Discordant effects of xenogeneic pig liver perfusion on function of sinusoidal endothelial cells and parenchymal cells.
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Biomedical subjects
Publications and source records attributed to T Inamoto.
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The apical canal sealing ability of newly developed calcium phosphate cement (TDM-C), an equimolar mixture of tetracalcium and dicalcium phosphate dihydrate kneaded with McIlvain's buffer solution containing sodium carboxymethyl cellulose, was evaluated in a relative comparison with several recognized available products. The latter included BONETRIX (alpha-tricalcium phosphate mixed with citric acid and tannic acid), ARS (alpha-tricalcium phosphate and hydroxyapatite mixed with polyacrylic acid), and zine oxide eugenol sealer (ZOE). Canals of forty extracted human maxillary central incisors were prepared to the file size #70, divided into four groups, and obturated with each of the four above materials. Setting of all sealers took place either in 30% bovine serum solution or in phosphate buffered saline solution. Teeth were then decalcified and cleared to measure linear penetration of India ink from the apex into the canal. Results were statistically analyzed by Student's t-test. Surfaces of all materials, after setting, were also observed by scanning electron microscopy (SEM). We found that TDM-C, BONETRIX, and ZOE all showed excellent apical sealing ability whereas ARS was less effective in this regard. SEM analysis revealed micropores on the surface of every apatitic product, but ZOE was free of such porosity. It was concluded that TDM-C and BONETRIX may prove clinically useful as root canal sealant materials.
To evaluate the roles of extracellular FKBP12, we examined the effect of extracellular FKBP12 on the immunosuppressive activity of FK506 in vitro and clinically. The ability of FK506 to suppress phytohemagglutinin-induced proliferative response of human peripheral blood mononuclear cells was inhibited in the presence of recombinant FKBP12 dose-dependently. We measured plasma levels of FKBP12 using a newly developed enzyme-linked immunosorbent assay system in 34 patients receiving FK506 after liver transplantation. In 7 patients with acute cellular rejection, plasma FKBP12 increased significantly at the onset of rejection compared with 1 week before onset (P < 0.05) and further increased to or remained at more than 250 ng/ml 1 week after onset. In 22 of 27 patients without acute cellular rejection, plasma FKBP12 was less than 70 ng/ml during the 4 weeks after transplantation. In the other 5 of 27 patients without acute cellular rejection, plasma FKBP12 exceeded 250 ng/ml. Rapid increase of plasma FKBP12 was observed in only one of these 5 patients, at the onset of high fever due to a liver abscess. There was no significant difference in whole blood trough levels of FK506 between the patients with or without acute cellular rejection. These results suggest that the rapid increase in plasma levels of FKBP12 may contribute to the occurrence and progress of acute cellular rejection probably by inhibiting the immunosuppressive activity of FK506.
The safety and lack of undue operative stress on the donor are documented from an analysis of 100 parental donors, whose children (3 months to 17 years old), received LRLTx at our institution between June 1992 and May 1994. Survival rate of recipients was 86%. No primary nonfunctioning liver was observed. The donors were 56 mothers and 44 fathers. Their ages ranged from 19 to 51 years and their weight ranged from 44 to 80 kg. They received partial liver resections to harvest the grafts. With regard to the liver graft, the left lobe was used in 24 cases (group L) and the left lateral segment was used in 75 cases (group S). The right lobe was used in one case. In the two groups, blood losses were 242 +/- 5 (S) and 312 +/- 14 ml (L); operation times were 6.22 +/- 0.11 (S) and 7.15 +/- 0.21 hr (L), respectively; in both groups, the postoperative hospital stay was 11 days (S, L). No significant differences between the two groups were observed in peripheral RBC and WBC count or serum AST. An increase in total bilirubin was not observed. In the exceptional case using the right lobe, blood loss of 2300 ml necessitated a blood transfusion of 1000 ml, and the total bilirubin increased up to 4.0 mg/dl on the third postoperative day, which prolonged the postoperative hospital stay to 17 days. These results conclusively suggest that safety is guaranteed when the left lobe or the left lateral segment is used as the liver graft for LRLTx.
Hepatic resection promotes such a high degree of surgical stress that it induces deterioration of various vital functions, which may involve the breakdown of signal transduction systems. To investigate the influence of surgical stress on signal transduction, we studied ligand-receptor specific binding activity after hepatic resection, focusing on lymphocyte beta 2-adrenoceptors. The maximum binding capacity (Bmax) and the dissociation constant (KD) were determined by radioligand binding assay using (-)3H-CGP12177 as a ligand. In the hepatectomy group, Bmax significantly decreased from 1380 +/- 109 to 799 +/- 49 receptors/cell on postoperative day (POD) 3 and to 802 +/- 93 receptors/cell on POD 7 (P < 0.05). In the control group, however, it did not significantly change after the operation. No significant changes in KD were found in either of these groups. The Bmax alteration was not due to the redistribution of lymphocyte subsets or receptor down regulation, but to the decrease in the Bmax of the individual subset. The hepatectomy group was divided into two groups according to the postoperative arterial ketone body ratio (AKBR): Group A, AKBR maintained at 0.7 or more; and Group B, AKBR decreased to below 0.7. The Bmax decrease, a percentage of the preoperative value, of Group B was significantly smaller than that of Group A (48.4 +/- 3.9 and 72.3 +/- 7.3%, respectively, P < 0.05). These results suggest that intense surgical stress, produced by hepatic resection, may influence even ligand-receptor binding parameters, and the decrease in AKBR can indicate the magnitude of surgical stress.
It has been shown that the progression of hepatic fibrosis in intrahepatic cholestasis (IHC) is not so prominent as in extrahepatic biliary atresia (EHBA), and that the biosynthetic activity of collagen increases along with the fibrotic disease process. We conducted immunohistochemical and ultrastructural studies on the distribution of collagen types III and IV and of alpha-actinin in the smooth musculature in liver specimens obtained from 4 patients with IHC and 14 patients with EHBA in liver transplantation from living related donors (LRLT). A recently developed sandwich enzyme immunoassay (EIA) was used to determine serum concentrations of type IV collagen, laminin and prolyl 4-hydroxylase (PH) in the patients before and after LRLT. Pathological study showed that the excessive deposition of type IV collagen in the perisinusoidal walls resulted in a clearly developed basal membrane beneath the sinusoidal endothelial cells, so-called sinusoidal capillarization. In the fibrous septa of IHC, fibrogenesis was apparently lower than in EHBA, since collagen deposition and myofibroblast proliferation were not so prominent compared to EHBA. Serum type IV collagen, laminin and PH increased in IHC, although not so markedly as in EHBA, and returned to normal within 5 weeks after successful LRLT. In conclusion, it is suggested that an increased level of serum type IV collagen reflects the de novo synthesis of basal membrane components, and that the determination of their serum levels by EIA can be utilized for the differentiation of the specific fibrogenic activity in each disease, and for monitoring patients before and after liver transplantation.
Adult T cell leukemia-derived factor (ADF), which is identical to a disulfide reducing enzyme human thioredoxin (TRX), is produced and released by activated or virus-infected lymphocytes. Here we report that, in peripheral blood mononuclear cells (PBMC) stimulated with phytohemagglutinin (PHA), ADF/TRX mRNA was induced within 8 h after stimulation as detected by in situ hybridization study. To analyze the mechanism of ADF/TRX induction during T cell activation, the effects of immunosuppressants including FK506, rapamycin (Rap) and cyclosporin A (CsA) on ADF/TRX expression were investigated by immunoblot analysis. ADF/TRX induction in PBMC by PHA, Con A or OKT3 mAb was almost completely suppressed by FK506. Whereas CsA also inhibited ADF/TRX expression in OKT3 mAb-stimulated PBMC, Rap failed to affect it in spite of exhibiting growth inhibition. In addition, exogenous IL-2 could not increase ADF/TRX production in FK506-treated PBMC or in PHA blasts. These results indicate that ADF/TRX induction in T cell activation depends on calcineurin-dependent events in the early phase and that IL-2 production is not directly involved in ADF/TRX induction. Furthermore, when recombinant ADF (rADF) was added to a culture of PBMC 1 h before the addition of PHA and FK506, the action of FK506 was partially reversed as determined by [3H]thymidine incorporation and viable cell counts. These results suggest that ADF/TRX produced and released from PBMC may be a crucial event in lymphocyte activation, and that FK506 and CsA may exert the immune suppression partly through inhibiting the induction of the endogenous reducing factor ADF/TRX.
Biochemical and antigenical characteristics of tannin-protein complex degrading enterobacteria (T-PCDE) isolated from Koalas, Phascolarctos cinereus, were investigated. T-PCDE had a specific profile of characteristics, and T-PCDE was distinguished from those of 12 type strains of Enterobacteriaceae used.
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Functional abnormalities of the liver uncovered during preoperative routine evaluation were analyzed in 109 donor candidates for 100 cases of living-related liver transplantation (LRLT) performed during the period from June, 1990 to May, 1994 at the Second Department of Surgery, Kyoto University Hospital. High serum transaminase (GOT, GPT) levels were noted in 10 (9.2%) cases among 109 candidates, high alkaline phosphatase in 4 (3.7%), hyperbilirubinemia in 3 (2.8%), anemia in 3 and high choline esterase in 3 cases. Positive hepatitis C antibody (HCV) was also noted in 1 case. Fatty liver was detected in 10 (9.2%) cases, cholecystitis in 2 cases, 1 case each of cyst and calcification in the liver by diagnostic imaging (ultra sonograph and/or computed tomography). These abnormalities of the liver necessitated replacing the initial candidate with the other parent in 9 cases, including 1 case without any functional abnormality whose graft liver was too large to fit the recipient abdominal cavity. There were 14 cases of ABO blood type incompatible combination. Switching the initial candidate due to these abnormalities mentioned above resulted in incompatible combinations in 4 of these 14 cases. Although the advantages of the LRLT are the superior viability of the donor graft and the genetic histocompatibility between recipient and donor, to optimize the advantage of LRLT, all donor candidates should be strongly advised to make every effort preoperatively to improve their physical condition in preparation for the LRLT protocol, since many of these abnormalities are typically reversible.
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The low-affinity Fc receptor for IgE (Fc epsilon RII/CD23) and its soluble form (sCD23, IgE-binding factor) have multiple functions, and enhanced levels of these are associated with various immunological diseases. We established two sensitive ELISA systems using enzyme-conjugated mAb and biotinylated mAb. The detection limits of the ELISA systems were 0.03 and 1.0 ng/ml, which showed good correlation in the range 1.0-10 ng/ml. In the ELISA system using enzyme-conjugated mAb, the average sCD23 concentration in 303 normal healthy volunteers was 1.4 +/- 0.3 ng/ml. In the ELISA system using biotinylated mAb, sCD23 levels in normal healthy volunteers showed almost the same values. In patients with autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, Sjögren syndrome, progressive systemic sclerosis, and mixed connective tissue disease, the sCD23 levels were significantly higher than those in normal individuals. Furthermore, in Epstein-Barr virus-related disorders after liver transplantation with immunosuppression, plasma levels of sCD23 rapidly increased to more than 12 ng/ml when clinical symptoms were evident. In addition, the sCD23 values remained high, although elevated GOT levels gradually decreased to standard values and EBV hepatitis improved. These data suggest that sCD23 levels are a sensitive marker of autoimmune diseases and EBV-related disorders in addition to allergic disorders. The ELISA system for sCD23 may be an additional diagnostic tool in estimating the clinical courses of these diseases.
Adult T cell leukemia-derived factor (ADF), originally defined as an interleukin 2 receptor/alpha (alpha) chain inducer produced by human T-lymphotropic virus type-I transformed cells, is identical to human thioredoxin (TRX). In this study, the protective effect of ADF/TRX on the cytotoxicity of endothelial cells caused by phorbol myristate acetate (PMA)-activated neutrophils or hydrogen peroxide (H2O2) was examined. When murine endothelial F-2 cells established from an ultraviolet light-induced tumor on a nude mouse were incubated with PMA-activated neutrophils or with 1 mM H2O2 for 6 hours, the cytotoxicity of F-2 cells was respectively 51 +/- 4% or 40 +/- 8% by the 51Cr releasing assay. Recombinant ADF/TRX (rADF/TRX) inhibited this cytotoxicity in a dose-dependent manner, although mutant ADF/TRX (cysteine 31 to serine), 2-mercaptoethanol and dithiothreitol did not. On a molar basis, rADF/TRX was more effective than glutathione but less effective than catalase. Immunoblotting analysis showed that treatment with 0.1 mM H2O2 induced murine TRX on F-2 cells. These findings indicate that ADF/TRX is an oxidative stress-inducible endogenous protein and rADF/TRX plays a protective role against activated neutrophils- or H2O2-induced endothelial cytotoxicity.
The antibiotic susceptibility of thirty-nine strains of Mycoplasma hyopneumoniae isolated from swine between 1970-1981 and 1989-1990 was investigated. From the present results, it is suggested that the susceptibility to chlortetracycline has been decreasing in Japan. On the other hand, all the strains were sensitive to lincomycin, thiamphenicol and macrolides. Newly developed macrolides such as tilmicosin, acetyl-isovaleryl-tylosin and mirosamycin had equal or higher activity than general macrolides.
Sixty one strains of Pasteurella multocida and 35 strains of Actinobacillus pleuropneumoniae isolated from pneumonic lesions of porcine lungs during the period from 1985 to 1989 in Japan were tested for antibiotic susceptibility to chlortetracycline (CTC), thiamphenicol (TP), tylosin (TS), acetylisovaleryl-tylosin (AIV-TS), tilmicosin (TMS), mirosamycin (MRM). Most strains of both species were sensitive to CTC, TP and TMS. Growth of fifty-one strains (83.6%) and forty-six strains (75.4%) of P. multocida were inhibited with 3.13 micrograms/ml of CTC and 0.78 micrograms/ml of TP, respectively. TS showed low activity against almost all strains (MIC > or = 6.25 micrograms/ml). Fifty-eight (95.1%), twenty-three (37.7%) and fifty (82%) of P. multocida showed MICs of > or = 6.25 micrograms/ml against AIV-TS, TMS and MRM, respectively. The MICs of A. pleuropneumoniae against CTC were less than 1.56 micrograms/ml. Thirty-two strains (91.4%) and 33 strains (94.3%) of A. pleuropneumoniae were inhibited with 3.13 micrograms/ml of TP and TMS respectively. However, TS, AIV-TS and MRM showed low activity against all of A. pleuropneumoniae (MIC > or = 6.25 micrograms/ml). Three different resistance patterns were observed in P. multocida and two in A. pleuropneumoniae isolates, respectively.
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The presence of activated lymphocytes in the blood of transplant recipients is considered to be a marker of an ongoing immune process. In this study, the clinical utility of spontaneous proliferation (SP) of peripheral blood mononuclear cells (PBMC) as a marker of in vivo activation was evaluated by in vitro [3H]thymidine incorporation in 22 patients in the 1st month after liver transplantation. Also immune activation in the graft was monitored by transplant aspiration cytology (TAC-A). In the study period, there were only 2 mild episodes of clinical acute rejection, where SP of PBMC was 1290 and 1541 cpm (vs. 99 cpm averaged in healthy controls). Though SP of PBMC was also increased in systemic infections, a significant positive correlation was observed between SP of PBMC and TAC-A score. In all episodes where TAC-A score was elevated to above 2, SP of PBMC was simultaneously increased to more than 1000 cpm. In 8 patients prophylactically treated with the anti-interleukin-2-receptor antibody, BT563, SP of PBMC was significantly lower compared to 7 patients treated with antithymocyte globulin. It is suggested that SP can be a reliable parameter of the in vivo activation of lymphocytes, which accompanies immune activation in liver graft, and is potentially useful as a sensitive, although rather non-specific, and non-invasive monitoring of intragraft alloresponse.