Induction of lymphocyte apoptosis in rat liver allograft with ongoing rejection by FTY720.

The action mechanism of FTY720, a novel immunosuppressant, is completely different from conventional immunosuppressants. The drug, which triggers apoptosis in murine and human lymphocytes, has a potent immunosuppressive activity to prevent allograft rejection without any severe side-effect. The present study was designed to determine whether FTY720 induces apoptotic cell death in activated lymphocytes infiltrated into liver grafts with ongoing rejection. FTY720 was orally administered at 5 mg/kg to the recipients on day 3 and day 4 after grafting, when the graft rejection was histologically confirmed. The intragraft patterns of IL-2, interferon-gamma (IFN-gamma), perforin, and granzyme B gene expression were detected by reverse transcriptase-polymerase chain reaction. The treatment reversed ongoing rejection and significantly prolonged recipient survival time compared with the control group. Light microscopic observation of the graft sections stained with the DNA nick-end labelling method showed that the apoptosis in the control allografts was mainly induced in hepatocytes, while that in the FTY720-treated allografts was in infiltrated lymphocytes. The rejection therapy with FTY720 did not alter the expression of IL-2, IFN-gamma, and perforin mRNAs, but slightly decreased granzyme B expression. Our results suggest that FTY720 does not alter the intrinsic lymphocyte function to produce the rejection-related cytokines, but strongly induces apoptotic cell death in the activated lymphocytes. Thus, FTY720 affords new insight into the mechanisms underlying improvements in immunosuppressive treatments.

Nitrosyl hemoglobin detected by near-infrared spectroscopy in rat liver allografts.

Near-infrared spectroscopy (NIRS) is a noninvasive biomeasurement system with rays in the near-infrared region that possess high permeability to biological tissues. NIRS was applied to liver allografts undergoing rejection in rats treated with deoxyspergualin (DSG) or tacrolimus (FK506). The nitrosyl hemoglobin (Hb) levels detected in the liver grafts increased 3 days and 5 days after grafting in both allogeneic and syngeneic transplantation. The levels on day 8 remained high in the allogeneic graft, but markedly decreased in the syngeneic graft. Although the serum levels of nitrite and nitrate were extremely low 8 days after grafting in allografted recipients treated with DSG or FK506, the nitrosyl-Hb level in DSG-treated graft was much higher than that in FK506-treated graft. There was no significant difference in survival time between DSG-treated and FK506-treated recipients. In conclusion, DSG and FK506 have a different effect on NO production in allografted liver with ongoing rejection, and circulating nitrite and /nitrate levels do not reflect the local levels of NO in the graft.

[Functions and the regulation of cytokines and growth factors in the thrombotic event of arteries and veins].

Recent progress of molecular biology has elicited the pathogenesis of arterial and venous thrombosis at the cellular level. The pathogenesis, as well as the etiology of the sequelae, has become clear to be closely relevant to inflammatory cytokines and growth factors such as tumor recrosis factor (TNF) alpha, interleukin-1 beta, or vascular endothelial growth factor (VEGF). From this point of view, the anti-cytokine or the anti-growth factor therapy including the use of some proteins, antibodies, drugs, and gene delivery systems has every possibility of improving the therapeutic outcome for the thrombosis.

An immunosuppressive regimen using FTY720 combined with cyclosporin in canine kidney transplantation.

FTY720 induces apoptosis, specifically in lymphocytes, and prolongs allograft survival in rats and dogs. The purpose of this study was to define an effective range of FTY720 doses that could be combined with a suboptimal dose (10 mg/kg) of cyclosporin for canine kidney allograft recipients. The combination significantly prolonged allograft survival in all groups receiving FTY720 at a dose of 0.1, 0.3, 1.0, or 3.0 mg/kg. None of the recipients died due to notable side effects of the drug. In peripheral blood, the number of lymphocytes was extremely low, whereas the percentage of granulocytes increased during FTY720 administration. No significant difference in cyclosporin trough levels was observed between the cyclosporin-alone group and the combination groups. We conclude from the present study that FTY720 has a potent effect at an extremely low dose and a wide therapeutic window when combined with cyclosporin in canine kidney transplants.

[A case of inoperable advanced gastric cancer remarkably responding to combined chemotherapy with UFT-E, MMC and PSK].

A 55-year-old male consulted a local doctor with the complaint of epigastralgia. Examination of the upper gastrointestinal tract revealed gastric cancer (Borrmann Type II) and he was referred to our hospital for operation. A few lymph nodes were palpable in the left supraclavicular fossa, and the biopsy of those lymph nodes revealed metastatic adenocarcinoma. The CT scan of the abdomen showed enlargement of paraaortic lymph nodes. Then, the patient was determined inoperable (T3, N4, H02 P01, M1 stage IVb). He was treated as an outpatient with UFT-E (300 mg/day, orally), Krestin (PSK 3.0 g/day, orally) and Mitomycin C (MMC 6 or 8 mg once a week, intravenously repeated interval of 4 weeks). The total dose of UFT-E, PSK and MMC was 219 g, 1,095 g and 136 mg, respectively. One month later, lymph nodes in the supraclavicular fossa disappeared, and the lesion in the stomach completely responded. We have followed the patient for more than one year. He visits our the outpatient department and has kept working until now.

Prevention of graft rejection and graft-versus-host reaction by a novel immunosuppressant, FTY720, in rat small bowel transplantation.

In the present study, we examined the immunosuppressive effect of a new drug, FTY720, on small bowel transplantation (SBT) in rats. Grafts from (LEW x BN) F1-to-LEW rats treated with FTY720 at 0.5 mg/kg from day 0 to 14 post-SBT survived significantly longer than untreated grafts. In addition, the administration of FTY720 combined with cyclosporin (CyA; 5 mg/kg per day) had a synergistic effect on allograft survival. The graft-versus-host reaction (GVHR) that occurred in the LEW-to-F1 rats was markedly reduced after the administration of FTY720. FTY720 combined with a low dose of CyA completely abrogated GVHR without any adverse reaction. FTY720 treatment resulted in a significant decrease in the number of lymphocytes in the peripheral blood and the spleen, but the number of peripheral neutrophils was unchanged. Thus, FTY720 would appear to be an ideal drug to combine with CyA in order to control the immune reaction after SBT.

Examination of 7-ethoxycoumarin deethylation and ammonia removal activities in 31 hepatocyte cell lines.

Aiming the selection of possible candidates for the reactor cells in a hybrid artificial liver, we investigated the activities of 7-ethoxycoumarin deethylation and ammonia removal in 31 hepatocytes cell lines established from the human, rat, mouse, and chicken, compared with freshly isolated rat hepatocytes. Rat liver homogenate and hepatocytes showed 7-ethoxycoumarin deethylation activity of 0.925 and 1.840 nmol/mg protein/min, respectively. Two human hepatoblastoma cell lines, Hep G2 and HuH 6, showed the activity of 0.190 and 0.027 nmol/mg protein/min, respectively. While the rat hepatocytes efficiently removed added ammonia form the culture medium, no ammonia removal activity was detectable in any cell line with the assay conditions in this study. Ammonia removal activity, possibly catalyzed by the urea cycle, is likely to be harder to restore than the drug metabolizing system in the course of cell immortalization.

Induction of selective cell death targeting on mature T-lymphocytes in rats by a novel immunosuppressant, FTY720.

A novel immunosuppressant, FTY720, induces a rapid and marked decrease of peripheral lymphocytes, and prolong allograft survival in rats. Its mechanism of action is mediated by apoptotic cell death. In this study, we determined the time-related changes in the numbers of total lymphocytes, and the ratios of lymphocyte subpopulations in peripheral blood and lymphomyeloid organs in rats after the single oral administration of FTY720 (10 mg/kg), comparing with the effects of cyclophosphamide (80 mg/kg, ip). Total number of peripheral lymphocytes decreased significantly 3 h after the administration of the drug, while that of polymorphonuclear cells increased. T-cells were markedly decreased in number and reached a minimum of 2.3% of the control 3 days after the treatment, while B-cells reached 19.7%. T-cells decreased in spleen and liver but there was no notable change in thymus, lymph nodes, and bone marrow. The susceptibility of the cells against the drug was variant based on the type and the source of cells in vitro. Polymorphonuclear cells were the most resistant and lymph node cells the most sensitive to FTY720 after 3 h incubation with different concentration of the drug (1, 10,100 mumol/l). When incubated with 10 mumol/l of FTY720, B-cells were significantly higher in viability than the whole T- or CD4-cells. These results demonstrated that FTY720 induces cell death selectively in mature T-lymphocytes, especially CD4-lymphocytes, in peripheral blood without the depression of bone marrow.

A novel immunosuppressant, FTY720, with a unique mechanism of action, induces long-term graft acceptance in rat and dog allotransplantation.

A new compound with an immunosuppressive property was purified from culture filtrates of Isaria sinclairii and was chemically modified to FTY720. Rat spleen cells incubated with FTY720 demonstrated features characteristic of apoptosis--such as the absence of surface microvilli, chromatin condensation, and the formation of apoptotic bodies--by electron microscopy, and genemic DNA fragmentation by agarose gel electrophoresis. When FTY720 was administered in liver-allografted rats at a dose of 0.5 mg/kg from day 1 to day 14 after transplantation, the recipients survived significantly longer than the control group. Pretransplant treatment with 5 mg/kg of FTY720 one day before and on the day of grafting induced a remarkable prolongation of recipient survival, and three of 10 recipients survived for longer than 50 days. Furthermore, administration of FTY720 at 5 mg/kg on days 3 and day 4 after grafting also prolonged survival. In canine kidney allografting, a pretransplant 2-day course of FTY720 at 5 mg/kg prolonged graft survival. Daily administration of FTY720 in combination with CsA resulted in a significant prolongation of graft survival in a synergistic manner. In addition, FTY720 appeared to be nontoxic in canine recipients. These results demonstrated that FTY720, having a unique mechanism of action, induces long-term graft acceptance in rat and dog allotransplantation.

[Hemodynamic changes in acute pancreatitis].

Hemodynamics studies were carried out from days 1-5 following the onset of illness in 7 patients with severe pancreatitis (group A) and in 7 patients with moderate pancreatitis (group B). Patients in both groups had a higher cardiac index (CI) and a lower systemic vascular resistance (SVR) than normal patients during 5 days of illness, and patients in group A had a higher CI of 5.38 +/- 0.841 x min/m2 and lower SVR of 889 +/- 234dyn.sec/cm5 than those in group B on day 4. Patients in group A had a lower pulmonary vascular resistance than normal patients on days 1, 3, and 4, but those in group B did not show this hemodynamic change. Group A patients had a higher pulmonary wedge pressure of 11.9 +/- 8.4mmHg and depressed left ventricular stroke work index of 59.8 +/- 17.8g.m/m2 as compared with group B (5.6 +/- 3.4mmHg, 77.7 +/- 23.6g./m2, respectively). These findings indicate that a hyperdynamic hemodynamic state may exist in the early stages of moderate and severe pancreatitis and myocardial depression may be evident in severe pancreatitis.

Reactivity of fecapentaene-12 toward thiols, DNA, and these constituents in human fibroblasts.

Micromolar concentrations of fecapentaene-12, a mutagen found in human feces, decrease survival measured as colony-forming efficiency and membrane integrity of cultured human fibroblasts. Fecapentaene-12 also decreases the content of cellular free low-molecular-weight thiols including glutathione. Fecapentaene-12 reacts directly with glutathione by causing both decreased levels of free thiol and some concomitant formation of oxidized glutathione, indicating that thiol depletion is a result of both alkylation and oxidative reactions. Exposure of cells to 2 or 5 microM fecapentaene-12 causes significant amounts of DNA-interstrand cross-links and DNA-single strand breaks, respectively, whereas exposure to a higher concentration of fecapentaene-12, i.e., 10 microM, also causes significant DNA-protein cross-links. Results from the reaction of fecapentaene-12 with isolated plasmid DNA parallel the cellular pattern of DNA damage; primarily interstrand cross-links and strand breaks occur also in plasmid DNA. Taken together, these studies show that fecapentaene-12 is a potent cytotoxic and genotoxic agent which can react with cellular thiols and cause several types of DNA damage.