Cardiac intervention using high-intensity focused ultrasound: creation of interatrial communication in beating heart of an anesthetized rabbit.

OBJECTIVE: Current fetal cardiac intervention for restrictive atrial septum is invasive and technically demanding. We investigated the feasibility of computer-assisted high-intensity focused ultrasound (HIFU) for cardiac intervention on the atrial septum of a beating heart. METHODS: To create an interatrial communication in the beating heart of nine anesthetized rabbits, the heart was exposed surgically and placed under a water-filled tank, in contact with the tank's membranous base. A HIFU transducer (3.3 MHz) coupled with a diagnostic ultrasound probe (10.0 MHz) was placed in the tank over the beating heart. The focus of the HIFU transducer was set so that it could create a hole in the target area on the atrial septum during the early diastolic phase. HIFU delivery was controlled based on ultrasound images captured with real-time image-recognition software. We attempted to create interatrial communication using HIFU and assessed the cardiac tissue specimen pathologically. RESULTS: In eight of nine rabbits, small holes in the atrial septum were successfully created. Serious complications occurred in two animals: a fatal atrioventricular block in one and a cardiac tamponade in the other. CONCLUSION: HIFU combined with a computer-assisted imaging system might be useful to create interatrial communication in beating hearts. This procedure may be helpful for making current fetal cardiac intervention less invasive.

Human CYP3A4-introduced HepG2 cells: in vitro screening system of new chemicals for the evaluation of CYP3A4-inhibiting activity.

1. The aims were to attest whether HepG2-GS-3A4, a cell line into which the human CYP3A4 gene was introduced, can be used for a screening of chemicals that will inhibit CYP3A4 activity. 2. The capacity of the cells for metabolizing CYP3A4 substrates in vitro was evaluated. Also determined was the effect of CYP3A4 inhibitors and non-inhibitors on nifedipine hydroxylation. Western blot, immunohistochemostry and determination of beta-nicotinamide adenine dinucleotide phosphate (NADPH)-reductase activity were performed. 3. HepG2-GS-3A4 selectively metabolized substrates of CYP3A4 (diazepam, nordiazepam, lidocaine, atorvastatin, and nifedipine) to a greater degree than control. The metabolites were easily detected in the culture medium. Values of V(max) of HepG2-GS-3A4 were about 30- to 100-fold higher than those of the control, while values of K(m) were comparable. Pre-incubation of cimetidine and ketoconazole significantly inhibited nifedipine hydroxylation, while addition of inhibitors specific to other isoforms of CYPs had no substantial effect. The HepG2-GS-3A4 expressed a higher amount of CYP3A4 protein and mRNA than control. Most NADPH reductase activity was detected in microsomal fractions. 4 In conclusion, HepG2-GS-3A4 sufficiently and selectively metabolize substrates of CYP3A4, and inhibitors of CYP3A4 reduced the metabolism. Because the metabolites were easily detected in the culture medium, this cell might be useful for the new and easy screening of new drugs for the evaluation of CYP3A4-inhibiting activity in vitro.

Ex Vivo Reperfusion Model to Evaluate Utility of Machine Preservation for Porcine Liver Donated After Cardiac Death.

BACKGROUND: Machine perfusion (MP) techniques are expected to prove useful for preserving the organ viability and recovering organ function for organ transplantation. Furthermore, an accurate assessment of organ viability using MP is important for expanding the donor criteria. In this study, an ex vivo reperfusion model (ERM) simulating transplantation using diluted autologous blood under normothermic conditions was evaluated for its utility of MP under subnormothermic conditions for livers donated after cardiac death (DCD). METHODS: The liver preservation methods for DCD porcine livers were evaluated using the ERM. This investigation was performed using a novel perfusion system developed by our research group. Porcine livers were procured with a warm ischemia time (WIT) of 60 minutes. The organs were then preserved using subnormothemic machine perfusion (SNMP) or static cold storage (CS) for 4 hours. We also compared these tissues with SNMP livers procured under a WIT of 0 minutes. After the preservation, the livers were reperfused for 2 hours using the ERM with diluted autologous blood oxygenated by a membrane oxygenator under NMP conditions. Reperfusion was evaluated based on perfusion flow dynamics and outflow of deviating enzymes. RESULTS: In the early stages of reperfusion, pressure in the blood vessels increased sharply in the CS group. Furthermore, the amount of aspartate aminotransferase accumulation was lower in the SNMP group than in the other groups. These results suggest ischemia-reperfusion injury is suppressed in SNMP conditions. CONCLUSION: An ERM has use in evaluating the utility of MP for the DCD liver.

Optimum Perfusate Volume of Purified Subnormothermic Machine Perfusion for Porcine Liver Donated After Cardiac Death.

INTRODUCTION: Subnormothermic machine perfusion (SNMP) shows some advantages for the preservation of grafts donated after cardiac death (DCD) and improvements in machine perfusion (MP) technology are important to enhance organ preservation outcomes for liver transplantation. In this study, we focused on purified subnormothermic machine perfusion (PSNMP) and volumes of perfusate removed to substitute for purification and replaced by modified University of Wisconsin-gluconate after the start of perfusion and investigated, in particular, the optimum perfusate purification volume. Several purification volumes under SNMP were compared. In addition, the perfusate purification during MP was indicated as a potential technique to enhance the organ quality of DCD grafts and extended-criteria donors. METHODS: The PSNMP at several volumes (0.5 L, 1.5 L, and 3 L) were compared with regular SNMP without any purification treatment (untreated control). In the PSNMP group, all perfusate was removed to substitute for purification of the perfusate by modified University of Wisconsin-gluconate solution after the start of perfusion. After removing the perfusate, new perfusate with the same components was perfused to preserve the porcine livers obtained under warm ischemia for 60 minutes using SNMP at 22°C porcine liver for 4 hours. RESULTS: The concentrations of aspartate aminotransferase and lactate dehydrogenase in the untreated group were significantly higher during perfusion compared to those of the intervention group. There are no significant differences among the volume conditions of the purification groups. CONCLUSIONS: The optimal volume of perfusate purification was confirmed with a simple experimental comparison between untreated and PSNMP conditions.

Enhancing Survival of Human Hepatocytes by Neonatal Thymectomy and Partial Hepatectomy in Micro-miniature Pigs.

BACKGROUND: With the goal of in vivo cultivation of human hepatocytes that have not been sufficient in full differentiation in vitro, the advantage of neonatal thymectomy was verified on expansion of xenogeneic human hepatocyte in the micro-miniature pig (MMP). METHODS: The thymus was excised immediately after the birth of the MMPs via cesarean section. Newborns were fed by artificial feeding under specific pathogen-free conditions. The thymectomized and nonthymectomized littermates were transplanted with human hepatocytes via a portal vein with or without partial hepatectomy at the MMP adult stage. RESULTS: The growth of thymectomized MMPs and the sham operated littermates was not significantly different; the former weighed 1.98 ± 0.30 kg (average ± standard deviation, n = 4) and the latter weighed 2.28 ± 0.39 kg (n = 4) at 1 month of age, and 17.48 ± 1.92 kg and 16.75 ± 2.68 kg at 12 months of age. Blood thymosin α1 concentrations in the thymectomy group were significantly lower than in the control group (0.22 ± 0.05 ng/mL vs 0.46 ± 0.16 ng/mL; n = 4, 12 months old, P = .029). After human hepatocyte transplantation, human albumin levels were detectable on day 28 in the peripheral blood of the thymectomy plus hepatectomy group (14.3 ± 4.9 ng/mL [± range, n = 2]) but were not detectable even on day 21 in the control group. CONCLUSIONS: Neonatal thymectomy was successfully achieved in infantile MMPs born via cesarean section. These pigs were considered to be an ideal in vivo bioreactor for human hepatocytes.

Regulated Oxygenation of Rewarming Machine Perfusion for Porcine Donation After Cardiac Death Liver Transplantation.

INTRODUCTION: Machine perfusion (MP) is particularly expected to preserve and resuscitate an organ obtained from extended criteria donors or donation after cardiac death to expand the donated organ pool for organ transplantation. This method requires to be investigated an optimal preservation condition. The aim of this study is investigation of the optimal oxygenation conditions under rewarming MP (RMP). METHODS: Porcine livers were perfused with an RMP system developed by our research group. All livers were procured under warm ischemia time of 60 minutes, and preserved in static cold storage for 2 hours, and perfused for 2 hours using the RMP. For group 1, the livers were supplied with oxygen constantly through perfusate. For group 2, the livers were supplied with oxygen increasingly with controlling flow rates and oxygen concentration. Effluent enzymes were obtained during perfusion preservation. RESULTS: The average levels of alanine aminotransferase were lower in group 2 than in group 1 during RMP, and also decreasing the hepatic artery pressures after 60 minutes. CONCLUSIONS: Regulated oxygenation of RMP has possibility to improve the graft preservation for liver transplantation.

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.

A novel immunomodulator KRP-203 combined with cyclosporine prolonged graft survival and abrogated transplant vasculopathy in rat heart allografts.

To find more effective and less toxic immunosuppressive strategies in long-term treatment for organ transplantation patients, we examined the effects on rat heart allograft survival of a novel sphigosine-1-phosphate receptor agonist, KRP-203, combined with a subtherapeutic dose of cyclosporine (CsA). Rat heart transplantation was performed across a major histocompatibility complex-incompatible (DA to LEW) rat combination. KRP-203 alone showed little or no effect on heart allograft survival. In contrast, KRP-203 combined with a subtherapeutic dose of CsA led to prolonged allograft survival. Histologic analyses showed that the combination completely suppressed acute rejection, as characterized by allograft vasculopathy, mononuclear cell infiltration, and myocardial necrosis in the heart allografts. RT-PCR analysis showed that the allografts treated with CsA or KRP-203 alone showed no suppression of IL-10, IFN-gamma, and TNF-alpha mRNA expression, but when combined with a subtherapeutic dose of CsA it completely suppressed their mRNA expressions. Furthermore, the combination treatment reduced donor-specific antibody production. KRP-203 combined with a subtherapeutic dose of CsA synergistically prolonged rat heart allograft survival. The combination of CsA with KRP-203 may provide an option to prevent allograft rejection and reduce adverse effects.

A technique for complete thymectomy in adult rats.

50 open thymectomies were performed in adult rodents using intubation combined with a fibrin glue able to prevent hemorrhage and pulmonary air leakage. This method had a 100% success rate and lower mortality than the ordinal suction procedure. Although the conventional suction thymectomy has been widely used, the open thymectomy method would permit more complete thymectomies for immunological studies.

Sex-associated differences in the survival of skin grafts in rats. Enhancement of cyclosporine immunosuppression in male compared with female recipients.

Focusing on sex-difference in prolongation of allograft survival time, we have performed skin grafts between fully allogeneic rat strains, AO (RT1u) and DA (RT1a) with an immunosuppressant, cyclosporine. Isografted skins survived indefinitely, whereas allografts were severely rejected at days 7-9 without immunosuppressive treatment. When adult male DA rats received CsA (15 mg/kg/day, i.m., for 14 days postoperatively), allogeneic skin was accepted from either male or female AO rats for 38.8 +/- 20.5 days (mean survival time [MST] +/- SD) and for 44.7 +/- 43.3 days (MST +/- SD), respectively, with normal hair growth at around day 17. Additional CsA administration every 5 days after the initial short course treatment was also effective in preventing chronic rejection. Male AO rat skin grafted onto adult male DA rats survived for over 50 days as long as the treatment was carried out. In contrast, when adult female DA rats were used as recipients, only a few days' prolongation was observed in comparison with a non-treated group. The rejection always occurred, even during the initial course of treatment (MST +/- SD): 10.9 +/- 1.6 days). Younger male DA recipients, 5 and 10 weeks old, rejected AO skin within a shorter time, depending on the age. The maximal graft survival was observed when male adult rats more than 14 weeks old were used as recipients. On the other hand, the CsA serum level of female recipients at day 14 was considerably lower than that of males. However, even when the level of females was adjusted to that of males by the administration of a double dosage (30 mg/kg/day), the female recipients consistently rejected the skins (MST +/- SD: 14.5 +/- 1.9 days). Therefore, these results clearly indicate that this male-associated immunosuppressive effect depends upon the sex and age of the recipient animals.

Effects of sex steroid hormones on sex-associated differences in the survival time of allogeneic skin grafts in rats. Evidence that testosterone enhances and estradiol reverses the immunosuppressive activity of cyclosporine.

Three-week-old DA (RT1a) male and female rats were pretreated with either orchiectomy or ovariectomy and administration of the opposite-sex steroid hormones, estradiol or testosterone. Skin from same-sex AO (RT1a) rats was then grafted when these pretreated rats reached 14 weeks of age, with a short course of immunosuppressive treatment of cyclosporine. The survival times of the grafts were reversed in that the male recipients pretreated to be like females acutely rejected the graft within 10 days as do normal adult female recipients. On the other hand, the female recipients pretreated to be like males accepted the graft as do normal adult male recipients. In addition, the synergistic effects of either testosterone or estradiol with CsA on the survival time of the graft were examined. Testosterone successfully prolonged graft survival on normal adult female and young male recipients, but negligible prolongation was observed on young females. In contrast, estradiol abrogated the immunosuppressive activity of CsA and accelerated graft rejection in both male and female recipients.

Evidence that changes in expression of major histocompatibility complex antigens may underlie the immunosuppressive effect of heat-treated cells in vivo and in vitro.

The prior transfusion of heat-treated (60 degrees C for 1 hr) allogeneic spleen cells is known to bring about specific prolongation of the survival of subsequent donor strain heart allografts. In this communication we show that some polymorphic and monomorphic class 1 determinants on spleen cells are heat denatured so that they no longer provoke antibody formation in naive allogeneic hosts. By contrast, the heated cells remain able to provoke the formation of anti-class 2 antibodies. When measured in a binding assay, the levels of anti-class 2 antibodies are similar irrespective of whether the immunizing inoculum consists of normal or heated cells. In a cytotoxic assay, the antibodies produced following exposure to normal cells are cytotoxic; this activity is substantially reduced when the cellular immunizing inoculum is heated. Cells heated to 60 degrees C for 1 hr are unable to stimulate in a mixed lymphocyte reaction, but reactivity can be partially restored by the addition of exogenous IL-2 to the culture. From previous evidence it seems unlikely that suppressor cells play a major role in the immunosuppression effected by cells heated to 60 degrees C. The results presented in this communication suggest a possible role for anticlass 2 antibodies and also imply the defective production of a costimulatory signal that normally follows the presentation of allogeneic MHC antigens.

Induction of natural chimerism after retransplantation of the liver in rats.

Immunological aspects after orthotopic rat liver retransplantation (re-OLT) were examined in association with cell migration and mixed chimerism. At day 2 after the first orthotopic liver transplantation (day 0) in the combination of DA (MHC haplotype, RT1a) donor into PVG (RT1c) recipient, the grafted DA liver was removed and a new PVG liver was implanted into the same PVG recipient (re-OLT). In the PVG recipient at various times after the re-OLT, DA-derived antigen and cells were detected using a DA-specific anti-class I mAb R3/13 in conjunction with ELISA, immunoblotting, and immunohistochemistry. The level of soluble class I antigen, which had risen to 270 ng/ml after the first OLT, substantially decreased within 24 hr after re-OLT. Using immunoblotting, DA class I antigen was detected in the PVG recipient's lymphoid organs at day 3 after DA liver grafting and persisted for up to 21 days after the DA liver was replaced by a new PVG liver. Immunohistochemistry on sections of spleen from re-OLT rats showed that the level of migratory cells expressing DA class I correlated with the findings obtained by immunoblotting. While the DA-derived antigen and cells were detected in the re-OLT recipient, the DA-specific inhibition of mixed lymphocyte reaction was observed in re-OLT serum. Our results suggest that the implanted DA liver graft was the source of DA soluble class I antigen, but DA-derived antigen and cells detected in the re-OLT recipient organs could persist for a relatively long time under immunosuppression after the implanted DA liver was removed by re-OLT.

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.

Prevention by liver transplantation of the graft-versus-host reaction and allograft rejection in a rat model of small bowel transplantation.

In the rat combination of DA (MHC haplotype RT1av1) donor into PVG(RT1c) recipient, liver grafts are not rejected and allow the acceptance of other organ grafts from the same donor strain. Here we show that an existing DA liver graft allows the acceptance of a DA small bowel graft in the PVG; the liver graft also prevented the graft-versus-host reaction normally associated with small bowel grafting. In addition, a liver graft could suppress the GVHR in F1 hybrid recipients of parental lymphoid cells, a classic GVHR model. GVHR suppression was immunologically specific and required that donor lymphocytes and liver be of the same strain. Besides their clinical implications, these results demonstrate the capacity of the liver for tolerance induction and suggest that it may play a physiological role in negative selection of T cells.

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.

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.

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.

Changes in cytochrome P450 molecular species in rat liver in chloroform intoxication.

The effect of CHCl3 on the composition of hepatic microsomal cytochrome P450 species was compared with that of CCl4 in rats pretreated with phenobarbital (PB) or 3-methylcholanthrene (3MC). The administration of CHCl3 hardly affected cytochrome P450 content in non-treated rat liver, but caused a similar degree of depletion in the content as observed after CCl4 administration in PB-pretreated rats. In the pretreatment with 3MC, the administration of CHCl3 brought about a marked decrease in the content to 24% of control after 12 hr, while CCl4 reduced the content only to one-half of control. It was demonstrated by SDS-polyacrylamide gel electrophoresis and Whatman DE-52 anion-exchange chromatography that 3MC-induced P450 species decreased with CHCl3, while it was affected little by CCl4 treatment. The activity of benzo[a]pyrene hydroxylase was altered together with the change in the content of cytochrome P450 species. The administration of CHCl3 to PB-pretreated rats caused the depletion in PB-induced P450. These findings indicate that cytochrome P450 species induced with 3MC as well as PB are highly susceptible to CHCl3 intoxication, whereas the administration of CCl4 depletes the PB-induced species without affecting the 3MC-induced species.

The proliferative response of p53 knock-out mouse-derived vascular smooth muscle cell line, P53LMAC01, to PDGF, when compared with human aortic smooth muscle cells.

To develop an in vitro experimental model of vascular smooth muscle cell hyperplasia, a major feature in chronic cardiac rejection, we studied a novel vascular smooth muscle cell line, P53LMAC01 (AC01), which was established from aortic smooth muscles of p53 knock-out mice, to determine its response to a platelet-derived growth factor (PDGF) and to Cyclosporin A (CsA). The responses were compared with those of human aortic smooth muscle cells (AOSMC). The AC01 exhibited a distinct proliferative response to PDGF similar to that of AOSMC under serum-free conditions. 10 ng/ml of PDGF-BB increased by a factor of 4.5 and PDGF-AB doubled the thymidine uptake, but PDGF-AA caused only a slight increase. The proliferation was markedly inhibited by 10(-6) M of CsA but less affected by 10(-7) M. These results indicate that the AC01 cell line could provide a convenient experimental system for investigating chronic rejection in vitro and that the system might work as a screening model of agents for treating transplant-related arteriosclerosis.