis↗
PubMed2001

TNF-alpha-induced cell death in feline immunodeficiency virus-infected cells is mediated by the caspase cascade.

TNF-alpha induced apoptosis in a feline fibroblastic cell line (CRFK) infected with FIV but not in its uninfected control. In this study, to understand the molecular basis of the different susceptibilities to TNF-alpha between FIV-infected and uninfected cells, we examined the expression of TNF receptors and the activation of the caspase and NF-kappaB pathways. Expression levels of TNFR I and TNFR II mRNAs were similar between uninfected and FIV-infected CRFK cells. To understand the role of caspases in TNF-alpha-induced apoptosis, we examined the effect of three different classes of caspase inhibitors, Z-VAD-FMK, Ac-YVAD-CMK, and Z-DEVD-FMK, on the TNF-alpha-induced apoptosis in FIV-infected cells. Pretreatment with each of these caspase inhibitors protected FIV-infected CRFK cells from TNF-alpha-induced cell death. Moreover, one of the caspase substrates, poly(ADP-ribose) polymerase, was shown to be cleaved after TNF-alpha treatment in FIV-infected CRFK cells but not in uninfected CRFK cells. Electrophoretic mobility shift assay using an NF-kappaB motif oligonucleotide and promoter assay using an NF-kappaB luciferase reporter construct indicated that TNF-alpha treatment had induced activation of NF-kappaB in both FIV-infected and uninfected CRFK cells. The present study indicates that TNF-alpha-induced apoptosis in FIV-infected CRFK cells is mediated by the activation of the caspase cascade, but not by either upregulation of TNF receptor or inhibition of NF-kappaB.

Amino Acid Chloromethyl Ketones↗
PubMed2001

Molecular cloning and expression of the cDNA encoding feline granulocyte colony-stimulating factor.

Both genomic DNA and cDNA of the feline granulocyte colony-stimulating factor (G-CSF) gene were cloned from CRFK cells. Southern blot analysis showed that the haploid genome contains a single copy of the G-CSF gene. The RT-PCR analysis of several feline cell lines revealed expression of G-CSF mRNA in response to lipopolysaccharide stimulation. Sequence analysis of genomic and cDNA clones indicated that the intron-exon junction structure is conserved between the human and the feline G-CSF genes. The G-CSF coding region encodes a predicted protein of 195 amino acids including a signal sequence of 21 amino acids. The feline G-CSF amino acid sequence shares a high degree of identity with the canine (90.8%), human (87.4%), ovine (83.9%), bovine (82.8%), porcine (80.5%), murine (70.7%) and rat (66.8%) G-CSF. The feline G-CSF expressed in insect cells using recombinant baculovirus vector was biologically active as measured in a proliferation assay using NFS-60 cells and an induction assay of leukocytes in cats.

Amino Acid Sequence↗
PubMed2001

Detection of differentially expressed genes in HeLa x fibroblast hybrids using subtractive suppression hybridization.

To understand genetic differences and similarities between tumorigenic and nontumorigenic HeLa x fibroblast hybrid cells, subtractive suppression hybridization (SSH), based on suppression PCR and a combination of normalization and subtraction in a single procedure, was used. Using the nontumorigenic CGL1 and tumorigenic CGL3, forward (CGL1-CGL3) and reverse (CGL3-CGL1) subtracted libraries were constructed. Among 192 clones, seven were identified as differentially expressed genes specific for either CGL1 or CGL3. All seven were not reported previously as differentially expressed genes in this hybrid system. In the forward subtraction, p16 was isolated, indicating the involvement of the loss of tumorigenic phenotype. Subsequent transfection of wild-type p16 to the tumorigenic CGL3 showed growth suppression in colony formation assay; however, no tumor suppression was observed when the transfectant was inoculated into nude mice. These results indicate that: (a) SSH is a suitable method to identify differentially expressed genes in two types of cells; and (b) although p16 plays some roles in growth suppression, the p16-transfected CGL3 is still capable to proliferate in vivo.

Animals↗
PubMed2001

Molecular cloning of feline interferon-gamma-inducing factor (interleukin-18) and its expression in various tissues.

Interleukin-18 (IL-18) is a cytokine with potent interferon-gamma-inducing activity, and plays an important biologic role in the enhancement of the activity of natural killer cells and cytotoxic T-lymphocytes. In this study, feline IL-18 cDNA was cloned and characterized to establish a basis for the prospective cytokine therapy in small animal practice. The nucleotide sequence of feline IL-18 cDNA obtained in this study was 712bp long and contained its entire open reading frame encoding 192 amino acid residues. The predicted amino acid sequence of feline IL-18 cDNA showed 77.2, 84.8, 60.2 and 62.6% similarity with those of human, dog, rat and mouse counterparts, respectively. The feline IL-18 cDNA included a putative cleavage site of IL-1beta-converting enzyme (ICE) and IL-1 signature-like sequences identified in human and mouse IL-18 cDNAs. Expression of IL-18 mRNA was detected in various tissues including spleen, liver and cerebrum in the cat.

Amino Acid Sequence↗
PubMed2001

Oligonucleotide microarray expression analysis of genes whose expression is correlated with tumorigenic and non-tumorigenic phenotype of HeLa x human fibroblast hybrid cells.

In order to understand the differences and similarities between tumorigenic and non-tumorigenic HeLaxhuman fibroblast hybrids, gene expression profiles were examined with synthetic oligonucleotide arrays containing nearly 7000 gene probe sets. We used two pairs of genetically related hybrids, each pair representing individual clones of non-tumorigenic and tumorigenic segregant hybrids, respectively. Analysis of six possible comparisons, utilizing two algorithms, identified 204 genes with differential expression. The greater number of differentially expressed genes was observed when non-tumorigenic hybrids were compared with tumorigenic segregants. Fifteen and 14 genes, respectively, were consistently found to be differentially expressed in non-tumorigenic and tumorigenic cells. Among those 29 differentially expressed genes, three (intestinal alkaline phosphatase, caveolin-1, and solute carrier family2, member3) have been reported previously to be associated with the tumorigenic phenotype, using the same hybrid pairs. In addition, among the genes previously detected by differential display, 78% of them exhibited more than 5-fold change, demonstrating a high consistency between the two methods of differential gene expression. These findings suggest that synthetic oligonucleotide arrays are a powerful and highly reproducible tool to identify those genes whose expression is associated with certain phenotypes.

Algorithms↗
PubMed2001

Analysis of the canine IgE-binding epitope on the major allergen (Cry j 1) of Japanese cedar pollen with anti-Cry j 1 monoclonal antibodies.

In our previous study [Immunology 91 (1997) 161] using monoclonal antibodies (mAbs) specific to Cry j 1, a major allergen in Japanese cedar (Cryptomeria japonica) pollen, we identified five independent epitopes (EP-1-EP-5) on the molecule and found that EP-1 and EP-5 are the predominant allergic epitopes for humans and monkeys, respectively. In this study, we analyzed the epitopes recognized by IgE in the sera of 10 dogs sensitive to C. japonica pollen allergen using an IgE-ELISA inhibition method with these mAbs. The IgE reaction patterns varied among dogs. In eight of the 10 dogs, IgE recognized EP-5 which is a predominant allergic epitope for monkeys with the pollenosis. In four dogs, IgE recognized EP-1 which is a predominant allergic epitope for human patients with the pollenosis. In three dogs, IgE recognized EP-4 which is a heat-stable epitope. EP-5 is a predominant allergic epitope for dogs and some, but not all, dogs have IgE reaction patterns to the epitopes similar to those of humans.

Allergens↗
PubMed2001

Severe sepsis induces deficient interferon-gamma and interleukin-12 production, but interleukin-12 therapy improves survival in peritonitis.

BACKGROUND: After severe sepsis, there is an increase of Th2 cytokine and a decrease in Th1 cytokine that may account for impaired cellular immunity. The aim of this study is to evaluate the Th1, Th2 cytokine balance in the serum, peritoneal lavage fluid (PLF) and liver mononuclear cells (MNC) of experimental peritonitis mice, and determine the effect of interleukin-12 (IL-12), a cytokine stimulating Th1 cytokine production, when administered to septic mice. METHODS: Experimental bacterial peritonitis mice was induced by cecal ligation and puncture (21-gauge needle, mild peritonitis) or cut (5 mm, severe peritonitis). Serum and PLF levels and liver MNC production of interferon (IFN)-gamma, IL-10, and IL-12 were measured after the procedure. Mild and severe peritonitis mice were treated intraperitoneally with recombinant IL-12 (r-IL-12) either 6 hours before or 6 and 24 hours after the procedure. The survival rates were then compared with nontreated mice. RESULTS: Serum and PLF IFN-gamma, IL-12 levels in severe peritonitis mice were significantly lower than those in mild peritonitis mice at 6 and 12 hours after the procedure. On the other hand, serum and PLF IL-10 levels in severe peritonitis mice were significantly higher than those in mild peritonitis mice at 6 hours after the procedure. Furthermore, liver MNC IFN-gamma production in severe peritonitis mice was significantly higher than that in mild peritonitis mice at 6 hours after the procedure, but liver MNC IL-12 production in severe peritonitis mice was significantly lower than that in mild peritonitis mice at 12 hours after the procedure. Severe peritonitis mice treated with r-IL-12 at 6 hours before the procedure improved survival rate, and mild peritonitis mice treated with r-IL-12 at 24 hours after the procedure showed significantly improved survival rates. CONCLUSIONS: Change in the Th1, Th2 cytokine balance in peritonitis mice might induce a shift toward a Th2 dominant phenotype according to the severity of peritonitis, and the capacity to produce IFN-gamma and IL-12 by liver MNC is reduced. Therapies designed to augment the production of Th1 cytokines, such as IL-12, may thus prove to be beneficial in the treatment of severe sepsis after peritonitis.

Animals↗
PubMed2001

IgE reactivity and cross-reactivity to Japanese cedar (Cryptomeria japonica) and cypress (Chamaecyparis obtusa) pollen allergens in dogs with atopic dermatitis.

The natural occurrence of Japanese cedar (Cryptomeria japonica) pollinosis has been reported in dogs with atopic dermatitis. However, the reactivity to Japanese cypress (Chamaecyparis obtusa) pollen allergens in these dogs has not been reported. The present study was designed to investigate the reactivity to Japanese cypress pollen allergens in dogs sensitized to Japanese cedar pollen allergens. In 19 dogs with specific IgE to C. japonica pollen allergen, we measured the specific IgE to C. obtusa pollen allergen and examined the reactivity to the allergen by intradermal test. Of the 19 dogs, 18 had specific IgE to crude and purified major allergens (Cha o 1) of C. obtusa pollen. Most of the dogs showed a positive reaction to C. obtusa pollen allergens in the intradermal test. Allergenic cross-reactivity between Cha o 1 and Cry j 1 (a major allergen in C. japonica pollen) was observed by the ELISA inhibition method. Dogs sensitized to Japanese cedar pollen allergens demonstrate reactivity to Japanese cypress pollen allergens.

Allergens↗
PubMed2001

A tandem repetitive sequence located in the centromeric region of common wheat (Triticum aestivum) chromosomes.

Although Tail-family sequences are present in the subtelomeric region of Leymus racemosus, it became apparent in the present study that such sequences are also present in the centromeric region of common wheat (Triticum aestivum). These sequences hybridized to all chromosomes with various degrees of signal strength. FISH using Tail and Ty3/gypsy, a conservative sequence in cereal centromeres, revealed a complicated arrangement of both sequences in all wheat chromosomes at once. Unlike the Arabidopsis centromeres characterized by massive tandem arrays of 180-bp family with flanking paracentromeric retrotransposons in all chromosomes, wheat chromosomes showed various arrangement patterns of Tail and Ty3/gypsy sequences depending on the chromosome; Tail-family sequences were scattered in many wheat centromeres as isolated colonies instead of forming uninterrupted solid tandem arrays. This pattern may have resulted from retrotransposon insertion within pre-existing Tail-tandem arrays or a two-step amplification mechanism of the Tail family where each Tail colony was amplified to form arrays independently after the insertion of Tail-family sequences along the entire centromere. Although sequence analysis of centromeric Tail repeats in wheat and subtelomeric Tail repeats in L. racemosus showed variable and conservative regions between the two repeats, they did not show a distinctive difference phylogenically. The widespread presence of tandem repetitive sequences in the eucaryotic centromere suggests a significant role for them in centromeric formation.

Arabidopsis↗
PubMed2001

Elevated serum levels of matrix metalloproteinase-9 (MMP-9) in Kawasaki disease.

Matrix metalloproteinases (MMPs) play an important role in the progression of tumour cells and the invasion of inflammatory cells by degrading the extracellular matrix. In the MMP family, MMP-9 gelatinase is thought to contribute to the pathogenesis of inflammatory arteritis by disrupting the elastic lamina. The aim of the present study is to investigate the potential role of MMP-9 in Kawasaki disease (KD), an acute type of systemic vasculitis in children. We studied the total levels of MMP-9 (free proMMP-9 and free MMP-9) in the sera using a new assay system and the expression of MMP-9 mRNA in the leucocytes using reverse transcription-polymerase chain reaction in 18 patients with KD, 10 patients with sepsis and 10 healthy children (HC). The serum MMP-9 levels were significantly higher (P < 0.01) in the acute phase of KD than in the acute phase of sepsis and HC. In the time course of KD, the serum MMP-9 levels decreased significantly (P < 0.01) from the subacute through the convalescent phases. In the acute phase of KD, the serum MMP-9 levels showed a significantly positive correlation (P < 0.05) with the circulating leucocyte counts, especially the neutrophil counts. Furthermore, the expression of MMP-9 mRNA in the circulating leucocytes increased in the acute phase of KD and decreased from the subacute through the convalescent phases. These findings indicate that an excessive amount of MMP-9 is present in the plasma during the acute phase of KD, thus suggesting that circulating leucocytes may be a source of the MMP-9 secreted into the circulation.

Acute Disease↗
PubMed2001

Delayed apoptosis of circulating neutrophils in Kawasaki disease.

Circulating polymorphonuclear neutrophils (PMNs) are known to increase in number and are functionally activated in the acute phase of Kawasaki disease (KD). In the present study, we investigated whether the apoptosis of PMNs is deregulated in KD. When the isolated PMNs were cultured in vitro, the proportions of spontaneous apoptotic PMNs (annexin V+ cells and cells with fragmented DNA) were found to be significantly lower (P < 0.01) in the patients with KD (n = 25) than in the patients with a bacterial infection (n = 20) or a viral infection (n = 20), or in healthy children (n = 20). The proportion of circulating Fas-positive PMNs was also significantly lower (P < 0.01) in the acute KD patients than in the other groups. In the acute phase of KD, the proportion of spontaneous apoptotic PMNs showed a significant positive correlation (P < 0.01) with the proportions of circulating Fas-positive PMNs. Furthermore, the agonistic anti-Fas MoAb (CH-11) induced a significant increase in the proportion of apoptotic PMNs in the patients with a viral infection and healthy children, but not in either the patients with KD or the patients with a bacterial infection. In the intracellular expression of anti- and pro-apoptotic proteins, the A1/Bax ratio was significantly higher in acute KD than in the other groups. These findings indicate that PMN apoptosis is inhibited during the acute phase of KD and also suggest that both the resistance against the Fas-mediated death signal and the down-regulation of the mitochondrial apoptotic signalling pathway due to an altered balance of Bcl-2 protein expression are responsible for the delayed PMN apoptosis.

Acute Disease↗
PubMed2001

Mechanism of the inhibitory effect of protease inhibitor on tumor necrosis factor alpha production of monocytes.

If the inflammatory response becomes excessive or uncontrolled by some stimuli, inappropriate inflammatory responses occur. Monocytes are extremely important cells for regulating the cytokine network and tumor necrosis factor alpha (TNFalpha) and interleukin- (IL) 10, which are mainly synthesized by monocytes, are representative cytokines that play a central role in the cytokine network. Protease inhibitors such as gabexate mesilate (GM) and ulinastatin (UTI) have been shown to have various beneficial effects by inhibiting the activation of leukocytes, but the mechanism for this has yet to be fully elucidated. In this study we investigated the mechanism of the inhibitory effect of protease inhibitors on the proinflammatory cytokine production of lipopolysaccharide- (LPS) stimulated monocytes. LPS-stimulated monocytes were treated with GM or UTI. The value of TNFalpha and IL-10 in the culture medium of monocytes was measured and each mRNA expression was assayed. The inhibitory effect of protease inhibitors on the activity of intracellular signal transduction pathways such as protein kinase C (PKC) and nuclear factor kappa B (NFkappaB) were also evaluated. GM decreased the TNFalpha production of LPS-stimulated monocytes as shown by the inhibition of mRNA expression and increased the IL-10 production of LPS-stimulated monocytes. GM also suppressed the NFkappaB activity of LPS-stimulated monocytes. UTI decreased the TNFalpha production of LPS-stimulated monocytes, but did not inhibit the TNFalpha mRNA expression. The present study shows that the inhibitory effect of GM on the TNFalpha production of activated human monocytes is mediated by the suppression of NFkappaB activation, while the mechanism of UTI inhibiting TNFalpha production of human monocytes may be due to the inhibition of either the translation or secretion of TNFalpha.

Cells, Cultured↗
PubMed2001

Screening of differentially expressed genes in 5-fluorouracil-resistant human gastrointestinal tumor cells.

To identify genes differentially expressed in association with resistance to 5-fluorouracil (5FU), an mRNA differential display (DD) analysis was used to compare transcripts from the NUGC-3 human gastric tumor cell line and the NUGC-3 / 5FU / L line, which had acquired 208-fold resistance as a consequence of repeated exposure to escalating concentrations of 5FU. The 110 cDNA fragments differentially expressed in the DD analysis of either the NUGC-3 or NUGC-3 / 5FU / L cells were sequenced and subjected to a homology search, and 29 overexpressed and 22 underexpressed genes were identified in NUGC-3 / 5FU / L as a result. To confirm whether the changes in the gene expression levels in the NUGC-3 / 5FU / L cells were shared by other 5FU-resistant cells, 35 genes were analyzed by northern hybridization in 3 pairs of parent / 5FU-resistant human gastrointestinal tumor cell lines. The analysis revealed 20 overexpressed and 10 underexpressed genes in at least one of the three 5FU-resistant cells as compared with those in the parent cells. Among them, P-glycoprotein, equilibrative nucleoside transporter 1, and methylenetetrahydrofolate dehydrogenase were highly expressed in two of the three 5FU-resistant cells and cytidine deaminase and integrin alpha3 were underexpressed. The acquisition of resistance to 5FU by tumor cells may result from multiple changes in cellular functions.

Antimetabolites, Antineoplastic↗
PubMed2001

Increased monocyte activation in elderly patients after surgical stress.

OBJECTIVE: To investigate age-related changes in the host response to surgical stress. The clinical course, serum interleukin-6 (IL-6) levels, monocyte production of tumor necrosis factor-alpha (TNF-alpha), and monocyte expression of CD11b/CD18 were used as markers of the systemic response. METHODS: Patients with gastric cancer, undergoing distal gastrectomy were divided into 2 groups: >75 years of age (elderly group) and < or =75 years of age (young group). Serum IL-6 levels, TNF-alpha production and CD11b/CD18 expression by monocytes, and the postoperative clinical course were compared between the 2 groups. RESULTS: TNF-alpha production by lipopolysaccharide-stimulated monocytes and CD11b/CD18 expression on monocytes after surgical stress were significantly higher in the elderly than in the young group. Moreover, serum IL-6 levels on the first postoperative day in the elderly group were significantly higher than those in the young group. The incidence and duration of systemic inflammatory response syndrome (SIRS) were significantly greater in the elderly than in the young group. CONCLUSIONS: The activation of monocytes and hypercytokinemia occur readily after surgical stress in the elderly and may therefore contribute to SIRS and increased susceptibility to postoperative complications.

Aged↗