Hepatobiliary phase hypointense nodule without arterial phase hyperenhancement as a risk factor for late recurrence (>1 year) of hepatocellular carcinoma after surgery.

AIM: To evaluate the value of magnetic resonance imaging (MRI) features, including liver stiffness measured by magnetic resonance elastography (MRE) and the presence of hepatobiliary phase (HBP) hypointense nodule without arterial phase hyperenhancement (APHE), for predicting late recurrence (>1 year) after surgery for hepatocellular carcinoma (HCC). MATERIALS AND METHODS: This retrospective study included 124 consecutive patients who had undergone surgery for HCC and preoperative MRI. After excluding patients with early recurrence within 1 year after surgery, 89 patients were analysed. Preoperative MRI images were reviewed by a radiologist to record imaging findings, including (1) liver stiffness by MRE, (2) size of the HCCs, (3) number of HCCs, and (4) presence of HBP hypointense nodule without APHE. Pathological findings included tumour grade, vascular/biliary/capsule invasion, and fibrosis stage of the liver. Considering imaging/pathological findings and patients' characteristics as dependent variables, Cox proportional hazards model analysis was performed to identify independent factors associated with late recurrence after surgery. RESULTS: The median follow-up period was 37.3 months. During follow-up, 29 patients (32.5%) developed late recurrence after surgery. In multivariate analysis, underlying liver disease (viral hepatitis) and presence of HBP hypointense nodules without APHE (p=0.010 and 0.033, respectively) were independently associated with disease-free survival (DFS). Kaplan-Meier analysis revealed that patients with HBP hypointense nodules without APHE had a significantly lower DFS rate than those without the nodule (39.2% versus 74.1% at 3 years after surgery, p=0.008). CONCLUSION: The presence of HBP hypointense nodules without APHE was an indicator of late recurrence after surgery for HCC.

Cellular responses of rat periodontal ligament cells under hypoxia and re-oxygenation conditions in vitro.

BACKGROUND AND OBJECTIVE: The aim of this study was to investigate the responses of periodontal ligament cells under hypoxia and re-oxygenation conditions in vitro. MATERIAL AND METHODS: Periodontal ligament fibroblasts were isolated from rat incisors. In the hypoxia group, cells were incubated in 2% O(2) for 1-3 d. In the re-oxygenation group, cells were first incubated under the same conditions as the hypoxia group for 24 h and then were returned to normoxic conditions and cultured for 1-2 additional days. RESULTS: Proliferation ratios increased in all groups in a time-dependent manner. Proliferation ratios in both the hypoxia and re-oxygenation groups were significantly higher than in the control group on days 2 and 3. Alkaline phosphatase activity was significantly higher in the hypoxia group than in the control and the re-oxygenation groups. The expression of bone sialoprotein mRNA was significantly higher in the hypoxia group than in the control group on days 1 and 2. The expression of vascular endothelial growth factor mRNA was significantly higher in the hypoxia group than in the control group on days 1 and 2. In the re-oxygenation group, the level of expression of bone sialoprotein mRNA and vascular endothelial growth factor mRNA were similar to those of the control group. The expression of heat shock protein 70 mRNA in the hypoxia group was similar to that in the control group, whereas in the re-oxygenation group it was statistically higher than in the other groups. CONCLUSION: These results suggest that periodontal ligament cells maintain their osteogenic ability in hypoxia and re-oxygenation conditions in vitro.

Evaluation of microencapsulated islets in agarose gel as bioartificial pancreas by studies of hormone secretion in culture and by xenotransplantation.

Islets were microencapsulated in agarose gel for examination of the possible use of microencapsulated islets as a bioartificial pancreas. Microencapsulated islets secreted insulin into the culture medium (RPMI-1640) and could rapidly increase their insulin release in response to a glucose challenge even after greater than 100 days. Hamster islets in groups of 400-1000 encapsulated in microbeads containing 11-14% (wt/wt) agarose were xenogenically transplanted into the peritoneal cavity of five diabetic mice. The longest normoglycemic period in these mice was 53 days, which was markedly longer than the normoglycemic period obtained by nonencapsulated islets. Agarose seems to be a suitable basic material for encapsulating islets, because the islets can easily be microencapsulated without any adverse effect on the islet function.

Elevated caspase-3 activity in peripheral blood T cells coexists with increased degree of T-cell apoptosis and down-regulation of TCR zeta molecules in patients with gastric cancer.

To evaluate the mechanisms of T-cell dysfunction in patients with gastric cancer, we investigated the caspase activity of T cells, the induction of spontaneous T-cell apoptosis, the expression of T-cell receptor (TCR) zeta molecules, and the ability of T cells to produce cytokines in peripheral blood lymphocytes from patients (n = 22) and healthy controls (n = 14). The caspase-3 activity of T cells was studied as the protease activity of caspase-3 using the cell-permeable substrate of PhiPhiLux G1D2. Flow cytometric analysis was performed with triple staining by annexin V-FITC, propidium iodide, and CD3-R-phycoerythrin-Cy5 for the detection of T-cell apoptosis and with intracellular staining using permeabilized cells for the expression of TCR-zeta molecules. IFN-gamma and tumor necrosis factor alpha production from T cells was evaluated in response to anti-CD3 stimulation. Caspase-3 activity of peripheral blood T cells from patients with advanced disease was significantly increased compared with that from controls [15.5 +/- 3.6 mean fluorescence intensity (MFI) versus 11.5 +/- 3.3 MFI; P = 0.0068]. Parallel to this, the apoptosis of peripheral blood T cells from patients with advanced disease was significantly higher than for those from controls (16.5 +/- 15.5% versus 4.8 +/- 2.7%; P = 0.010). Furthermore, the expression of TCR-zeta molecules in patients with advanced disease was significantly decreased in comparison with that of the controls (41.0 +/- 13.9 MFI versus 56.7 +/- 16.3 MFI; P = 0.014), and this decreased expression coexisted with impaired IFN-gamma (42.4 +/- 43.2 pg/ml versus 1,757.4 +/- 2449.0 pg/ml; P = 0.031) and tumor necrosis factor alpha (682.6 +/- 519.3 pg/ml versus 1,686.0 +/- 1,533.7 pg/ml; P = 0.041) production of T cells. Thus, peripheral blood T cells from gastric cancer patients simultaneously exhibit an elevated caspase-3 activity, an increased degree of T-cell apoptosis, a down-regulation of TCR-zeta molecules, and impaired cytokine production. These observations suggest that induction of T-cell apoptosis coexisting with a down-regulation of TCR-zeta molecules may be responsible for T-cell dysfunction in patients with gastric cancer.

Characterization of new monoclonal antibodies against porcine lymphocytes: molecular characterization of clone 7G3, an antibody reactive with the constant region of the T-cell receptor delta-chains.

A battery of mouse monoclonal antibodies (mAbs) reactive with porcine peripheral blood (PB) leukocytes was generated. Among the mAbs, 6F10 was found to react probably with cluster of differentiation (CD)8 alpha-chain, while 7G3 and 3E12 were found to recognize gammadelta T-cells, as revealed by two-color flow cytometric and immunoprecipitation studies. 7G3 was shown to react with the constant (C) region of the T-cell receptor (TCR) delta-chain by the following facts: (1) 7G3 immunoprecipitated full-length TCR delta-chain protein fused with glutathione S-transferase (GST) produced by Esherichia coli and (2) 7G3 reacted with TCR delta-chain expressing Cos-7 cells transfected with either full-length or N-terminal deleted mutant cDNA, but did not react with Cos-7 cells transfected with C-terminal deleted mutant TCR delta-chain cDNA. All three mAbs produced high-quality immunostaining results on frozen sections, revealing a distinct distribution of gammadelta T-cells and CD8(+) cells. This report precisely characterizes mAbs against porcine TCR for the first time, facilitating molecular biological investigations of the porcine immune system.

Assessment of severity of cardiac rejection in heterotopic heart transplantation using indium-111 antimyosin and magnetic resonance imaging.

Seven canine donor hearts in which atrial septal defect and tricuspid regurgitation had previously been produced were heterotopically transplanted into the recipients' chest cavities. Indium-111 antimyosin myocardial imaging of the excised heart was performed using a scinticamera. Magnetic resonance imaging was also performed and the T2 relaxation time calculated. Subsequently, these data were correlated with pathological findings, which indicated the degree of rejection. Indium-111 antimyosin uptake was high in moderate and severe rejection, but the T2 relaxation time was prolonged even in mild rejection. Thus indium-111 antimyosin uptake was specific, and the T2 relaxation time was sensitive, for detecting the severity and extent of cardiac rejection. Although ex vivo experimental results have been reported, these new methods allow characterisation and accurate evaluation of myocardial tissue undergoing cardiac rejection.

Sodium nuclear magnetic resonance imaging of acute cardiac rejection in heterotopic heart transplantation.

Nuclear magnetic resonance (NMR) imaging was used to measure tissue sodium-23 in the myocardium undergoing cardiac rejection. In six dogs, the donor heart was heterotopically transplanted into the recipient's chest cavity. The dogs were then killed and sodium-23 images of the excised hearts were obtained using a high field (1.5 Tesla) NMR imaging system. Proton NMR imaging of each excised heart was also performed and T1, T2 relaxation times were calculated. Subsequently, these data were correlated with pathological findings of mild, moderate and severe rejection. The correlation coefficients between the rejection score and the T1, T2 relaxation times and sodium NMR signal intensity were 0.79, 0.70 and 0.84, respectively. Severely rejected areas of the myocardium were visualised by increased sodium NMR signals. These findings suggest that an increase of sodium NMR intensity is mainly caused by an increase of intracellular sodium content due to irreversible myocardial necrosis. Sodium NMR allows evaluation of the location and extent of rejection of myocardium after heart transplantation.

Mechanism of o-aminophenol metabolism in human erythrocytes.

o-Aminophenol was found to be rapidly metabolized to a brown compound in the presence of purified human oxy- and methemoglobin, coupled with the oxidation and reduction of these hemoglobins by o-aminophenol. The final product of o-aminophenol was identified as 2-aminophenoxazine-3-one, by using spectrophotometry and HPLC. The metabolism of o-aminophenol was also observed in human erythrocytes. The production rates of 2-aminophenoxazine-3-one in the cells were very fast, but these were strongly decreased by bubbling carbon monoxide into the cell suspension when intracellular hemoglobin was in the ferrous state. The production of 2-aminophenoxazine-3-one from o-aminophenol in the cells was completely suppressed by cyanide and azide when intracellular hemoglobin was in the ferric state. These results suggest that oxy- and methemoglobin are involved in metabolism of o-aminophenol to 2-aminophenoxazine-3-one in human erythrocytes.

Apolipoprotein(a) and pentanucleotide repeat polymorphisms are associated with the degree of atherosclerosis in coronary heart disease.

To evaluate whether a high level of lipoprotein(a) (Lp(a)) is a risk for the development of coronary heart disease (CHD), 94 Japanese patients and 64 age-matched Japanese controls, diagnosed after coronary angiography (CAG), were analyzed with special reference to the relations between the degree of atherosclerosis, Lp(a) levels and the apolipoprotein(a) (apo(a)) genotypes. the degree of atherosclerosis was evaluated based on CAG findings in the following three ways: the number of diseased vessels, the Gensini score, and the presence or absence of vascular ulcers and/or irregular outlines of coronary stenotic lesions. Apo(a) protein sizes and the pentanucleotide (TTTTA) repeat polymorphism in the 5' control region of the apo(a) gene were analyzed. Multivariate predictors for the number of diseased vessels were, in decreased order of significance, plasma Lp(a) levels, history of smoking, hypertension, diabetes mellitus, and body mass index (BMI). Independent factors associated with the Gensini score were Lp(a) levels, BMI, hypertension, and diabetes mellitus. A negative association of Lp(a) levels with apo(a) protein sizes, and higher Lp(a) levels in those homozygous for an allele with 8 8 (TTTTA)-repeats, was found in both the controls and patients. In decreasing order of significance, apo(a) protein sizes, the degree of atherosclerosis, the genotype of the pentanucleotide repeat, and gender were independent predictors of Lp(a) levels in stepwise regression models. Apo(a) protein sizes were a significant predictor, and the genotype homozygous for the 8 (TTTTA)-repeats was a possible predictor, for the degree of atherosclerosis in CHD. These findings support the notion that a high Lp(a) level is a risk for the development of atherosclerosis in CHD.

Methylenetetrahydrofolate reductase and apolipoprotein E polymorphisms are independent risk factors for coronary heart disease in Japanese: a case-control study.

A missense variant of the C677T (Ala --> Val) polymorphism in the methylenetetrahydrofolate reductase gene (MTHFR) (the T allele) may increase levels of plasma homocysteine. Apolipoprotein E4 increases plasma LDL-cholesterol levels. Increased levels of homocysteine and LDL-cholesterol have been recognized as risk factors for coronary heart disease (CHD). To examine whether the polymorphisms in the MTHFR gene and the APOE gene are associated with CHD in the Japanese, we analyzed 214 CHD patients with an onset age before 65 and 310 apparently healthy persons. In the controls, significantly higher plasma concentrations of homocysteine were observed in the MTHFR TT genotype (15.1+/-6.0 mmol/l) compared with the CT genotype (11.2+/-1.9 mmol/l) and the CC genotype (10.5+/-3.3 mmol/l). The MTHFR TT genotype was significantly more frequent in the CHD patients (28.5%) compared with the control subjects (13.5%); the odds ratio was 2.54 (P < 0.00003). Subjects with the apo E4 allele were significantly more frequent in the CHD group (22.9%) than in the control group (10.0%); the odds ratio was 2.74 (P < 0.00004). Multivariate analysis showed that the TT genotype of MTHFR and the apoE4 allele are independent risk factors for CHD in the Japanese.

Effect of a new immunosuppressant, 15-deoxyspergualin, on heterotopic rat heart transplantation, in comparison with cyclosporine.

In this study, 15-deoxyspergualin (DSG) or cyclosporine (CsA) was administered to heterotopically heart-grafted rats for 15 days, commencing on the day of transplantation. In addition, a 31P nuclear magnetic resonance (NMR) technique was applied to investigate the in vivo energy metabolism of the graft. A significant prolongation of graft survival was observed in groups treated with 2.5 mg/kg and 5 mg/kg of DSG, when compared with the control group not treated with an immunosuppressant. One graft in the DSG 2.5 mg/kg-treated group and one in the 5 mg/kg-treated group survived for more than 100 days after grafting. The 31P NMR study demonstrated that, although rejection occurred in the rats treated with 2.5 mg/kg of DSG during the early period after transplantation, 5 mg/kg of DSG inhibited rejection completely. As for CsA, while 2 mg/kg of the drug did not affect graft survival, 5 mg/kg and 14 mg/kg significantly prolonged survival. It was revealed by 31P NMR, however, that CsA 5 mg/kg did not quite inhibit rejection by itself, and 14 mg/kg of CsA, which was the tolerogenic dose, exerted a cardiotoxic effect. In consequence, DSG seems to be a powerful immunosuppressant with a low toxic effect.

Therapeutic effect of 15-deoxyspergualin on acute graft rejection detected by 31P nuclear magnetic resonance spectrography, and its effect on rat heart transplantation.

We investigated the effect of 15-deoxyspergualin (DSG) on graft rejection, starting administration at the onset of rejection and on the induction of immunologic unresponsiveness. Hearts from WKAH rats were transplanted into the neck of ACI rats. The energy metabolism of the grafted hearts was followed by 31P nuclear magnetic resonance spectroscopy. The day that energy metabolism started to fall was defined as the onset of rejection, and intraperitoneal administration of DSG was initiated at 5 mg/kg/day for 15 days from this day. The grafted heart arrested in 2 of 10 rats 9 and 11 days after transplantation, respectively, but the remaining 8 recovered from rejection and 5 of them showed evidence of immunologic unresponsiveness. Of 10 rats treated with DSG from the day of transplantation, only 1 rat showed evidence of unresponsiveness. The initiation of DSG treatment from the onset of rejection resulted in a higher percentage of induction of unresponsiveness. Therefore, DSG was considered to specifically inhibit lymphocyte clone expansion at the onset of rejection. Spleen cells obtained from recipients 7-10 days after the end of DSG treatment were administered to syngeneic ACI rats grafted with WKAH hearts. Graft survival was significantly prolonged, but long-term unresponsiveness could not be transferred. However, immunologic unresponsiveness could be adoptively transferred in 3 of 5 rats receiving spleen cells from syngeneic rats that had recovered from rejection after DSG treatment and had acquired long-term unresponsiveness. These results suggest that suppressor cells are resistant to DSG and are spared and participate in the maintenance of immunologic unresponsiveness.

Transplantation tolerance mediated by suppressor T cells and suppressive antibody in a recipient of a renal transplant.

This is a report of a patient who underwent cadaveric renal transplantation in spite of the presence of three HLA-A, B and two DR antigen mismatches between the recipient and donor. The recipient received more than 20 units of blood before transplantation. The crossmatch between the recipient's serum and the T and B cells of the donor was negative. The patient exhibited hepatic dysfunction from the early posttransplant period, which eventually led to discontinuation of azathioprine or Bredinin at one year posttransplantation. Thereafter, only betamethasone was administered once every 3 days. The patients has maintained good renal function for more than one year following withdrawal of the immunosuppressants. It appeared that transplantation tolerance was established in this patient. Therefore, we examined the mechanisms sustaining the tolerance. Both nylon-wool-adherent, alloantigen-specific suppressor T cells and nonadherent, nonspecific suppressor T cells were observed in the lymphocytes of the patient after transplantation. It was also shown that suppressive antibody was present in the serum directed toward the clone of autologous lymphocytes that reacted with the mixed lymphocyte reaction (MLR) antigen of the donor. In the inhibition test against various types of MLR antigens using this suppressive antibody, it was found that the reaction against the donor cells was suppressed when the responding cells shared the same class I antigen with the recipient. When the stimulating cells had the class II antigen of the donor, the reaction of the specific responding cells was also inhibited. These inhibiting effects were only seen when the responding cells were pretreated with the antibody, but not when stimulating cells were pretreated.

Simultaneous evaluation of nitric oxide synthesis and tissue oxygenation in rat liver allograft rejection using near-infrared spectroscopy.

Near-infrared (NIR) spectroscopy was applied to rat liver allografts for assessing nitric oxide (NO) synthesis and tissue oxygenation as a means of monitoring the rejection response following liver transplantation. Orthotopic liver transplantation was performed in rats, which were assigned to three groups as follows: group 1, a syngeneic combination (lewis to Lewis); group 2, an allogeneic combination (ACI to Lewis); and group 3, an allogeneic combination treated with 15-deoxyspergualin. NIR spectroscopy was performed on the grafts in recipients, and the relative changes in nitrosyl-Hb (NO bound to erythrocyte hemoglobin), oxy-Hb, and oxidized cytochrome oxidase (Cyt.aa3) were obtained. The level of nitrosyl-Hb was significantly elevated from postoperative day (POD) 3 in group 2 compared with that in group 1, which remained constant (P < 0.05). In group 3, the elevation was significantly suppressed. These data indicate that the alloimmune response is associated with a dramatic change in NO synthesis in grafted livers. In a separate experiment, NO synthesis was also increased after long cold preservation (24 hr) in syngeneic liver transplants. However, the increase was transient and subsided on POD 3. Levels of oxy-Hb and oxidized Cyt.aa3 in group 2 were significantly decreased when parenchymal disorder was confirmed histologically (POD 6 and 8), compared with those in group 1, which remained constant (P < 0.05). In group 3, both of these levels showed improvement. Thus, our NIR spectroscopy technique was shown to be capable of assessing simultaneously both the immune response and the degree of immune-induced destruction of allograft tissue following liver transplantation through monitoring of NO synthesis and tissue oxygenation.

The significant effect of HLA-DRB1 matching on long-term kidney graft outcome.

Serotyping and genotyping (polymerase chain reaction with sequence-specific oligonucleotide probes method) were conducted on 520 unrelated individuals to determine the linkage disequilibrium of HLA-B and HLA-DRB1. Analyses of 511 kidney transplants (300 related and 211 cadaver recipients) were carried out at 4 transplant centers using the linkage disequilibrium of HLA-B and HLA-DRB1 established previously. All transplant recipients received CsA immunosuppression and were transplanted from June 1983 to December 1991. There were 51 significant linkages formed between HLA-B and HLA-DRB1 alleles (P < 0.05). DRB1-compatible transplants experienced a comparable 5-year graft success rate of 94% as did the HLA-identical recipients with a 100% 5-year success rate. However DRB1-incompatible recipients displayed a significantly reduced 5-year graft survival rate of 73% (73% vs. 94% P < 0.01). The 5-year graft survival rate of HLA-DR-incompatible recipients of 71% was compatible to the 73% for HLA-DRB1-incompatible recipients. No variation of rejection rate for DRB1-compatible grafts was seen in any of the 4 transplant centers. The results also indicated that HLA-DRB1 compatibility was essential for optimal success rate, regardless of HLA class I mismatches. The overall conclusion was that matching for HLA-DR was important to achieve optimal kidney graft survival on the molecular level but not on the serotyping level.

Prolonged cardiac allograft survival in rats systemically injected adenoviral vectors containing CTLA4Ig-gene.

BACKGROUND: CTLA4Ig, a soluble recombinant fusion protein that contains the extracellular domain of the CTLA4 and Fc portion of IgG1, strongly adheres to the B7 molecule to block CD28-mediated costimulatory signals and inhibits in vitro and in vivo immune responses. In vivo gene transfer using adenovirus vector achieves a high transfection rate into organ cells that usually contain adenoviral receptors. In this study, we investigated expression levels of the transfected gene and the survival times of the allografts in cardiac recipients systemically administered adenoviral vectors containing CTLA4Ig. METHODS: Hearts from DA rats (RT-1a) were transplanted into a cervical location in LEW recipients (RT1(1)). The adenoviral vectors containing CTLA4Ig was injected via a recipient vein immediately after grafting. RESULTS: The serum level of CTLA4Ig reached to maximum at 51-93 microg/ml 3 to 7 days after gene-transfection and declined after 14 days, although detectable levels were observed up to 49 days. The median survival time of the allografts in the gene-transfected group were significantly prolonged (27 days) in compared to the control group (6 days). In addition, down-regulation of IL-2 and IFN-gamma mRNAs and persistence of IL-4 and IL-10 transcripts were observed in the graft infiltrating cells. CONCLUSION: The adenovirous-mediated CTLA4Ig gene transfer into a recipient liver by systemic administration resulted in remarkable prolongation of cardiac allograft survival. Its action mechanisms may be mediated by inhibition of CD28-associated signal transduction, reduction of Th1-type cytokine production, and continuous expression of Th2-type cytokines in the activating lymphocytes.

Prolonged survival of rat liver allografts transfected with Fas ligand-expressing plasmid.

BACKGROUND: Transplantation of Fas ligand (FasL) gene-transfected tissues can have opposite effects. For example, cotransplantation of pancreas islets with myoblasts transfected with FasL-expressing plasmid vector (pFasL) prevented graft rejection, whereas the expression of FasL directly within islets using adenovirus vector led to graft destruction. It was also reported that FasL expression on pancreas islets led to neutrophilic infiltration and rapid destruction of the islets. From these results, overexpression of FasL in transfected tissues may lead directly to self destruction through an autocrine Fas-FasL pathway or graft destruction through neutrophil recruitment. To date there have been no reports of successful transplantation of FasL gene-transfected solid organs. METHODS: Rat pFasL was transfected at a dose of 90, 180, 270, or 360 microg into rat liver with an inactivated hemagglutinating virus of Japan conjugated to liposome vesicles (HVJ-liposome), and the gene-transfected livers were transplanted to allogeneic rats. RESULTS: In 18 rats transfected with 180 microg of pFasL, 14 (78%) did not develop fulminant hepatitis. FasL-mRNA was detected in these livers at 3, 5, 7, and 14 days after transfection. The expression of FasL protein was also observed in the transfected liver, and the transfection rate by this method was 11.1+/-1.9%. The livers were then transplanted to allogeneic recipients, resulting in significant (P<0.01) prolonged recipient survival times. Histological observation showed that the pFasL-transfected liver allografts caused apoptotic cell death in infiltrating activated T cells. In contrast, transfection of pFasL higher than 180 microg resulted in lethal hepatitis in all rats, and its low dose (90 microg) did not induce the hepatitis or prolong recipient survival. CONCLUSION: Our results indicate that rat liver allografts can be protected to host immune responses by an adequate level (approximately 10%) of FasL expression in the livers using HVJ-liposome incorporating pFasL.

A novel rescue drug, 15-deoxyspergualin. First clinical trials for recurrent graft rejection in renal recipients.

The present multicentral clinical study performed in 6 institutes demonstrated that the novel immunosuppressive agent, 15-deoxyspergualin (DSG), is very effective on rejection. In 34 cases of rejection, 30 were treated with DSG at 40 mg/m2 (1 case), 80 mg/m2 (7 cases), 120 mg/m2 (9 cases), 180 mg/m2 (9 cases), and 220 mg/m2 (8 cases). The overall remission rate was 79% in 34 cases of rejection including accelerated, acute, and chronic rejection in different periods after transplantation. Analyzing the remission rates of early phase acute rejection occurring within 3 months after transplantation according to treatment pattern, the remission rate was 100% in 3 cases treated with DSG alone (using DSG 1 week or longer after other agents), 88% in 8 cases treated by rescue use of DSG (using DSG within 1 week after other agents), and 86% in 7 cases treated by combined use of DSG with other agents. Adverse reactions included reductions in WBC and platelets, anemia, perioral numbness, gastrointestinal troubles, and others. However all these symptoms were so mild that DSG treatment was not discontinued. Further studies are necessary on the effect of DSG, especially in acute rejection under conditions that reduce the many influences of other agents as much as possible.

Fulminant hepatitis by Fas-ligand expression in MRL-lpr/lpr mice grafted with Fas-positive livers and wild-type mice with Fas-mutant livers.

BACKGROUND: Fulminant hepatitis in mice could be induced by gene-transfection of Fas ligand (FasL). However, the mechanisms of this event still remain controversial as to whether it is mediated by direct Fas/FasL interaction and/or neutrophil migration. To investigate the role of exogenous FasL-expression, we established a simple but clear mouse model on which we performed liver transplantation between Fas-mutant mice (MRL-lpr/lpr) and wild-type mice (MRL+/+). METHODS: The controls were nontransplanted wild-type (group 1) and MRL-lpr/lpr (group 2) mice. We obtained recipients with a Fas defect only in the liver (group 3; MRL-lpr/lpr liver graft in wild-type mice) and Fas-defected recipients with Fas-positive livers (group 4; wild-type graft in MRL-lpr/lpr). We successfully expressed FasL in the liver by cotransfection of two types of adenoviral vectors, AxCALNFasL and AxCANCre, with a Cre-loxP switching system. RESULTS: FasL-expression in the livers in groups 3 and 4 resulted in animal death due to fulminant hepatitis within 48 hr after administration of the vectors. We obtained similar findings in group 1, whereas the mice in group 2 survived without any evidence of hepatitis. Immune staining revealed a marked infiltration of CD11b-positive cells in group 1 and group 3. Despite the number of apoptotic cells, a few infiltration of CD11b-positive cells were seen in group 4. We observed no remarkable findings in the FasL-expressed livers in group 2. CONCLUSION: The results indicated that exogenous FasL-expression induces hepatocyte apoptosis both by direct interaction with Fas and by recruiting Fas-positive inflammatory cells. These findings are important for generating a new strategy to prevent hepatitis as well as for understanding the role of the Fas/FasL interaction in the pathophysiology of hepatitis.

Microchimeric cells from the peripheral blood associated with cardiac grafts are bone marrow derived, long-lived and maintain acquired tolerance to minor histocompatibility antigen H-Y.

BACKGROUND: Although it has been well established that the microchimerism occurs in the peripheral blood of the recipients after various settings in both clinical and experimental organ transplantation, nevertheless, their roles in inducing and maintaining acquired transplantation tolerance are controversial. Furthermore, regarding the cell lineages, kinetics, and functions of the cells that constitute the microchimerism after organ transplantation, solid information is not available. METHODS: Using rat heterotopic heart isografts from bone marrow chimeras between cross-sex and applying polymerase chain reaction with specific primers to rat sex determining region of Y chromosome, a relationship between a state of microchimerism and induction as well as maintenance of acquired tolerance to H-Y antigen were examined. RESULTS: Microchimeric cells of the peripheral blood (MCPB) after cardiac grafting contain bone marrow-derived and radiation-sensitive cells. Furthermore, removal of the primary cardiac grafts revealed that microchimeric cells in the peripheral blood are long-lived cells, i.e., more than 6 months. When the female rats that had contained long-lasting MCPB, were innoculated with syngeneic male dendritic cells, failure to sensitize female toward male specific antigen H-Y was found to occur. CONCLUSIONS: Thus it was suggested that radiation-sensitive, bone marrow derived, long-lived MCPB play a significant role in maintaining acquired transplantation tolerance to minor histocompatibility antigen H-Y.