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Biomedical subjects

Y Yokomizo

Publications and source records attributed to Y Yokomizo.

At least 37 records · Page 2Linked to original sources
PubMed2001

Effects of long-term administration of recombinant bovine tumor necrosis factor-alpha on glucose metabolism and growth hormone secretion in steers.

OBJECTIVE: To investigate the effects of long-term administration of recombinant bovine tumor necrosis factor-alpha (rbTNF) on plasma glucose and growth hormone concentrations, and to determine whether treatment with rbTNF causes insulin resistance in steers. ANIMALS: 5 steers treated with rbTNF and 5 steers treated with saline (0.9% NaCl) solution (control). PROCEDURES: In experiment 1, rbTNF (5.0 microg/kg of body weight) or saline solution (5 ml) was administered SC daily for 12 days. Blood samples were obtained before treatment, and plasma was harvested for determination of glucose, insulin, and growth hormone (GH) concentrations. In experiment 2, insulin, glucose, or growth hormone-releasing hormone (GHRH) was administered IV on days 7, 9, and 11, respectively, after initiation of rbTNF or saline treatment in experiment 1. Plasma glucose and insulin concentrations were measured before and at various times for 4 hours after insulin or glucose administration. Plasma GH concentrations were measured at various times for 3 hours after GHRH administration. RESULTS: In experiment 1, administration of rbTNF resulted in hyperinsulinemia without hypoglycemia and decreased plasma GH concentrations. In experiment 2, plasma glucose concentrations were higher in steers treated with rbTNF and insulin than in controls. Plasma GH concentrations were lower in steers treated with rbTNF and GHRH than in controls. CONCLUSIONS AND CLINICAL RELEVANCE: Prolonged treatment with rbTNF induced insulin resistance and inhibited GHRH-stimulated release of GH in steers. Results indicate that rbTNF is a proximal mediator of insulin resistance and inhibits release of GH during periods of endotoxemia or infection.

Animals↗
PubMed2001

Inflammatory cytokines and antigen-responsive mononuclear cells in peripheral blood of cattle infected with Salmonella takoradi.

To determine the immunological response in lactating dairy cows infected with Salmonella (S.) Takoradi, the relationships among distributions of peripheral blood mononuclear cell (PBMC) subpopulations, endotoxin concentrations and dynamics of inflammatory cytokines in blood were investigated. The ratio of CD4+ T cells to CD8+ T cells was significantly lower in the affected cattle than in the control cattle (p<0.05) to decrease in the number of CD4+ T cells in the infected cattle. In contrast, the numbers of gammadeltaT cells, MHC class II-positive cells were significantly higher in the affected cattle than in the control cattle (p<0.01 respectively). Endotoxemia was found in all but one of the affected cattle. Serum IL-1 and IL-6 bioactivities were significantly higher in the affected cattle than in the control cattle (IL-1, p<0.05; IL-6. p<0.01). Serum TNF-alpha activities and levels were not detected in the control and affected cattle. The activities of proinflammatory cytokines determined by the bioassay are important to the relationships between concentration of endotoxin, cytokines and clinical signs. such as leukocytosis, leukopenia, fever or bacterial shedding. Serum IL-2 levels were lower in the affected cattle than in the control cattle. Serum IFN-y was not detected in the affected cattle except one. These results by the ELISA seemed to reflect the condition of subpopulation in the PBMCs from the shedding cattle. The present results suggest that cellular immunity is suppressed while the humoral immunity is activated in acute bovine salmonellosis.

Animals↗
PubMed2000

Expression of a bioactive bovine interleukin-12 using baculovirus.

Recombinant baculoviruses that express recombinant bovine interleukin-12 (rboIL-12) subunits, p35 and p40 subunits were constructed. A recombinant virus containing the p40 subunit gene expressed the p40 subunit as a 40kDa monomer and an 80kDa disulfide-linked homodimer in the infected insect cells and in the culture supernatant. The p35 subunit was expressed in a 30kDa monomer in the infected cells but not in the supernatant. Superinfection of both recombinant viruses into the cells in a spinner flask resulted in the formation of a 70kDa disulfide-bonded heterodimer detected in the supernatant by immunoblotting using anti-p40 and anti-p35 subunits antibodies. The superinfected culture supernatant showed induction of IFNgamma mRNA synthesis and IFNgamma production in bovine peripheral blood mononuclear cells. Thus, the bioactive rboIL-12 was produced in large scale using a baculovirus expression system.

Animals↗
PubMed2000

Production of monoclonal antibodies to porcine interleukin-18 and their use for immunoaffinity purification of recombinant porcine interleukin-18.

We have recently reported the cloning and expression of porcine interleukin-18 (IL-18). In this study, we describe the production of anti-porcine IL-18 monoclonal antibodies (mAb) and their use in the purification of a large amount of recombinant porcine IL-18 by immunoaffinity column chromatography. Five monoclonal antibodies (2-2-B, 2-5-B, 2-13-C, 3-1-C and 5-3-B) were established and characterized. Three (2-2-B, 3-1-C and 5-3-B) of them were of IgG1 subclass, and the other two were IgMs. Epitope analysis of the three IgG1 mAbs showed that they recognized the same epitope. All five mAbs demonstrated reactivity with baculovirus generated porcine IL-18 by immunoblot analysis. Biologically active porcine IL-18 was obtained by immunoaffinity chromatography using anti-porcine IL-18 mAb at more than 85% purity from culture supernatants of Trichoplusia ni (Tn5) derived cells infected with recombinant baculovirus containing the coding sequence of porcine mature IL-18. These results suggest that the anti-porcine IL-18 mAbs established in this study are useful for one-step purification of porcine mature IL-18 as well as the detection of porcine IL-18 by immunoblotting.

Animals↗
PubMed2000

Porcine interleukin 18: cloning, characterization of the cDNA and expression with the baculovirus system.

We have isolated and sequenced a cDNA that contains the coding sequence of porcine interleukin 18 (IL-18) and the recombinant protein of porcine IL-18 was expressed using the baculovirus system. The open reading frame (ORF) of the porcine IL-18 cDNA is 579 base pairs (bp) in length and encodes 192 amino acids. The predicted amino acid sequence is 76.7%, 64.7% and 61.6% homologous to the predicted human, murine and rat amino acid sequences, respectively. The porcine precursor and mature IL-18 protein were expressed respectively in Trichoplusia ni -derived (Tn5) cells using the baculovirus Autografha californica nuclear polyhedorosis virus (AcNPV) as a vector. Tn5 cells infected with recombinant virus containing a whole IL-18 protein coding region sequence secreted porcine precursor IL-18 into the culture medium. On the other hand, Tn5 cells infected with recombinant virus containing a mature IL-18 protein coding region sequence expressed several proteins in the cell lysates, but did not secrete mature protein into the culture medium efficiently. Immunoblotting analysis of recombinant protein showed cross-reactivity with anti-human IL-18 polyclonal antibody. The mature form of porcine IL-18 protein induced IFN-gamma production in suboptimal doses of anti-CD3 antibody and concanavalin A- (ConA) stimulated porcine peripheral blood mononuclear cells (PBMC), but the precursor form had little effect.

Amino Acid Sequence↗
PubMed2000

Cyclosporin A, but not bursectomy, abolishes the protective immunity of chickens against Leucocytozoon caulleryi.

Chickens that survive primary infection with Leucocytozoon caulleryi show strong resistance to reinfection. Using bursectomized (BX) or cyclosporin A (CsA)-treated chickens, we performed experiments to determine which type of immunity, humoral or cellular immunity, plays an important role in the resistance of chickens against reinfection with L. caulleryi. BX chickens were inoculated with 2, 20 or 200 sporozoites of L. caulleryi intravenously at 3 weeks of age. Some BX chickens which were inoculated with 2 or 20 sporozoites survived the primary infection. These birds had no parasites in their peripheral blood after challenge infection with 5 x 10(3) sporozoites, even though they had no antibody to L. caulleryi. In contrast, CsA-treated chickens had parasitemia, serum-soluble antigen and antibodies, as did untreated chickens during primary infection. After secondary infection, CsA-treated chickens had parasitemia and serum-soluble antigen, even though they had specific antibodies to L. caulleryi whereas untreated chickens showed no parasitemia. The number of CD4(+), CD8(+), T cell receptor (TCR) alpha ss-bearing cells and TCRgamma delta-bearing cells decreased markedly in the peripheral blood of CsA-treated chickens compared to those of untreated chickens. Lymphocyte proliferation in response to concanavalin A, and T cell growth factor production, were also markedly suppressed in CsA-treated chickens. These results suggest that cell-mediated immunity plays an important role in the development of resistance of chickens against reinfection with L. caulleryi.

Animals↗
PubMed2000

Effect of intramammary infusion of tumour necrosis factor-alpha on milk protein composition and induction of acute-phase protein in the lactating cow.

The present study was designed to evaluate the effects of tumour necrosis factor-alpha (TNF-alpha) on lactating bovine mammary functions such as milk protein secretion and the integrity of the milk-blood barrier. The effect on the induction of the systemic inflammatory response was also examined using concentrations of serum haptoglobin (Hp), a major inflammatory acute-phase protein, as an index. One hundred micrograms per mammary gland of recombinant bovine (rBo) TNF-alpha or placebo saline was individually infused into a rear mammary gland of each of four lactating cows, and milk and blood samples were collected before and 4, 8, 24, 32, 48, 96 and 168 h after infusion. In the rBoTNF-alpha-infused gland, increases of somatic cell counts were observed at 4-48 h. Although concentrations of total milk protein were not changed, compositions of milk proteins varied following rBoTNF-alpha infusion. Concentrations of caseins, alpha-lactalbumin and beta-lactoglobulin were significantly decreased at 4 and 8 h. Lactoferrin concentrations were significantly increased at 4 h. Significant infiltrations of serum albumin, immunoglobulin G1 (IgG1) and IgG2 were observed at 4 and 8 h. Elevations of the serum concentration of Hp were detected at 8-32 h, but were very small in comparison with those reported in inflammatory diseases. Changes in rectal temperature and white blood cell counts were not significant. These results show that single rBoTNF-alpha infusion into the lactating mammary gland suppresses the lactogenic function of the gland and influences the function of the milk-blood barrier, with little effect on the generalized inflammatory response.

Acute-Phase Proteins↗
PubMed2000

Detection of porcine interleukin-18 by sandwich ELISA and immunohistochemical staining using its monoclonal antibodies.

We describe here the development of sandwich enzyme-linked immunosorbent assay (sandwich ELISA) and immunohistochemical staining for porcine interleukin-18 (PoIL-18) and their application to detection of PoIL-18 in vivo. Ten anti-PoIL-18 monoclonal antibodies (mAb), all of which were reactive with recombinant PoIL-18 by Western blotting, were established. Four (2-C-4, 9-H-6, 11-H-5, and 12-C-12) of 10 neutralized the biologic activity of PoIL-18 to induce interferon-y (IFN-gamma) from porcine peripheral blood mononuclear cells (PBMC). Four (2-C-4, 5-F-6, 9-H-6, and 12-C-12) of 10 were shown to be useful in immunohistochemical staining and detected PoIL-18 in Kupffer cells and macrophages in hepatic focal necrosis and macrophages in interstitial pneumonia in piglets with experimental endotoxemia using formalin-fixed, paraffin-embedded sections. A sandwich ELISA was developed using mAb 7-G-8 as a capture antibody and biotinylated mAb 5-C-5 as a detection antibody. This ELISA detected PoIL-18 with a minimum detectable concentration of 20 pg/ml and did not show cross-reactivity against PoIL-1beta, IL-8, IL-12, and IFN-gamma or murine and human IL-18. Using this ELISA, PoIL-18 was detected in the plasma and the bronchoalveolar lavage fluid (BALF) of pigs experimentally infected with Actinobacillus pleuropneumoniae. The availability of this ELISA and immunohistochemical staining for PoIL-18 may contribute to a further understanding of the role of this cytokine in various porcine immune responses and diseases.

Animals↗
PubMed2000

The inhibitory effect of interferon gamma and tumor necrosis factor alpha on intracellular multiplication of Neospora caninum in primary bovine brain cells.

Primary culture of bovine brain cells was examined for its susceptibility to Neospora caninum infections, and this model was used to investigate the effects of bovine interferon gamma (IFN-gamma) and tumor necrosis factors alpha (TNF-alpha) on tachyzoite growth. Tachyzoites of N. caninum grew well in this culture, and tachyzoite growth in astroglia and microglia were confirmed by immunocytochemical staining. IFN-gamma inhibited the tachyzoite growth, and this inhibition was not reversed by the addition of nitric oxide antagonist. TNF-alpha, to a lesser extent, also inhibited the tachyzoite growth. Th-1 type cytokines may play an important role in host defense mechanisms in N. caninum infection.

Animals↗
PubMed2000

Cloning and chromosomal assignment of the porcine interleukin-2 receptor alpha (IL-2Ralpha) gene.

Porcine genomic DNA encoding a 55 kDa subunit of interleukin-2 receptor (IL-2R), which is termed alpha chain (IL-2Ralpha), was cloned by repeated plaque hybridization using IL-2Ralpha cDNA as a probe. Two different lambda phage clones, one of which encoded exon 1 and the 5'-upstream flanking region of IL-2Ralpha gene and another encoded the sequence from exon 2 to exon 8, were isolated. By analysis of the 5'-upstream region of the gene, putative binding motifs for transcription factors such as GATA family proteins, Ikaros, NF-kappaB, NF-IL2Ralpha and SRF, were found as described in human, murine and bovine genes. Two additional motifs for STAT4 binding were also found in this region. Moreover, using the FISH technique, we assigned the porcine IL-2Ralpha locus to the distal end of the long arm of chromosome 10 (10q6-qter) where the vimentin gene had been assigned nearby.

Animals↗
PubMed2000

Reduced formation of granulomata in gamma(delta) T cell knockout BALB/c mice inoculated with Mycobacterium avium subsp. paratuberculosis.

The role of gamma(delta) T cells in the bovine immune response to Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) infection is poorly understood. Accordingly, using BALB/c mice that are innately susceptible to M. paratuberculosis, we compared wild-type and gamma(delta) T cell knockout BALB/c mice to study the protective roles of gamma(delta) T cells in M. paratuberculosis infection. Ten-week-old mice were inoculated intraperitoneally with either a low dose (4 x 10(6) colony-forming units [CFU]/mouse) or a high dose (4 x 10(9) CFU/mouse) of M. paratuberculosis strain ATCC 19698. Histopathologic and morphometric examinations showed reductions in the number and area of granulomatous lesions in the liver of the knockout mice at 18 weeks after inoculation with either the low or the high dose of the mycobacteria. Furthermore, at 18 weeks after inoculation, the bacterial load in the spleens of the knockout mice inoculated with the high dose was significantly lower than that of wild-type mice. No differences were found in bacterial load between the knockout and the wild-type mice in the low-dose groups. In contrast, in the livers of wild-type mice inoculated with either the low or high mycobacterial dose, increased areas of epithelioid granulomata were observed and the granulomata became disseminated widely during the experimental period. These findings in model mice suggest that gamma(delta) T cells, rather than restricting mycobacterial growth, may play a crucial role in development of epithelioid granulomata similar to those seen consistently in bovine paratuberculosis. The results of this study may have relevance to our understanding of the pathogenesis of paratuberculosis in ruminants, in which a prominent number of gamma(delta) T cells exist in the lymphoid system.

Animals↗
PubMed2000

Administration of recombinant bovine tumor necrosis factor-alpha affects intermediary metabolism and insulin and growth hormone secretion in dairy heifers.

Four experiments were conducted to clarify the effect of intravenous (i.v.) administration of recombinant bovine tumor necrosis factor alpha (rbTNF) on selected metabolites and on hormone secretion in Holstein heifers (n = 6; 347.0 kg average BW). In Exp. 1, rbTNF was injected at three dosage levels in a Latin square; 0 (CONT), 2.5 (TNF2.5), or 5.0 (TNF5) microg/kg BW. Plasma glucose and triglyceride concentrations were at first elevated (P < .05) by rbTNF treatment and then were decreased (P < .05) by TNF2.5 and TNF5. Plasma NEFA concentrations were increased (P < .05) in rbTNF-treated groups. The injection of rbTNF resulted in an increase in plasma insulin levels (P < .05 with TNF5) during the period between 2 and 24 h, except for the period between 6 and 8 h, after the treatment. In Exp. 2, 3, and 4, each heifer received i.v. injections of glucose (.625 mM/kg BW) + rbTNF (5 microg/kg) or glucose + saline (10 mL) (Exp. 2), insulin (0.2 U/kg) + rbTNF or insulin + saline (Exp. 3), and GHRH (0.25 microg/kg) + rbTNF or GHRH + saline (Exp. 4) at 1-wk intervals. In Exp. 2, rbTNF inhibited (P < .05) glucose-stimulated insulin secretion during the initial phase. Thereafter, plasma insulin was higher (P < .01) with the glucose + rbTNF treatment than with the glucose + saline treatment. Treatment with rbTNF inhibited the insulin-stimulated glucose utilization (Exp. 3) and GHRH-stimulated GH secretion (Exp. 4) during the initial phase. These results suggest that rbTNF directly and(or) indirectly affects the intermediary metabolism and hormone secretion in Holstein heifers.

Animals↗
PubMed2000

Cerebral ischemia as a causative mechanism for rapid progression of brain atrophy in chronic hemodialysis patients.

BACKGROUND: It has been found that brain atrophy develops more rapidly in patients with end-stage renal failure after initiation of dialysis therapy. The present study was designed to analyze the relationship between brain atrophy and asymptomatic ischemic brain lesions. PATIENTS AND METHODS: Magnetic resonance imaging (MRI) was performed for the evaluation of brain atrophy and ischemic lesions. Brain atrophy was assessed by the ventricular-brain ratio (VBR), calculated as the ratio of the ventricular area to the whole brain area on the maximum MRI slice. The severity of periventricular hyperintensity (PVH) and the number of lacunae were also regarded as ischemic brain lesions. Fifty-five patients undergoing maintenance hemodialysis (HD) without clinically overt neurological signs and symptoms, with a mean age of 52 +/- 11 (SD) years and a mean HD duration of 7 +/- 6 (SD) years were subjected. VBR and its relationship to ischemic brain lesion data were compared to those in 35 non-HD patients (controls), with a mean age of 42 +/- 14 (SD) years. RESULTS: The VBR, the number of lacunae and the severity of PVH tended to increase with age in HD. The VBRs at all age groups were significantly higher in HD than in controls (7.0 vs 3.7% at the 4th decade, p < 0.05; 8.4 vs 5. 9% at the 5th decade, p < 0.05; 9.6 vs 5.4% at the 6th decade, p < 0.05; and 11.6 vs 6.3% at the 7th decade, p < 0.05). HD patients had significantly higher number of lacunae and had more advanced PVH than did controls. Both the number of lacunae and the severity of PVH were significantly correlated to VBR in HD. CONCLUSION: In conclusion, the rapid progression of brain atrophy was related to the asymptomatic ischemic brain lesions in our HD patients. Such data indicated that cerebral ischemia might be a causative mechanism of brain atrophy in chronic hemodialysis patients.

Atrophy↗
PubMed1999

Production of biologically active, heterodimeric porcine interleukin-12 using a monocistronic baculoviral expression system.

A baculoviral expression system for the production of biologically active, heterodimeric interleukin (IL)-12 was developed by utilizing foot-and-mouth disease virus (FMDV) self-cleaving peptide, 2A. Recombinant porcine IL-12 (rpoIL-12) was produced by insect cells after infection with recombinant baculoviruses expressing the gene encoding a fusion protein of p35 and p40 subunits of IL-12 connected with 2A. By reducing and non-reducing SDS-PAGE analyses, it was demonstrated that rpoIL-12 had a heterodimeric structure which was resulted from 2A-dependent cleavage of the precursor fusion protein. In contrast, uncleaved, monomeric rpoIL-12 was produced by infection with baculoviruses expressing the gene lacking the 2A sequence. To assess the biological activities of these recombinants, we performed the proliferation assays of PHA-activated human PBMCs. The heterodimeric rpoIL-12 induced proliferation in a dose-dependent manner, whereas the uncleaved rpoIL-12 did not. Moreover, such biological activity was specifically inhibited by addition of anti-IL-12 antibodies or rpoIL-12 p40. These observations suggest that FMDV 2A can exert its self-cleaving activity even in a heterologous system, and that biologically active, heterodimeric rpoIL-12 can be generated by monocistronic expression of the p35/p40 fusion gene in combination with the 2A sequence.

Amino Acid Sequence↗
PubMed1999

Biological functions of recombinant bovine interleukin 6 expressed in a baculovirus system.

The cDNA encoding bovine interleukin 6 (IL-6) was obtained from messenger RNA extracted from lipopolysaccharide-stimulated bovine Kupffer cells by the reverse transcription polymerase chain reaction (RT/PCR), and cloned into the baculovirus vector pVL 1392. Insect cells (Sf21AE derived from Spodoptera frugiperda) infected with the recombinant baculovirus secreted a large amount of 23.7 kD protein into the culture medium. This protein was capable of causing increased haptoglobin production and decreased albumin production in primary cultured bovine hepatocytes. The swine and human IL-6s were also able to decrease albumin production in bovine hepatocytes. This recombinant IL-6 did not stimulate the proliferation of 7TD1 cells (a murine IL-6-dependent cell line), whereas the recombinant swine IL-6 which was expressed in the same baculovirus system, and recombinant human IL-6 derived from Escherichia coli were each capable of stimulating proliferation of 7TD1 cells, respectively. This suggests a species restriction between bovine IL-6 and murine IL-6 dependent cell lines.

Acute-Phase Proteins↗
PubMed1999

Up-regulation of tumor necrosis factor alpha mRNA is associated with bovine-leukemia virus (BLV) elimination in the early phase of infection.

Protective immune responses were analyzed in eight sheep vaccinated with BLV envelope peptides and experimentally infected with bovine-leukemia virus (BLV). Five of eight peptide-immunized sheep showed a high T-cell proliferative response to the BLV peptides and all of these were protected from the infection. The other three peptide-immunized sheep showed no T-cell proliferative responses to any BLV antigens similar to control sheep, though they also exhibited resistance to BLV challenge. To investigate other mechanisms which suppress BLV expansion in these non-responding sheep, we measured the levels of the cytokine expressions before, and after, BLV challenge using competitive reverse-transcriptase polymerase chain-reaction systems. It was revealed that the expression of tumor necrosis factor alpha (TNFalpha) was higher in BLV-resistant sheep than in BLV-susceptible sheep. Thus, TNFalpha expression rather than specific T-cell activity may play an important role in the protective mechanism against BLV infection, at least during the primary viremia phase.

Amino Acid Sequence↗
PubMed1999

Renal complications in patients with diabetes mellitus associated with an A to G mutation of mitochondrial DNA at the 3243 position of leucine tRNA.

The substitution of guanine for adenine at position 3243 of the leucine tRNA gene of mitochondrial DNA was originally described in association with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes). Diabetes mellitus associated with the mutation (mitochondrial diabetes) is a different phenotype from MELAS. We identified 11 patients with the mutation among 385 Japanese diabetic patients: two had MELAS and nine had mitochondrial diabetes. We present data on a male patient with mitochondrial diabetes who developed the nephrotic syndrome at the age of 23. Light microscopy revealed mesangial expansion, PAS-positive deposits and segmental sclerosis in the glomeruli. Scattered mesangial electron-dense deposits and thickening of the basement membrane were found on electron microscopy, suggesting that diabetic glomerulosclerosis accompanied by focal glomerulosclerosis (FGS). Mitochondrial diabetes may pre-dispose patients to renal complications, including forms of glomerulonephritis, such as FGS.

Adult↗