Liver Xenografts Research Articles

Quercetin Aglycone Is Bioavailable in Murine Pancreas and Pancreatic Xenografts

Quercetin is a potential chemopreventive and chemotherapeutic agent for pancreatic and other cancers. This study examined the distribution of quercetin in plasma, lung, liver, pancreas, and pancreatic cancer xenografts in a murine in vivo model and the uptake of quercetin in pancreatic cancer MiaPaCa-2 cells in a cellular in vitro model. Mice were randomly allocated to control or 0.2 and 1% quercetin diet groups utilizing the AIN93G-based diet (n = 12 per group) for 6 weeks. In addition, 6 mice from each group were injected weekly with the chemotherapeutic drug gemcitabine (120 mg/kg mouse, ip). MiaPaCa cells were collected from culture medium after cells were exposed to 30 muM quercetin for 0.5, 1, 2, 4, 8, and 24 h. Levels of quercetin and 3-O'-methylquercetin in mouse tissues and MiaPaCa-2 cells were measured by high-pressure liquid chromatography following enzymatic hydrolysis and then extraction. The study showed that quercetin is accumulated in pancreatic cancer cells and is absorbed in the circulating system, tumors, and tissues of pancreas, liver, and lung in vivo. A higher proportion of total quercetin found in tumors and pancreas is aglycones. Gemcitabine cotreatment with quercetin reduced absorption of quercetin in the mouse circulatory system and liver. Results from the study provide important information on the interpretation of the chemotherapeutic efficacy of quercetin.

Abstract 366: Changes in gene expression profiles in human breast & liver cancer cells and xenografts acquiring resistance to sunitinib

Abstract Background: Deciphering acquired mechanisms of resistance to sunitinib, a multitarget receptor tyrosine kinase inhibitors blocking VEGFR, PDGFR, KIT, and RET, remains a major clinical challenge for developing a second generation of compounds, simultaneous and sequential combination strategies. This study aimed characterizing molecular changes associated with acquired cellular resistance to sunitinib in two human cancer models. Materials and Methods: Parental MCF7 breast carcinoma and SK-HEP1 hepatocellular carcinoma cells primarily sensitive to sunitinib were exposed to stepwise increasing doses of sunitinib for >4 months and >7 months, respectively. MCF7-suni and SK-HEP1-suni were shown to grow in the presence of 3-fold IC50 concentrations as compared to parental cells. Mouse xenograft bearing MCF7 were treated with subtoxic doses of sunitinib 2-20mg/kg/day. mRNA levels of a panel of 75 selected genes were evaluated in sensitive and resistant cell lines using q-RT-PCR. Protein phoshorylation (p) was assessed by western-blot analysis. Results: MCF-7 cells expressed PDGFRA and SK-HEP1 cells expressed PDGFRB, both having low/no baseline levels of VEGFR1-3 and RET mRNA expression. Exposure to 48-hour sunitinib led to IC50s of 5.5µM and 4.5µM in MCF-7 and SK-HEP1, respectively. In SK-HEP1 cells, exposure to sunitinib was associated with a decreased p-AKTS473, p-70S6KT389 and p-ERKT202/Y204. IC50s of sunitinib were 10µM in MCF7-suni and 11 µM in SK-HEP1-suni. Sustained exposure to sunitinib led to >3-fold increased mRNA expression of VEGFC, VEGFR1, VEGFR3, Neuropilin-1, CXCL12, CXCR4, HIF1-alpha, PDGFRA, endothelin-1, RET, and in a lesser extend EG-VEGF in MCF7-suni as compared to parental MCF7. Protracted exposure to sunitinib in SK-HEP1-suni led to a >3-fold increase mRNA expression of VEGFA-D, VEGFR3, CXCL12, CXCR4, PDGFRA, and RET as compared to parental SK-HEP1 cells. At IC50 concentration, p-ERKT202/Y204 was decreased in SK-HEP1-suni cells. To mimic acquired resistance to sunitinib in human, mice bearing MCF7 xenografts were treated using continuous daily dosing of sunitinib until occurrence of tumor progression under therapy occurring around day 20. mRNA expressions were compared in tumors at baseline and at the time of progression. Increased >3-fold mRNA expressions of CXCR4, CXCL12, PDGFRA were observed in progressive xenograft as compared to untreated control tumors. Conclusions: Our results suggest that acquired in vitro & in vivo resistance of human cancer cells to sunitinib may involve the increased expression of several known targets of sunitinib as well as the expression of other family of receptor involved in tumor angiogenesis, invasion, and metastasis. Further works shall focus in understanding the intriguing role of CXCR4/CXCL12 (SDF-1) in the development of resistance to sunitinib. Partially granted by FNAB. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 366.

Abstract 1389: Low dose metronomic chemotherapy of S-1 and Vandetanib produces nontoxically good therapeutic results for hepatocellular carcinoma (HCC)

Abstract [Background and aim] A conventional chemotherapy for cancer, which is administered at the maximum tolerated dose (MTD), requires a break period to avoid adverse toxic events. The breaks sometimes allow tumor regrowth. On the other hand, low dose metronomic chemotherapy (LDM) means frequent low dose administration of the cytotoxic agent without breaks. Here, we investigated the therapeutic efficacies of LDM using S-1, a novel 5-FU based drug, and with Vandetanib (VEGFR-2 and EGFR inhibitors), an anti-angiogenic agent. [Materials and Methods] The inhibitory effects of 5-FU on the proliferation of HUVEC and human hepatoma cell lines were evaluated in vitro. We compared the anti-tumor effect, toxicity and survival in the following 6 groups; MTD and LDM using S-1, Vandetanib (25 mg/kg/day), S-1 MTD + Vandetanib and S-1 LDM + Vandetanib. In the S-1 MTD group, mice were administered orally at 15 mg/kg/day for 7 days followed by a break for 7 days. In the S-1 LDM group, S-1 of 5mg/kg/day was consecutively administered once a day without a break. In the subcutaneous tumor model, tumor volumes and toxicities were evaluated. In the liver xenograft model, tumor volumes and survival were evaluated. Micro vessel density (MVD) in tumor tissues was evaluated by immunohistochemistry using an anti CD-31 antibody. The expression of VEGF and thrombospondin-1 (an endogeneous anti-angiogenic factor: TSP-1) was analysed by Western blot. [Results] Cell proliferation of HUVEC and hepatoma cell lines was inhibited at apparently lower doses in the metronomic schedule compared with the MTD schedule. In the subcutaneous tumor model, S-1 LDM significantly inhibited tumor growth (control: 4,810 ± 1,440 mm3, S-1 MTD: 3,212 ± 1,364 mm3, S-1 LDM: 1,927 ± 652 mm3) and its effect was enhanced by the combination with Vandetanib (LDM S-1 + Vandetanib: 1,383 ± 697 mm3). In toxicities, S-1 MTD caused severe body weight loss, and myelo-suppression. On the other hand, S-1 LDM did not show any toxicities. In the liver xenograft model, S-1 LDM and the combination therapy with Vandetanib significantly inhibited tumor growth and S-1 LDM apparently prolonged survival (control: 28.9 ± 6.4 days, S-1 MTD: 29.6 ± 3.9 days, S-1 LDM: 34.3 ± 3.9 days). S-1 LDM + Vandetanib showed the most prolonged survival (49.6 ± 11.5 days). MVDs in the tumor of control, S-1 MTD, S-1 LDM, S-1 LDM + Vandetanib were 41.1 ± 9.2 /field, 35.8 ± 5.5 /field, 17.2 ± 4.1 /field, and 8.2 ± 1.6 /field, respectively. The expression of VEGF and TSP-1 was strongly upregulated in S-1 LDM group and S-1 LDM + Vandetanib group. [Conclusion] S-1 LDM significantly suppressed tumor growth and prolonged survival without severe toxicities. These anti-tumor effects were enhanced by the combination therapy with Vandetanib. These results indicate that S-1 LDM with an anti-angiogenic agent will become a novel therapeutic strategy for human hepatocellular carcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1389.

The selective Aurora B kinase inhibitor AZD1152 as a novel treatment for hepatocellular carcinoma

We previously identified that high Aurora B expression was associated with hepatocellular carcinoma (HCC) recurrence due to tumor dissemination. In this preclinical study, a novel inhibitor of Aurora B kinase was evaluated as a treatment for human HCC. AZD1152 is a selective inhibitor of Aurora B kinase. Twelve human HCC cell lines were analyzed for Aurora B kinase expression and the in vitro effects of AZD1152. The in vivo effects of AZD1152 were analyzed in a subcutaneous xenograft model and a novel orthotopic liver xenograft model. Aurora B kinase expression varied among the human HCC cell lines and was found to correlate with inhibition of cell proliferation, accumulation of 4N DNA, and the proportion of polyploid cells following administration of AZD1152-hydroxyquinazoline-pyrazol-anilide (AZD1152-HQPA). AZD1152-HQPA suppressed histone H3 phosphorylation and induced cell death in a dose-dependent manner. Growth of subcutaneous human HCC xenografts was inhibited by AZD1152 administration. In an orthotopic hepatoma model, treatment with AZD1152 significantly decelerated tumor growth and increased survival. Pharmacobiological analysis revealed that AZD1152 induced the rapid suppression of phosphohistone H3, followed by cellular apoptosis in the liver tumors but not in the normal tissues of the orthotopic models. Our preclinical studies indicate that AZD1152 is a promising novel therapeutic approach for the treatment of HCC.

Study of the microcirculation in hDAF transgenic rat livers xenoperfused with human blood

The microcirculation was assessed in the livers of human decay accelerating factors (hDAF) and wild-type transgenic rats by fluorescent intravital microscopy, histology and histomorphology to determine the benefits of hDAF expression for the microcirculation of a rat liver xenograft perfused with human blood. Male hDAF transgenic rats (group A; n = 20) and wild-type Sprague-Dawley rats (group B; n = 20) were xenoperfused with human blood, while other male wild-type Sprague-Dawley rats (group C; n = 10) were perfused with allogeneic blood. Following plasma and leukocyte staining with fluorescein sodium, and platelet staining with rhodamine, the right lobe of the liver was assessed by intravital microscopy, counting the numbers of perfused sinusoids and leukocytes adhering to the endothelium per mm(2), and calculating the acinar perfusion index (Pi). The liver underwent histological assessment at the end of each experiment. Mean +/- SEM values were calculated and the Mann-Whitney U-test was used for statistical analysis. The number of perfused sinusoids was higher in the group of hDAF rat livers (group A) and controls (group C) than in the group of non-transgenic rat livers perfused with human blood (group B) (P < 0.05), although only group C still had a significantly more perfused sinusoids than the other groups after 90 min of perfusion (P < 0.05). The acinar perfusion index was higher in groups A and C than in group B (P < 0.05); here again, only group C still had a significantly higher Pi than group B after 90 min of perfusion (P < 0.05). There was a massive accumulation of leukocytes that peaked after 5 min and persisted throughout the perfusion in all three groups. Histology showed portal and subendothelial hepatic vein hemorrhage, necrosis and inflammatory reaction, which were particularly evident in group B. In our study, rat livers transgenic for hDAF were better protected against early tissue damage by perfusion with human blood, but this did not result in a longer xenograft survival.

Rho-kinase 2 is frequently overexpressed in hepatocellular carcinoma and involved in tumor invasion #

Deregulation of Rho family small guanosine triphosphatases has been implicated in human carcinogenesis. Rho-kinases are downstream effectors of Rho guanosine triphosphatases in the regulation of cytoskeletal reorganization and cell motility. However, their functions in human cancers remain elusive. In this study, we aimed to investigate the role of Rho-kinases in hepatocellular carcinoma (HCC) tumor progression and invasion. We first examined the expression of the two Rho-kinases (ROCK1 and ROCK2) in human HCC, and found that ROCK2 was frequently overexpressed in primary HCCs (22/41 [53.66%]). Clinico-pathological analysis revealed that overexpression of ROCK2 was significantly associated with the presence of tumor microsatellite formation (P = 0.005), suggesting that deregulation of ROCK2 may contribute to the intrahepatic metastasis of HCC. Consistently, we demonstrated that stable overexpression of ROCK2 significantly enhanced cell motility and invasiveness in HCC cells. Conversely, stable knockdown of ROCK2 by short hairpin RNA approach remarkably reduced HCC cell migration and invasion. Moreover, orthotopic liver xenograft models provided further support that stable knockdown of ROCK2 suppressed HCC invasion in vivo. Stable knockdown of ROCK2 in HCC cells significantly inhibited Golgi reorientation, myosin phosphatase phosphorylation, and formations of stress fibers, filopodia, and lamellipodia; these molecular and cellular events are crucial for cell motility and cancer invasion. Our results indicate that ROCK2 was overexpressed in human HCCs, and this overexpression was associated with a more aggressive biological behavior. Our findings also demonstrate that ROCK2 played a significant role in regulating cytoskeletal events and contributed to the invasion of HCC.

Liver xenografts for the treatment of acute liver failure: Clinical and experimental experience and remaining immunologic barriers

A critical element restricting the application of liver transplantation is the shortage of human deceased donor organs. Xenotransplantation using pig organs might be a solution to this shortage. Although the problems that still require resolution include the immunologic barrier, the potential risk of transferring infectious agents with the transplanted organ, and uncertainty about whether the transplanted organ will function satisfactorily in the human environment, recent progress in the genetic manipulation of pigs has led to the prospect that clinical xenografting, at least as a bridge to allotransplantation, may be possible in the foreseeable future. Experience with clinical auxiliary and orthotopic liver xenotransplantation and experimental liver xenotransplantation in nonhuman primate and other large animal models is reviewed, and the remaining immunologic problems are discussed. Evidence suggests that, in patients with hepatic failure, the pig liver may be less susceptible to antibody-mediated injury than other pig organs, such as the heart or kidney. Pig Kupffer cells and other macrophages will recognize and phagocytose primate red blood cells, but this problem should be overcome by pretransplant depletion of macrophages from the organ-source pig. From the evidence currently available, it does not seem unduly optimistic to anticipate that a liver from an alpha1,3-galactosyltransferase gene-knockout pig would survive at least long enough to function as a successful bridge to allotransplantation.

Superparamagnetic iron oxide nanoparticles stabilized by alginate: Pharmacokinetics, tissue distribution, and applications in detecting liver cancers

The objectives of this study were to describe the pharmacokinetics and tissue distribution of superparamagnetic iron oxide nanoparticle (SPIO) stabilized with alginate (SPIO-alginate), and investigate its potential in detecting liver cancers as a newly developed magnetic resonance (MR) contrast agent. Pharmacokinetics and tissue distribution of SPIO-alginate were investigated in Sprague–Dawley rats. The results showed that SPIO-alginate was eliminated rapidly from serum with the half-life of 0.27 h at 109.5 μmol Fe/kg and accumulated dominantly in liver and spleen with a total percentage of more than 90% of dose after intravenous injection. The studies of pharmacokinetics and distribution of SPIO-alginate in rats indicated the MR contrast agent, based on SPIO, mainly accumulating in targeting organs that contain phagocytosing cells, i.e. liver and spleen. The efficacies in detecting hepatocellular carcinoma (HCC) of rat with primary liver cancer and xenograft liver cancers of rabbit were investigated before and after injection of SPIO-alginate. The signal intensity of liver parenchyma in rabbit with VX2 tumor after injection of SPIO-alginate was reduced sharply resulting in a significant contrast between liver parenchyma and tumor. Detection of the HCC in rat model was also demonstrated. The present study provides evidence that SPIO-alginate might have the ability to improve the detection of liver tumors as an MR contrast agent, and the efficacy is associated with the SPIO specifically located in Kupffer cells in hepatic sinusoid.

Oncolytic recombinant herpes simplex virus for treatment of orthotopic liver tumors in nude mice

Cell-specific, replicating viruses are being developed as a new class of oncolytic agents. A novel approach to viral gene therapy with the use of replication-competent herpes simplex virus has been described; G92A is a replication-competent, multimutant oncolytic herpes simplex virus (HSV) that has been evaluated for anticancer effects and selectivity in the treatment of subcutaneous tumors. G92A replicates efficiently in albumin-producing tumor cell lines but not in non-albumin-producing tumor cell lines, whereas both types are equally susceptible to a non-tissue-specific recombinant HSV, hrR3. In this study, we analyzed the antitumoral efficacy of a single intrasplenic G92A or hrR3 injection in nude mice. In vivo, G92A replicated well in liver xenografts of human albumin-producing hepatoma cells (Hep3B) but not in liver xenografts of a non-albumin-producing malignant colon tumor cell line (HT29), whereas hrR3 replicated well in both tumor types. G92A effectively and selectively replicated throughout liver tumors without apparent hepatotoxicity and inhibited tumor growth, leading to a significantly increased survival time. By monitoring lacZ histochemical staining, we determined the oncolytic potential of recombinant HSV against liver tumors. Our results indicate that G92A warrants further investigation as a clinical therapy against malignant liver tumors.

Simultaneous determination of capecitabine and its metabolites by HPLC and mass spectrometry for preclinical and clinical studies

A reverse-phase high-performance liquid chromatography method with electrospray ionization and detection by mass spectrometry is described for the simultaneous determination of capecitabine, its intermediate metabolites (DFCR, DFUR) and the active metabolite 5-fluorouracil in mouse plasma, liver and human xenograft tumours. The method was also cross-validated in human plasma and human tumour for clinical application. The method has greater sensitivity than previously published methods with an equivalent accuracy and precision. It uses less biological material (plasma, tissue) and should therefore be applicable to biopsies in patients treated with capecitabine.

Therapeutic Effect of Fe&lt;SUB&gt;2&lt;/SUB&gt;O&lt;SUB&gt;3&lt;/SUB&gt; Nanoparticles Combined with Magnetic Fluid Hyperthermia on Cultured Liver Cancer Cells and Xenograft Liver Cancers

The therapeutic effect of Fe2O3 nanoparticles combined with magnetic fluid hyperthermia (MFH) on human hepatocarcinoma SMMC-7721 cells in vitro and xenograft liver cancer in nude mice is studied. We examined growth and apoptosis of SMMC-7721 cells treated with MFH containing Fe2O3 nanoparticles at various concentrations (2, 4, 6, and 8 g/liter) for 30-60 min by using MTT, flow cytometry (FCM), and transmission electron microscopy (TEM). We also observed weight and volume inhibitory rates of the tumors of SMMC-7721-bearing nude mice by using animal experiments. The results showed that Fe2O3 nanoparticles combined with MFH could significantly inhibit the proliferation and increase the ratio of apoptosis of SMMC-7721 cells, and the effect was dose-dependent. The inhibitory rate was 26.5%, 33.53%, 54.4%, and 81.2%, respectively, and the apoptosis rate was 30.26%, 38.65%, 50.28%, and 69.33%, respectively. Animal experiments showed that tumors became small. The weight inhibitory ratio was 42.10%, 66.34%, 78.5%, and 91.46%, and the volume inhibitory ratio was 58.77%, 80.44%, 93.40%, and 98.30%, respectively. Compared with the control and experimental groups, each group had statistically significant difference (p < 0.05). So, Fe2O3 nanoparticles combined with MFH could inhibit the proliferation and induce apoptosis of SMMC-7721 cells and also has a significant inhibitory effect on the weight and volume of xenograft liver cancer. However, the mechanism remains to be further investigated.

Liposome transfected to plasmid-encoding endostatin gene combined with radiotherapy inhibits liver cancer growth in nude mice

To evaluate whether intratumoral injection of liposome-endostatin complexes could enhance the antitumor efficacy of radiation therapy in human liver carcinoma (BEL7402) model. Recombinant plasmid pcDNA3.End was transfected into human liver carcinoma cell line (BEL7402) with lipofectamine to produce conditioned medium. Then BEL7402 cells and human umbilical vein endothelial cells (HUVECs) were treated with the conditioned medium. Cell cycle and apoptosis were analyzed by flow cytometer and endothelial cell proliferation rates were determined by MTT assay. The antitumor efficacy of endostatin gene combined with ionizing radiation in mouse xenograft liver tumor was observed. Endostatin significantly suppressed the S phase fraction and increased the apoptotic index in HUVECs. In contrast, endostatin treatment had no effect on BEL7402 cell apoptosis (2.1+/-0.3% vs 8.9+/-1.3%, t = 8.83, P = 0.009<0.01) or cell cycle distribution (17.2+/-2.3% vs 9.8+/-1.2%, t = 4.94, P = 0.016<0.05). The MTT assay showed that endostatin significantly inhibited the proliferation of HUVECs by 46.4%. The combination of local endostatin gene therapy with radiation therapy significantly inhibited the growth of human liver carcinoma BEL7402 xenografts, the inhibition rate of tumor size was 69.8% on d 28 compared to the untreated group. The tumor volume in the pcDNA3.End combined with radiation therapy group (249+/-83 mm3) was significantly different from that in the untreated group (823+/-148 mm3, t = 5.86, P = 0.009<0.01) or in the pcDNA3 group (717+/-94 mm3, t = 6.46, P = 0.003<0.01). Endostatin or the radiation alone also inhibited the growth of liver tumor in vivo, but their inhibition effects were weaker than those of endostatin combined with radiation, the inhibition rates on d 28 were 44.7% and 40.1%, respectively. Endostatin not only significantly suppresses tumor growth but also enhances the antitumor efficacy of radiation therapy in human carcinoma xenograft.

Mechanisms of purified cobra venom factor in preventing hyperacute rejection following discordant liver xenotransplantation in rats

To investigate the mechanisms of purified cobra venom factor (CVF) in preventing hyperacute rejection (HAR) and prolonging recipient survival following discordant liver xenotransplantation in rats. All animals were divided into two groups, group I (n = 20), unmodified recipients as control; group II (n = 20), xenograft recipients were treated with 50 microg/kg CVF i.v. on day-1. The pathologic changes of liver were observed and TNF-alpha of blood serum was detected. Recipient survival after CVF treatment is significantly prolonged compared with unmodified recipients (1.8 +/- 0.6 vs.7.4 +/- 2.1 h, P < 0.01). Histologically, widespread thrombosis, interstitial haemorrhage, C3 deposits on sinusoids and central veins characterized xenografts of the control group. Xenografts of the CVF group showed endothelium swelling and cellular infiltrate, no deposit of C3 was detected. Purified CVF can prevent guinea pig-to-rat liver xenografts from HAR and extend recipient survival. Preconditioning with CVF, guinea pig-to-rat combination is a useful life-supporting model to explore further mechanisms of discordant liver xenotransplantation.

Conditionally replicative adenovirus driven by the human telomerase promoter provides broad-spectrum antitumor activity without liver toxicity.

The human telomerase reverse transcriptase (hTERT) promoter is known to selectively drive transgene expression in many human cancer cells expressing hTERT, the catalytic component of the telomerase ribonucleoprotein complex. We have created a conditionally replicative adenovirus where the viral E1A gene, which is required for viral replication, is under the control of the hTERT promoter (AdhTERTp-E1A). In vitro studies with AdhTERTp-E1A virus on a variety of normal and tumor cell lines have shown that viral genome replication and productive infection is primarily restricted to telomerase-positive tumor cells. Lytic replication was not observed in normal primary fibroblast and epithelial cell lines tested. In vivo administration of the virus into nude mice bearing human liver or prostate tumor xenografts produced significant tumor reduction and, in some cases, resulted in complete tumor regression. AdhTERTp-E1A virus did not actively express E1A in normal mouse liver, in contrast to a control oncolytic vector in which the CMV promoter (AdCMVp-E1A) was driving the E1A gene. In addition, AdhTERTp-E1A virus produced no apparent toxicity to the liver in systemically injected mice. The hTERT promoter-driven oncolytic virus also produced significantly less toxicity to freshly cultured human hepatocytes. These studies demonstrate that an oncolytic virus driven by the telomerase promoter can be used to effectively kill a wide variety of cancer cell types and has the potential to treat primary and metastatic cancer of diverse origins.

The long-term survival of baboon-to-monkey kidney and liver xenografts.

The present study was undertaken to develop an optimum immunosuppressive regimen in baboon-to-monkey life-supporting kidney xenografts. Baseline therapy for all groups include cyclosporine (CsA) and steroids. We compared adding (1) cyclophosphamide (CyP) at dose of 20 mg/kg/day given on post-operative day (POD) 0, 2, 5 and 7; (2) mycophenolic mofetil (MMF) at a dose of 40 mg/kg/day by daily gavage; or (3) CyP + rapamycin (Rap). The latter group was divided into high and low dose subgroups. Untreated xenografts were rejected on POD 6, CsA alone treated xenografts survived for 35 days and CsA + CyP treated xenografts survived for 45 days. Adding MMF significantly prolonged mean survival to 111 +/- 53 days, but the xenografts eventually developed rejection. Combination therapy including CsA, CyP and Rap reliably prevented xenogenic rejection and achieved a mean survival of 290 +/- 30 days. However, high dose CyP + Rap led to high incidence of post-transplant lymphoproliferation disorders (PTLD), while the incidence of PTLD was significantly less in the low dose subgroup (P < 0.01). Four animals in this subgroup survived for more than 300 days with normal renal function and histology. In addition, two liver recipients treated with CsA + CyP survived for 91 and 1,076 days. We conclude that long-term survival of kidney or liver xenografts can be achieved in a non-human concordant xenograft model using currently available immunosuppressive agents.

Modulation of the pathology of late xenograft rejection by PAF-antagonist UR-12670 in the hamster-to-rat liver xenotransplant model.

PAF antagonists have been used in xenotransplantation to alleviate the pathogenesis of hyperacute rejection. This study evaluated the ability of the PAF antagonist UR-12670 to improve graft function in late xenograft rejection (LXR) in an orthotopic liver xenotransplantation model, and the involvement of PAF (platelet activating factor) in this type of rejection. The recipients of a hamster xenograft received standard immunosuppression (tacrolimus 0.2 mg/kg/30 days, MMF 25 mg/kg/8 days). Study groups: group A, without UR-12670, group B, UR-12670 (20 mg/kg/8 d) and group C, continuous administration of UR-12670 (20 mg/kg/d). Serum levels of xenoantibodies were evaluated by flow cytometry and tissue deposits by immunofluorescence. Immunoblot and indirect immunofluorescence assessed specificity of xenoantibodies. Conventional histology was performed. Continuous administration of UR-12670 improved the histological pattern of liver xenografts, especially necrosis, loss of hepatocytes, hemorrhage, sinusoidal congestion and lymphocyte infiltration. There was not a shift in specificity of xenoantibodies at different times posttransplantation, as demonstrated by immunoblotting and indirect immunofluorescence. UR-12670 administration had a beneficial effect on graft function and considerably improved the histopathological pattern, but it failed to induce tolerance after withdrawal of immunosuppression. UR-12670 had an immunomodulatory effect on cellular response but not on antibody production. There was not a change in the specificity of xenoantibodies produced at LXR compared with pretransplant antibodies.

Potent inhibition of angiogenesis and liver tumor growth by administration of an aerosol containing a transferrin-liposome-endostatin complex.

To obtain an efficient delivery system for transporting endostatin gene to mouse liver tumor xenografts by administration of aerosol. Recombinant plasmid pcDNA3.0/endostatin containing human endostatin gene together with signal peptide from alkaline phosphatase were transferred into human umbilical vein endothelial cell (HUVEC) by transferrin(TF)-liposome-endostatin complex. Western blot was used to detect the expression of human endostatin in transfected HUVEC cells and its medium. After the tumor-bearing mice were administrated with TF-liposome-endostatin complex, the lung tissue was analyzed by immunohistochemical method for expression of endostatin and the tumors were treated with CD-31 antibody to detect the density of microvessels in tumor tissues. The inhibition of tumor growth was estimated by the weight of tumors from groups treated with different doses of TF-liposome-endostatin complex. DNA fragmentation assay was used to detect the apoptosis of the cells from primary liver tumor. Western blot analysis and immunohistochemical method confirmed the expression of endostatin protein in vitro and in vivo. After the tumor sections were treated with CD-31 antibody, the positive reaction cells appeared brown while the negative cells were colorless. The positively stained area of the TF-liposome-endostatin treated group was significantly smaller (P<0.01, 645.8+/-55.2 microm(2)) than that of the control group (1 325.4+/-198.5 microm(2)). The data showed a significant inhibition of angiogenesis. After administration of TF-liposome-endostatin, comparing with the control group administrated with TF-liposome-pcDNA3.0, liver tumor growth in the mice treated with 50, 250 and 500 mg DNA/kg was inhibited by 36.6 %, 40.8 %, and 72.8 %, respectively (P<0.01). And a typical DNA fragmentation of apoptosis was found in the cells from tumor tissues of the mice treated with TF-liposome-endostatin but none in the control group. Endostatin gene could be efficiently transported into the mice with TF-liposome-DNA delivery system by administration of aerosol. TF-liposome-mediated endostatin gene therapy strongly inhibited angiogenesis and the growth of mouse xenograft liver tumors. It also could promote the development of apoptosis of tumors without direct influence on tumor cells.

Prolongation of liver xenograft survival by adenovirus vector-mediated CTLA-4Ig gene transfer

Cytotoxic T lymphocyte-associated antigen-4/immunoglobulin fusion products (CTLA-4Ig), a structural homologue of CD28, has been shown to inhibit cellular and humoral immune responses. In this study, we investigated the efficacy of an adenovirus vector containing the CTLA-4Ig gene (AdCTLA-4Ig) on recipient survival after hamster-to-rat liver xenografting. AdCTLA-4Ig was administrated intravenously immediately after grafting. Gene expression was achieved a maximum of 7 days after vector injection and continued for more than 4 weeks. The proportion of CD25 + T-cells in recipient lymph nodes was significantly reduced 7 days after administration of AdCTLA-4Ig, compared to a group given an adenoviral vector containing LacZ gene (AdLacZ) or to an untreated control group. AdCTLA-4Ig markedly reduced CD2 + T-cell infiltration into the graft and significantly prolonged recipient survival time (9.2±4.12 days), compared to the untreated group (5.4±0.78 days) ( P<0.001) and the AdLacZ-treated group (5.2±0.28 days) ( P<0.001). These results indicate that a blockade of T-cell co-stimulation by AdCTLA-4Ig inhibited T-cell activation and attenuated CD2 + T-cell infiltration into the xenograft, resulting in significant prolongation of recipient survival time. Thus, AdCTLA-4Ig therapy may provide a novel approach to immune regulation.

Single vs Dual Vessel Porcine Extracorporeal Liver Perfusion

Background. The use of porcine extracorporeal liver perfusion (PECLP) to provide temporary hepatic support for patients in fulminant hepatic failure has been limited by the fact that individual perfusions can be sustained for only a few hours. Inadequate liver function and/or hemodynamic instability are the major contributing factors for early interruption of PECLP. Recent reports suggest that the choice of single (portal vein only) vs dual (portal vein and hepatic artery) vessel perfusion may influence the duration of perfusion. We hypothesize that PECLP with single vessel perfusion (SVP) is associated with worse liver function and greater hemodynamic instability than PECLP with dual vessel perfusion (DVP).Materials and methods. To eliminate the potentially confounding influences of liver failure and xenograft rejection, liver isografts procured from White-Landrace pig donors were perfused by either SVP or DVP via an extracorporeal circuit established with normal White-Landrace pig recipients. The function of perfused livers was evaluated by measuring production of bile and Factors V and VIII, clearance of ammonia and lactate, and extraction of O2 at baseline and at 0, 1, 3, 6, 12, and 24 h after initiation of PECLP. The impact of PECLP on recipient hemodynamic status was assessed by monitoring BP, heart rate, urine output, O2 saturation, etc. Among other parameters evaluated were serum albumin and total protein and hepatic release of IL-1β and nitric oxide to assess their possible contributions to hemodynamic instability.Results. DVP and SVP livers cleared ammonia and lactate similarly. Both approaches were associated with progressive hypoalbuminemia and hypoproteinemia. DVP livers produced more bile and Factor V and were associated with less recipient hypotension and IL-1β and NO release than SVP livers.Conclusions. Livers with DVP function better than livers with SVP. The duration of PECLP can be limited by recipient hypotension, although this complication is less severe with DVP than with SVP.

Aberrations in hemostasis and coagulation in untreated discordant hepatic xenotransplantation: studies in the dog-to-pig model.

Discordant liver xenotransplantation is a poorly explored entity. Data from the few large animal studies of hepatic xenotransplantation suggest that severe hemorrhage is encountered. The purpose of the studies described here is to characterize the nature of the hemorrhage that accompanies liver xenotransplantation. Canine livers were transplanted into porcine recipients, and lethal hemorrhage was encountered. Analysis of recipient blood showed that factors V, IX, and X were present in adequate levels until after the hemorrhage appeared, suggesting that coagulation factor loss was the result and not the cause of hemorrhage. Platelet counts decreased dramatically in recipients within minutes of graft reperfusion. There also was no evidence of clotting activity in the blood of recipients of liver xenografts within minutes of graft reperfusion. This loss of clotting activity was specific to liver xenografts, was not seen in renal xenografts with or without venovenous bypass, and also was absent in pig-to-pig liver allografts. In brief, the hemorrhage that accompanies liver xenotransplantation occurs because of a decrease in the number and function of circulating platelets in the recipient.