The role of donor hepatocytes and/or splenocytes pre-injection in reducing islet xenotransplantation rejection

OBJECTIVE: To observe the effect of tail vein injection with donor hepatocyte and/or splenocyte on islets xenotransplantation rejection. METHODS: New-born male pigs and BALB/C mice were selected as donors and recipients respectively. Islets xenotransplantation was performed in recipients just after the third time of tail vein injection with donor hepatocytes and/or splenocytes. Macrophage phagocytosis, NK killing activity, T lymphocyte transforming function of spleen cells, antibody forming function of B lymphocytes, and T lymphocyte subsets were taken to monitor transplantation rejection. The effects of this kind of transplantation were indicated as variation of blood glucose and survival days of recipients. RESULTS: Streptozotocin (STZ) succeeded in inducing diabetes mellitus models of mice. Pre-injection of donor hepatocytes, and splenocytes or their mixture via tail vein was effective in preventing donor islets transplantation from rejection, which was demonstrated by the mentioned immunological marks. And each group of transplantation could decrease the blood glucose of recipients and prolong the survival days. Pre-injection of mixture of donor hepatocytes and splenocytes was more effective in preventing rejection than pre-injection of donor hepatocytes or splenocytes separately. CONCLUSION: We propose that pre-injection of donor hepatocytes, splenocytes separately or their mixture before donor islets transplantation is a good way to prevent rejection.

Dexamethasone potentiates the insulin-induced Srebp-1c expression in primary rat hepatocytes

The impacts of dexamethasone(Dex)and thyroid hormone T3 on the insulin-stimulated Srebp-1c expression were studied in primary rat hepatocytes. Primary hepatocytes from Sprague-Dawley rats were isolated, cultured and treated with insulin in the presence or absence of the indicated reagents over time. The mRNA levels of indicated genes were determined using real-time PCR. Insulin treatment induced the Srebp-1c expression and suppressed the Pck1 expression in a time-dependent manner. Dex treatment alone reduced the Srebp-1c expression, whereas potentiated the insulin-induced its expression, which reached to a level that was higher than the insulin alone group. On the other hand, insulin treatment completely suppressed the Dex-induced Pck1 expression in the same cells. T3 treatment did not affect the expressions of Srebp-1c and Pck1 alone or in the presence of absence of insulin or Dex. Interestingly, insulin treatment induced the Rxrg m RNA expression level in the absence or presence of T0901317, a specific agonist for the liver X receptor. Dex and insulin mutually affect each other's ability to regulate the expression levels of hepatic genes involved in glucose and fatty acid metabolism. Insulin induced Rxrg expression in primary hepatocytes, which may contribute to the induction of Srebp-1c expression in the same cells.

Cyclic feeding regime may delay aging in animals by enhancing the hepatocytes nuclei structure

Background:The liver is fundamental for keeping up the entire body’s homeostasis.The liver hepatocytes have been shown to undergo genomic instability with aging.The stability of the hepatocytes depends on its nuclear architecture.Calorie restriction has been shown to extend life-span favorably and this may be through the reorganization of the nuclear structure.Objective:To study the effect of cyclic feeding regime on the chromatin assembly anchored to the nuclear membrane scaffold of rat models hepatocytes nuclei.Method:Rats models underwent cyclic feeding regime,after which nuclei were isolated;then,we investigated the chromatin decondensation and nuclear membrane disintegration of the hepatocytes using fluorescence imaging methods.Results:In 60 seconds,protease decondensed the chromatin and disintegrated the nuclear membrane structure of controls.After the first fasting,the time increased to 145 seconds in 3-month-old rats.The first refeeding increased the time to 156 seconds with a further rise to 340 seconds following the second fasting,then dropped to 116 seconds by the second refeeding.20 months old rats showed 186 seconds increase in the time of chromatin decondensation and nuclear membrane disintegration after the first fasting,with a decrease to 140 seconds observed after first refeeding.The second fasting increased the time to 165 seconds,which then slightly decreased to 163 seconds after the second refeeding.Conclusion:These results show that intermittent fasting may have acted on chromatin histone interactions and the structural lamin networks of the nuclear membranes in bringing about nuclear stability,which is essential for normal cellular function.

Effects of Fluoride on Lipid Peroxidation, DNA Damage and Apoptosis in Human Embryo Hepatocytes

Objective To investigate the effects of fluoride on lipid peroxidation, DNA damage and apoptosis in human embryo hepatocyte L-02 cells. Methods Lipid peroxide (LPO) level, reduced glutathione (GSH) content, DNA damage, apoptosis, and cell cycle analysis were measured after in vitro cultured L-02 cells were exposed to sodium fluoride at different doses (40 μg/mL, 80 μg/mL, and 160 μg/mL) for 24 hours. Results Fluoride caused an increase of LPO levels and a decrease of GSH content in L-02 cells. There appeared to be an obvious dose-effect relationship between the fluoride concentration and the observed changes. Fluoride also caused DNA damage and apoptosis and increased the cell number in S phase of cell cycle in the cells tested. There was a statistically significant difference in DNA damage and apoptosis when comparing the high dose of fluoride treated cells with the low dose of fluoride treated cells. Conclusion Fluoride can cause lipid peroxidation, DNA damage, and apoptosis in the L-02 cell experimental model and there is a significant positive correlation between fluoride concentration and these pathological changes.

Reversible immortalization of human hepatocytes mediated by retroviral transfer and site-specific recombination

AIM: To establish a method for the reversible immortalization of human hepatocytes, which may offer a good and safe source of hepatocytes for practical applications.

Induction of Heme Oxygenase-1 in Human Hepatocytes to Protect Them From Ethanol-induced Cytotoxicity

Background/Aim We investigated the relationship between ethanol exposure and heme oxygenase (HO-1) in human hepatocytes in order to ascertain if induction of HO-1 can prevent ethanol induced cellular damage. Methods Dose-dependent (25-100 mmol/L) and time-dependent (0-24 h) ethanol exposure were used in the present study. HO-1 mRNA and protein expression were detected by PT-PCR and Western blot respectively. HO-1 activity was indicated by bilirubin and Fe2+ formation. Cytotoxicity was investigated by means of lactate dehydrogenate (LDH) and aspartate transaminase (AST) level in culture supernatants, as well as the intracellular formation of malondialdehyde (MDA), cellular glutathione (GSH) status and CYP 2E1 activity. Results We first demonstrated a dose-dependent response between ethanol exposure and HO-1 mRNA and protein expression in human hepatocytes. We further observed a time-dependent increase of HO-1 mRNA expression using 100 mmol/L ethanol starting 30 minutes after ethanol exposure, reaching its maximum between 3 h and 9 h. Being similar to what had been demonstrated with the mRNA level, increased protein expression started at 6 h after ethanol exposure, and kept continuous elevated over 18 h. In addition, we found that ethanol exposure to hepatocytes markedly increased HO-1 enzyme activity in a time-dependent manner measured as bilirubin and Fe2+ formation in human hepatocytes. Our results clearly showed that ethanol exposure caused a significant increase of LDH, AST, and MDA levels, while the antioxidant GSH was time-dependently reduced. Furthermore, we demonstrated that pre-administration of cobalt protoporphyrin (CoPP) induced HO-1 in human hepatocytes, and prevented an increase of MDA and a decrease of GSH. These effects could be partially reversed by zinc protoporphyrin (ZnPP), an antagonist of HO-1 induction. Conclusion HO-1 expression in cells or organs could lead to new strategies for better prevention and treatment of ethanol-induced oxidative damage in human liver.

Establishment of Reproducible Xenotransplantation Model of T Cell Acute Lymphoblastic Leukemia in NOD/SCID Mice

T cell acute lymphoblastic leukemia(T-ALL) is an aggressive leukemia.However the poor prognosis and low morbidity restrict further analysis of the disease.Therefore there is an increasing demand to develop animal models for identifying novel therapeutic approaches.In this study,we inoculated the anti-mouse CD122 monoclonal antibody conditioned NOD/SCID mice with the leukemia cells from 9 T-ALL patients and 1 cell line via the tail vein.Four of the 9 patients and the cell line were successfully engrafted.Flow cytometry detected high percentage of human CD45 + cells in recipient mice.Immunohistochemistry showed infiltration of human CD45 + cells in different organs.Serial transplantation was also achieved.In vivo drug treatment showed that dexamethasone could extend survival,which was consistent with clinical observation.These results demonstrated that we successfully established 5 xenotransplantation models of T-ALL in anti-mCD122 mAb conditioned NOD/SCID mice,which recapitulated the characteristics of original disease.

Establishment of Xenotransplantation Model of Human CN-AML with FLT3-ITD^(mut)/NPM1 in NOD/SCID Mice

Summary： Patients with FLT3-ITD^mmutt/NPM1- cytogenetically normal acute myeloid leukemia （CN-AML）, as high-risk molecular group in CN-AML, are associated with a worse prognosis than other CN-AML patients. It is beneficial to generate xenotransplantation model of FLT3-ITD^mut/NPM1- CN-AML to better understand the pathogenesis and therapeutic strategies of such AML subtype. The purpose of present study was to establish the xenotransplantation model in NOD/SCID mice with FLT3-ITD^mut/NPM1- CN-AML primary cells. The FLT3-ITD^mut/NPM1- CN-AML primary cells from 3 of 7 cases were successfully transplanted into NOD/SCID mice, and human CD45 positive cells were detected in the peripheral blood, spleen and bone marrow of mice by using flow cytometry. Infiltration of human leukemia cells in various organs of mice was observed by using immunohistochemistry. Gene analysis confirmed sustained FLT3/ITD mutation without NPM1 mutation in mice. By performing serial transplantation, it was found that characteristics of the leukemia cells in secondary and tertiary genera- tion models remained unchanged. Moreover, in vivo cytarabine administration could extend survival of NOD/SCID mice, which was consistent with clinical observation. In conclusion, we successfully estab- lished xenotransplantation model of human FLT3-ITD^mut/NPM1- CN-AML in NOD/SCID mice. The model was able to present primary disease and suitable to evaluate the curative effects of new drugs or therapy strategies.

Response of porcine hepatocytes in primary culture to plasma from severe viral hepatitis patients

AIM： To observe the effects of plasma from patients with severe viral hepatitis （SVHP） on the growth and metabolism of porcine hepatocytes and the clinical efficiency of bioartificial liver device.METHODS： Hepatocytes were isolated from male porcines by collagenase perfusion. The synthesis of DNA and total protein, leakages of AST and LDH, changes in glutathione （GSH）, catalase and morphology of porcine hepatocytes exposed to SVHP were investigated to indicate the effect of plasma from patients with severe hepatitis on the growth, injury, detoxification, and morphology of porcine hepatocytes.RESULTS： The synthesis of DNA and protein was inhibited in the medium containing 100% SVHP compared to the controls. The leakages of LDH and AST increased in porcine hepatocytes following exposure to 100% SVHP for 5 h. The difference between 100% SVHP and 10% newborn calf serum （NCS） was significant in t-test （LDH： t = 24.552, P = 0.001; AST： t = 4.169, P = 0.014）. After exposure to SVHP for 24 h, alterations in GSH status were significant （F = 2.746, P〈0.05） between porcine hepatocytes in 100% SVHP and 10% NCS, but no alteration occurred in the culture medium after 48 h （F = 4.378, ,P〈0.05）. A similar profile was observed in catalase activity. Many round vacuoles were observed in porcine hepatocytes cultured in SVHR The membranes of these cells became indistinct and almost all the cells died on d 5.CONCLUSION： Plasma from patients with severe hepatitis inhibits the growth, injures membrane, disturbs GSH homeostasis and induces morphological changes of porcine hepatocytes, It is suggested that SVHP should be pretreated to reduce the toxin load and improve the performance of porcine hepatocytes in extracorporeal liver-support devices.

CYP2E1-dependent hepatotoxicity and oxidative damage after ethanol administration in human primary hepatocytes

AIM： To observe the relationship between ethanol-induced oxidative damage in human primary cultured hepatocytes and cytochrome P450 2E1 （CYP2E1） activity, in order to address if inhibition of CYP2E1 could attenuate ethanol- induced cellular damage. METHODS： The dose-dependent （25-100 mmol/L） and time-dependent （0-24 h） exposures of primary human cultured hepatocytes to ethanol were carried out. CYP2E1 activity and protein expression were detected by spectrophotometer and Western blot analysis respectively. Hepatotoxicity was investigated by determination of lactate dehydrogenase （LDH） and aspartate transaminase （AST） level in hepatocyte culture supernatants, as well as the intracellular formation of malondialdehyde （MDA）. RESULTS： A dose-and time-dependent response between ethanol exposure and CYP2E1 activity in human hepatocytes was demonstrated. Moreover, there was a time-dependent increase of CYP2E1 protein after 100 mmol/L ethanol exposure. Meanwhile, ethanol exposure of hepatocytes caused a time-dependent increase of cellular MDA level, LDH, and AST activities in supernatants. Furthermore, the inhibitor of CYP2E1, diallyl sulfide （DAS） could partly attenuate the increases of MDA, LDH, and AST in human hepatocytes. CONCLUSION： A positive relationship between ethanolinduced oxidative damage in human primary cultured hepatocytes and CYP2E1 activity was exhibited, and the inhibition of CYP2E1 could partly attenuate ethanol-induced oxidative damage.

Intracerebral xenotransplantation of semipermeable membrane- encapsuled pancreatic islets

AIM： To identify the decreasing effect of xenotransplantion in combination with privileged sites on rejection and death of biological semipermeable membrane-（BSM） encapsulated implanted islets. METHODS： After the BSM experiment in vitro, BSM- encapsulated SD rat＇s islet-like cell clusters （ICCs） were xenotransplanted into normal dog＇s brain. Morphological changes were observed under light and transmission electron microscope. The islets and apoptosis of implanted B cells were identified by insulin-TONEL double staining. RESULTS： The BSM used in our study had a favorable permeability, some degree of rigidity, lighter foreign body reaction and toxicity. The grafts consisted of epithelioid cells and loose connective tissue. Severe infiltration of inflammatory cells was not observed. The implanted ICCs were identified 2 mo later and showed typical apoptosis. CONCLUSION： BSM xenotransplantation in combination with the privileged site can inhibit the rejection of implanted heterogeneous ICCs, and death of implanted heterogeneous B cells is associated with apoptosis. 2005 The WJG Press and Elsevier Inc. All rights reserved.

Novel SLA class I alleles of Chinese pig strains and their significance in xenotransplantation

To lay background for studying rejection mechanisms in xenotransplantation and developing the strategies for intervention, class I genes of swine leukocyte antigens (SLA) of three Chinese pig strains Bm, Gz and Yn were cloned and sequenced. The cDNA of the class I loci P1 and P14 were amplified by RT-PCR and subjected to insert into sequencing vectors. All six allelic sequences we examined, each two for one Chinese strain, are not identical to those reported, which allows these novel sequences receiving their accession numbers AY102467- AY102472 from GenBank. This study further reveals that the homologies of MHC class I genes in their primary structures and the deduced amino acids between Chinese pigs (SLA) and human (HLA-A*0201) are better than those between pigs and mice (H-2Db/H-2Kb). The comparison also indicates that the amino acid residues critical for recognition by human KIRs are altered in the swine class I molecules. The amino acids responsible for binding human CD8 coreceptor are largely conserved although there are two critical residues substituted. A functional test indicated that the human T cells specific for the prokaryotically expressed SLA Plprotein could respond quite well in vitro to the class I-positive swine chon-drocytes and PBMCs in presence of human APCs. This implies that, due to the substitution of two critical residues, the inaccessibility of human CD8 coreceptor to swine class I molecule might be contributable to the indirect pathway that the human T cells have to use for recognizing the SLA class I xenogeneic antigens.

Expression of L amino acid transport system 1 and analysis of iodine-123-methyltyrosine tumor uptake in a pancreatic xenotransplantation model using fused high-resolution-micro-SPECT-MRI

BACKGROUND:The specificity in discriminating pancreatitis is limited in the positron emission tomography(PET)using Fluorine-18-fluorodeoxyglucose.Furthermore,PET is not widely available compared to the single photon emission computed tomography(SPECT).Since amino acids play a minor role in metabolism of inflammatory cells,the potential of the SPECT tracer,3-[ 123 I]iodo-L-α-methyltyrosine(123I-IMT),for detecting pancreatic cancer was examined in xenotransplantation models of human pancreatic carcinoma in mice. METHODS: 123 I-IMT was injected to eight mice inoculated with subcutaneous or orthotopic pancreatic tumors.Fused high-resolution-micro-SPECT(Hi-SPECT)and magnetic resonance imaging were performed.The gene expression level of L amino acid transport-system 1(LAT1)was analyzed and correlated with tumor uptake of 123 I-IMT. RESULTS:A high uptake of 123 I-IMT was detected in all tumor-bearing mice.The median tumor-to-background ratio (T/B)was 12.1(2.0-13.2)for orthotopic and 8.4(1.8-11.1)for subcutaneous xenotransplantation,respectively.Accordingly, the LAT1 expression in transplanted Colo357 cells was increased compared to non-malignant controls.CONCLUSIONS:Our mouse model could show a high 123 I-IMT uptake in pancreatic cancer.Fused MRI scans facilitate precise evaluation of uptake in the specific regions of interest.Further studies are required to confirm these findings in tumors derived from other human pancreatic cancer cells.Since amino acids play a minor role in the metabolism of inflammatory cells,the potential for application of 123 I-IMT to distinguish pancreatic tumor from inflammatory pancreatitis warrants further investigation.

Response of the xenograft endothelium in the concordant xenotransplantation

To investigate the response of the xenograft endothelium in the concordant hamster to rat cardiac xenotransplantation and the mechanism of acute vascular rejection. Methods： The animals were divided into 5 groups randomly： control group, CsA group, splenectomy group, DO splenectomy＋CsA group and D3 splenectomy＋CsA group. Hamster heart was heterotopicaly transplanted to rat abdominal cavity. The graft survival was monitored by palpation of the rat abdominal wall. The histological and ultrastructural changes of the xenogafts were investigated. NF-κB and P-selectin expression in the xenograft were detected. Heme Oxigenase-1 and Bcl-2 expression were also detected in the xenografts of different groups. Results： The mean survival time of the xenografts in control group, CsA group, splenectomy group, DO splenectomy＋CsA group and D3 splenectomy＋CsA group was 3.4±0.55, 3.8±0,45, 6.4± 1.52, 30 and 7.4 ±1.14 days. The rejected graft showed typical acute vascular rejection in control group, CsA group,splenectomy group and D3 splenectomy＋CsA group. Endothelial cells of the rejected xenograft showed dramatic assembly of ribosomes and expansion of the rough endoplasmic reticulum. However, the endothelium of the long-term survived grafts in DO splenectomy＋CsA group showed normal architecture. NF-κB and P-selectin expression were detected in the rejected xenografts. HO-1 expression was observed in the long-term survived xenografts in DO splenectomy＋CsA group. Conclusion： The endothelial cells of the xenograft might be activated during the acute vascular rejection. Expression of HO-1 might inhibit the upregulation of NF-κB and adhesion molecular which decreases the activation of the endotheliuna of the graft.

Protective effects of ACLF sera on metabolic functions and proliferation of hepatocytes co-cultured with bone marrow MSCs in vitro

AIM： To investigate whether the function of hepatocytes co-cultured with bone marrow mesenchymal stem cells （MSCs） could be maintained in serum from acute-on- chronic liver failure （ACLF） patients.METHODS： Hepatocyte supportive functions and cy- totoxicity of sera from 18 patients with viral hepatitis B-induced ACLF and 18 healthy volunteers were evalu- ated for porcine hepatocytes co-cultured with MSCs and hepatocyte mono-layered culture, respectively. Chemo- kine profile was also examined for the normal serum and liver failure serum.RESULTS： Hepatocyte growth factor （HGF） and Tumor necrosis factor; tumor necrosis factor （TNF）-a were re- markably elevated in response to ACLF while epidermal growth factor （EGF） and VEGF levels were significantly decreased. Liver failure serum samples induced a higher detachment rate, lower viability and decreased liver sup- port functions in the homo-hepatocyte culture. Hepato-cytes co-cultured with MSCs could tolerate the cytotoxic- ity of the serum from ACLF patients and had similar liver support functions compared with the hepatocytes cul- tured with healthy human serum in vitro. In addition, co- cultured hepatocytes maintained a proliferative capability despite of the insult from liver failure serum.CONCLUSION： ACLF serum does not impair the cell morphology, viability, proliferation and overall metabolic capacities of hepatocyte co-cultured with MSCs in vitro.

Xenotransplantation of embryonic pig pancreas for treatment of diabetes mellitus in non-human primates

Transplantation therapy for diabetes in humans is limited by the low availability of human donor whole pancreas or islets. Outcomes are complicated by immunosuppressive drug toxicity. Xenotransplantation is a strategy to overcome supply problems. Implantation of tissue obtained early during embryogenesis is a way to reduce transplant immunogenicity. Pig insulin is biologically active in humans. In that regard the pig is an appropriate xenogeneic organ donor. Insulin-producing cells originating from embryonic pig pancreas obtained very early following pancreatic primordium formation [embryonic day 28 (E28)] engraft long-term in rhesus macaques. Endocrine cells originating from embryonic pig pancreas transplanted in host mesentery migrate to mesenteric lymph nodes, engraft, differentiate and improve glucose tolerance in rhesus macaques without the need for immune suppression. Transplantation of embryonic pig pancreas is a novel approach towards beta cell replacement therapy that could be applicable to humans.

Replication of hepatitis B virus in primary duck hepatocytes transfected with linear viral DNA

AIM: To explore the expression and replication of hepatitis B virus (HBV) DNA in primary duck hepatocytes (PDHs).METHODS: Complete HBV genome was transfected into PDHs by electroporation (transfected group, 1.19×1012copies of linear HBV DNA/1×107 PDHs). After 1-5 d of transfection, HBsAg and HBeAg in the supernatant and lysate of PDHs were measured with the IMX System.Meanwhile, replicative intermediates of HBV DNA were analyzed by Southern blotting and Dot blotting. PDHs electroporated were used as control group.RESULTS: HBsAg in the hepatocyte lysates of transfected group was 15.24 (1 d), 14.55 (3 d) and 5.13 (5 d; P/N values, positive≥2.1) respectively. HBeAg was negative (＜2.1). Both HBsAg and HBeAg were negative in the supernatant of transfected group. Dot blotting revealed that HBV DNA was strongly positive in the transfected group and negative in the control group. Southern blot analysis of intracellular total DNA indicated that there were relaxed circular (rc DNA), covalently closed circular (ccc DNA), and single-stranded (ss DNA) HBV DNA replicative intermediates in the transfected group, there was no integrated HBV DNA in the cellular genome. These parameters were negative in control group.CONCLUSION: Expression and replication of HBV genes can occur in hepatocytes from non-mammalian species.HBV replication has no critical species-specificity, and yet hepatic-specific regulating factors in hepatocytes may be essential for viral replication.

Gene expression analysis of primary normal human hepatocytes infected with human hepatitis B virus

AIM： To find the relationship between hepatitis B virus （HBV） and hepatocytes during the initial state of infection by cDNA microarray. METHODS： Primary normal human hepatocytes （PNHHs） were isolated and infected with HBV. From the PNHHs, RNA was isolated and inverted into complement DNA （cDNA） with Cy3- or Cy5- labeled dUTP for microarray analysis. The labeled cDNA was hybridized with microarray chip, including 4224 cDNAs. From the image of the microarray, expression profiles were produced and some of them were confirmed by RT-PCR, immunoblot analysis, and NF-κB luciferase reporter assay. RESULTS： From the cDNA microarray, we obtained 98 differentially regulated genes. Of the 98 genes, 53 were up regulated and 45 down regulated. Interestingly, in the up regulated genes, we found the TNF signaling pathway-related genes： LT-α, TRAF2, and NIK. By using RT-PCR, we confirmed the up-regulation of these genes in HepG2, HuhT, and Chang liver cells, which were transfected with pHBV1.2x, a plasmid encoding all HBV messages. Moreover, these three genes participated in HBV- mediated NF-κB activation. CONCLUSION： During the initial state of HBV infection, hepatocytes facilitate the activation of NF-κB through up regulation of LT-α, TRAF2, and NIK.

Current advances in xenotransplantation

BACKGROUND: Transplantation of organs is a well-known and accepted life-saving procedure for end-stage kidney, liver, heart and lung diseases. The insufficient number of donor organs limits the application of this technique and leads to unnecessary loss of life. Experimental techniques such as xenotransplantation are extremely important to determine new methods of creating organ availability. DATA SOURCES; A literature search of Pubmed database was conducted and research articles reviewed. RESULTS: Xenotransplantation is a progressive field of research. Human complement regulatory protein ( hDAF) transgenic pigs and new immunosuppressive strategies that reduce xenoreactive αgal antibodies, have decreased rates of acute vascular rejection. Transplantation of α-1, 3-galac-tosyltransferase knock-out pig organs into baboons has resulted in the longest graft survival to date. Coagulation pathways have been identified as having a role in graft rejection. In vitro studies of porcine endogenous retroviruses (PERVs) show encouraging results that zoonosis will be less hindering to xenotransplantation than once thought. CONCLUSIONS: Several recent advances in xenotransplantation research have brought this technique closer to clinical application. The Ethics Committee of the International Xenotransplantation Association has made recommendations to ensure maintenance of ethical standards. Advancement will depend on the development of pig models, novel immunosuppressive strategies to target the innate immune system, and new ways to create donor specific tolerance. Prevention of rejection and transmission of infectious agents remain unresolved issues. In the future, it is feasible that xenotransplantation will be used to resolve this medical dilemma.

Protective Effects of Dimethyl-4,4'-Dimethoxy-5,6,5',6'-Dimethylene Dioxybiphenyl-2,2'-Dicarboxylate on Damages of Isolated Rat Hepatocytes Induced by Carbon Tetrachloride and D-galactosamine

The protective effect of biphenyl dimethyl dicarboxylate (DDB) on chemically induced damages was studied in isolated suspended rat hepatocytes. The experimental results showed that DDB (200μg/106 cells) efficiently protected the hepatocytes against carbon tetrachloride (CC14 10 mrnol.L-1) and D-galactosamine (1 mmol.L-1) induced damages. Membranal lipid peroxidation (malondialdehyde, MDA formation) and glutamic pyruvic transaminase (GPT) release from the hepatocytes were markedly decreased. The damage of the cell surfaces of the hepatocytes were also reduced as seen under a scanning electron microscope (SEM). Pretreatment with DDB (300 mg-kg-1) orally ameliorated the reduction of liver glycogen and blood glucose caused by ip injection of D-galactosamine (800 mg-kg-1) in mice. When normal rats were given DDB 300 mg-kg-1 once daily for 10 d, the free ribosomal protein and RNA in the liver increased significantly. These results indicate that DDB is of beneficial effects on both damaged and normal hepatocytes.