Vesicular stomatitis virus variants selectively infect and kill human melanomas but not normal melanocytes.

Metastatic malignant melanoma remains one of the most therapeutically challenging forms of cancer. Here we test replication-competent vesicular stomatitis viruses (VSV) on 19 primary human melanoma samples and compare these infections with those of normal human melanocyte control cells. Even at a low viral concentration, we found a strong susceptibility to viral oncolysis in over 70% of melanomas. In contrast, melanocytes displayed strong resistance to virus infection and showed complete protection by interferon. Several recombinant VSVs were compared, and all infected and killed most melanomas with differences in the time course with increasing rates of melanoma infection, as follows: VSV-CT9-M51 < VSV-M51 < VSV-G/GFP < VSV-rp30. VSV-rp30 sequencing revealed 2 nonsynonymous mutations at codon positions P126 and L223, both of which appear to be required for the enhanced phenotype. VSV-rp30 showed effective targeting and infection of multiple subcutaneous and intracranial melanoma xenografts in SCID mice after tail vein virus application. Sequence analysis of mutations in the melanomas used revealed that BRAF but not NRAS gene mutation status was predictive for enhanced susceptibility to infection. In mouse melanoma models with specific induced gene mutations including mutations of the Braf, Pten, and Cdkn2a genes, viral infection correlated with the extent of malignant transformation. Similar to human melanocytes, mouse melanocytes resisted VSV-rp30 infection. This study confirms the general susceptibility of the majority of human melanoma types for VSV-mediated oncolysis.

Anti-caspase-3 preconditioning increases proinsulin secretion and deteriorates posttransplant function of isolated human islets.

Human islet isolation is associated with adverse conditions inducing apoptosis and necrosis. The aim of the present study was to assess whether antiapoptotic preconditioning can improve in vitro and posttransplant function of isolated human islets. A dose-finding study demonstrated that 200 µmol/L of the caspase-3 inhibitor Ac-DEVD-CMK was most efficient to reduce the expression of activated caspase-3 in isolated human islets exposed to severe heat shock. Ac-DEVD-CMK-pretreated or sham-treated islets were transplanted into immunocompetent or immunodeficient diabetic mice and subjected to static glucose incubation to measure insulin and proinsulin secretion. Antiapoptotic pretreatment significantly deteriorated graft function resulting in elevated nonfasting serum glucose when compared to sham-treated islets transplanted into diabetic nude mice (p < 0.01) and into immunocompetent mice (p < 0.05). Ac-DEVD-CMK pretreatment did not significantly change basal and glucose-stimulated insulin release compared to sham-treated human islets but increased the proinsulin release at high glucose concentrations (20 mM) thus reducing the insulin-to-proinsulin ratio in preconditioned islets (p < 0.05). This study demonstrates that the caspase-3 inhibitor Ac-DEVD-CMK interferes with proinsulin conversion in preconditioned islets reducing their potency to cure diabetic mice. The mechanism behind this phenomenon is unclear so far but may be related to the ketone CMK linked to the Ac-DEVD molecule. Further studies are required to identify biocompatible caspase inhibitors suitable for islet preconditioning.

Do mesenchymal stem cells function across species barriers? Relevance for xenotransplantation.

BACKGROUND: Allogeneic mesenchymal stem (stromal) cells (MSC) are a promising therapy for various pathological conditions. Genetically modified pig MSC have been demonstrated to downregulate the human T-cell response to pig antigens in vitro. Before genetically modified pig MSC can be used clinically, however, evidence needs to be provided to indicate whether they will survive in a human (xenogeneic) host. LITERATURE SEARCH AND RESULTS: A literature search through the end of 2011 identified 94 reports of the in vivo cross-species administration of MSC in a variety of experimental models. The majority (n = 89) involved the use of human MSC in various other species, with an occasional study using pig, rat, or guinea-pig MSC. When human MSC were used, they were largely derived from the bone marrow, adipose tissue, or umbilical cord blood. The routes of administration were varied, although almost half of the studies utilized the intravenous route. In 88 experiments (93.6%), there was evidence that the MSC engrafted and functioned across the species barrier, and in only six cases (6.4%) was there evidence of failure to function. Importantly, MSC function was confirmed in several different cross-species models. For example, human MSC functioned in no fewer than seven different recipient species. CONCLUSIONS: The data provided by this literature search strengthen the hypothesis that pig MSC will function satisfactorily in a different species, for example, humans. The data also suggest that our own in vitro observations on the efficacy of pig MSC in downregulating the strength of the human T-cell response to pig antigens will likely be reproduced in vivo in pre-clinical large animal models and in clinical trials.

Up to 9-day survival and control of thrombocytopenia following alpha1,3-galactosyl transferase knockout swine liver xenotransplantation in baboons.

BACKGROUND: With standard miniature swine donors, survivals of only 3 days have been achieved in primate liver-transplant recipients. The recent production of alpha1,3-galactosyl transferase knockout (GalT-KO) miniature swine has made it possible to evaluate xenotransplantation of pig organs in clinically relevant pig-to-non-human primate models in the absence of the effects of natural anti-Gal antibodies. We are reporting our results using GalT-KO liver grafts. METHODS: We performed GalT-KO liver transplants in baboons using an immunosuppressive regimen previously used by our group in xeno heart and kidney transplantation. Post-operative liver function was assessed by laboratory function tests, coagulation parameters and histology. RESULTS: In two hepatectomized recipients of GalT-KO grafts, post-transplant liver function returned rapidly to normal. Over the first few days, the synthetic products of the donor swine graft appeared to replace those of the baboon. The first recipient survived for 6 days and showed no histopathological evidence of rejection at the time of death from uncontrolled bleeding, probably caused by transfusion-refractory thrombocytopenia. Amicar treatment of the second and third recipients led to maintenance of platelet counts of over 40 000 per µl throughout their 9- and 8-day survivals, which represents the longest reported survival of pig-to-primate liver transplants to date. Both of the last two animals nevertheless succumbed to bleeding and enterococcal infection, without evidence of rejection. CONCLUSIONS: These observations suggest that thrombocytopenia after liver xenotransplantation may be overcome by Amicar therapy. The coagulopathy and sepsis that nevertheless occurred suggest that additional causes of coagulation disturbance must be addressed, along with better prevention of infection, to achieve long-term survival.

Characterization of LHY-821, a novel moderately differentiated endometrial carcinoma cell line.

Endometrial cancer is a major problem for women but only a small number of comprehensively characterized cell models are available for studies. Here, we established a new cell line derived from a Stage IIIc(1) Grade 2 endometrial adenocarcinoma. The cell line, designated LHY-821, was characterized using growth curve, karyotyping, immunohistochemical staining, immunoblotting, drug sensitivity assay, invasion assay, and xenografting in nude mice. LHY-821 has a doubling time of about 46 h and a colony-forming efficiency of approximately 71 %. These cells expresse high levels of progesterone receptor but not estrogen receptor and are sensitive to medroxyprogesterone acetate (MPA). LHY-821 also expresses pan-cytokeratin, PTEN, p53, ß-catenin, IGF-1, and IGF-2. In addition, karyotype analysis revealed that LHY-821 possessed a near diploid karyotype including 6q-, 10p-, Xq-, 13q+, 17p+, and Triplo-12. LHY-821 showed highly tumorigenicity in nude mice (100 %) and weak invasiveness. Chemosensitivity tests showed that LHY-821 was sensitive to both carboplatin and paclitaxel. LHY-821 is an immortalized cell line which had survived more than 80 serial passages; it may provide a novel tool to study the molecular mechanism and potential treatment for endometrial cancer.

Binding of transcription factor activating protein 2 γ on the 5'-proximal promoter region of human porcine endogenous retrovirus subgroup A receptor 2/GPR172B.

BACKGROUND: Xenotransplantation is one of the solutions for the shortage of organ donors, and pigs have been considered to be the most suitable animal donors. Specific pathogen-free pigs are utilized in the xenotransplantation; however, pigs have infectious gammaretroviruses, named porcine endogenous retroviruses (PERVs) in their genome. Of them, PERV-A and PERV-B can infect human cells in vitro and potentially induce diseases like other gammaretroviruses. The human cellular receptors for PERV-A were identified and named human PERV-A receptor (HuPAR)-1 and HuPAR-2 (also called as GPR172A and GPR172B, respectively). We have recently reported that HuPAR-2 expression was regulated by epigenetic modification and preferentially expressed in placenta. However, the detailed mechanisms of HuPAR-2 expression have not been fully characterized. In this study, we analyzed molecular mechanisms associated with HuPAR-2 transcription through the identification of transcription factors that bind to the promoter region of HuPAR-2. METHODS: In situ hybridization was performed to identify the cells expressing HuPAR-2 in placental tissues. Transcriptional activities were measured by dual-luciferase reporter assay using serial deletion mutants of HuPAR-2 5'-flanking region. To identify the transcription factors bound to the promoter region, in silico analysis, electrophoresis mobility shift assay, and chromatin immunoprecipitation assay were conducted. The effect of the transcription factor transcription factor activator protein (TFAP)-2γ on the promoter activities was investigated by overexpression of the factor. RESULTS: We identified that HuPAR-2 was specifically expressed in villous trophoblast cells. We also identified that a region spanning from -126 to -32 had proximal promoter activities and TFAP-2γ bound to a region spanning from -58 to -35 in vitro and in vivo. The overexpression of TFAP-2γ also augmented the proximal promoter activity. CONCLUSION: We demonstrated that TFAP-2γ is one of the transcription factors involved in the HuPAR-2 expression in human villous trophoblast cells. By studying transcriptional factors involved in the expression of HuPAR-2, we may find a clue to control the potential risks caused by PERV-A infection in xenotransplantation.

Host response to xenograft ECM implantation is not different between the shoulder and body wall sites in the rat model.

In the context of tendon and ligament repair, mechanical loading and the presence of joint synovial fluid are known to profoundly influence the form and function of the repair tissue and potentially the host response to biomaterials. Previously, we demonstrated that a xenograft extra cellular matrix (ECM) scaffold implanted in the rat shoulder elicited a unique host response from that seen in the body wall. However, the host response to xenografts implanted in shoulders with a tendon/capsule injury was not different from xenografts implanted in shoulders with no injury. In the current study, we hypothesized that varying clinically relevant surgical and environmental factors would introduce significant differences in host response to xenograft implantation at the shoulder. Contrary to our hypothesis, we found no significant differences in host response between any shoulder implantation conditions or between shoulder and body wall implantation in the rat model. These findings suggest that there is no advantage to using an orthotopic shoulder model to investigate the host response to rotator cuff scaffold materials in the rat model, and due to the insensitivity of its host response to various clinically relevant surgical conditions, may suggest that the rat does not provide a surrogate for directly translating the host response to biomaterials to the human application.

Transgenic organs and xenotransplants.

Advances in immunosuppressive treatments reached in the last decades of the 20th century have made solid organ transplantation the treatment of choice for cases of irreversible organ failure. However, the availability of human cadaver organs is limited and the demand for transplants is still on the rise. Also, there is a recognised lack of cells and human tissues for generalised use in transplantation for the treatment of diseases that are characterised by failure of specialised cells (such as pancreatic cells to cure diabetes). Xenotransplantation, which is the transplant of cells, tissues or organs from other species, became the focus of attention in the nineteen-nineties as a solution to the lack of organs and tissues for transplantation. Previous clinical studies using nonhuman primates produced poor outcomes (survival from days to a few months) and confirmed the difficulty of obtaining organs from these species. Since then, progress in xenotransplantation has been slow and still now various immunological and non-immunological barriers need to be overcome. These barriers are reviewed in this chapter and the various approaches explored to date to overcome them, in particular those based on the genetic modification of pigs. Also, cell transplant studies such as those of pancreatic islets in monkeys have led to even more hopeful results. The range of possibilities offered by this technology will be unlimited, making it possible for xenotransplantation to be a clinical reality in a not very distant future.

Modeling the spatial distribution of chronic tumor hypoxia: implications for experimental and clinical studies.

Tumor oxygenation status is considered one of the important prognostic markers in cancer since it strongly influences the response of cancer cells to various treatments; in particular, to radiation therapy. Thus, a proper and accurate assessment of tumor oxygen distribution before the treatment may highly affect the outcome of the treatment. The heterogeneous nature of tumor hypoxia, mainly influenced by the complex tumor microenvironment, often makes its quantification very difficult. The usual methods used to measure tumor hypoxia are biomarkers and the polarographic needle electrode. Although these techniques may provide an acceptable assessment of hypoxia, they are invasive and may not always give a spatial distribution of hypoxia, which is very useful for treatment planning. An alternative method to quantify the tumor hypoxia is to use theoretical simulations with the knowledge of tumor vasculature. The purpose of this paper is to model tumor hypoxia using a known spatial distribution of tumor vasculature obtained from image data, to analyze the accuracy of polarographic needle electrode measurements in quantifying hypoxia, to quantify the optimum number of measurements required to satisfactorily evaluate the tumor oxygenation status, and to study the effects of hypoxia on radiation response. Our results indicate that the model successfully generated an accurate oxygenation map for tumor cross-sections with known vascular distribution. The method developed here provides a way to estimate tumor hypoxia and provides guidance in planning accurate and effective therapeutic strategies and invasive estimation techniques. Our results agree with the previous findings that the needle electrode technique gives a good estimate of tumor hypoxia if the sampling is done in a uniform way with 5-6 tracks of 20-30 measurements each. Moreover, the analysis indicates that the accurate measurement of oxygen profile can be very useful in determining right radiation doses to the patients.

Porcine alanine transaminase after liver allo-and xenotransplantation.

Aspartate transaminase (AST) and alanine transaminase (ALT) are measured following liver transplantation as indicators of hepatocellular injury. During a series of orthotopic liver allo-and xenotransplants, we observed that there was an increase in AST in all cases. The anticipated concomitant rise in ALT did not occur when a wild-type (WT) pig was the source of the liver graft, but did occur when a baboon or a genetically engineered (α1,3-galactosyltransferase gene-knockout [GTKO]) pig was the source of the graft. We hypothesized that the cience of Galα1,3Gal in GTKO pig livers may render pig hepatocytes similar to human and baboon hepatocytes in their response to hepatocellular injury. Reviewing the literature, after WT pig liver allotransplantation or xenotransplantation, in the majority of reports, although changes in AST were reported, no mention was made of changes in ALT, suggesting that there was no change in ALT. However, Ramirez et al. reported two cases of liver xenotransplants from hCD55 pigs, following which there were increases in both AST and ALT, suggesting that it is not simply the cience of expression of Galα1,3Gal that is the cause. We acknowledge that our observation is based on a small number of experiments, but we believe it is worth recording.

Protease inhibitor nafamostat mesilate attenuates complement activation and improves function of xenografts in a discordant lung perfusion model.

BACKGROUND: Anti-complement activity of nafamostat mesilate (FUT-175) is strong including its variety of pharmacological effects. The effect of FUT-175 for xenografts in an ex vivo guinea pig-to-rat lung perfusion model was evaluated. METHODS: Heparinized Lewis rat blood was used to perfuse the lungs in three groups (n = 6 each). Group I used Lewis rat left lung for donor, Group X used guinea pig left lung for donor, and Group XF used guinea pig left lung for donor, which was perfused with Lewis rat blood with 0.2 mg/ml of FUT-175. Complement activity causing 50% hemolysis (CH50) in the perfusion blood and pulmonary function either before or during perfusion were serially measured. Pathological assessments of the lungs were also carried out after perfusion. RESULTS: The duration of satisfactory pulmonary function was significantly increased in Group XF. Complement activity causing 50% hemolysis in Group XF decreased more significantly compared to Group X. FUT-175 suppressed both the increase in pulmonary arterial pressure and airway resistance, and the decrease in dynamic lung compliance. In Group X, pathology showed intra-alveolar hemorrhage, perivascular edema, and medial thickening with endothelial swelling of the pulmonary arteries. In Group XF, less changes were observed compared to Group X. Group X showed deposition of IgM, IgG, and C3 at the endothelium of arteries, which was fewer in Group XF, and even fewer in Group I. CONCLUSIONS: This study suggests that FUT-175 inhibited complement activation and improved lung xenograft function. FUT-175 ameliorates hyperacute rejection in a guinea pig-to-rat ex vivo xenogeneic lung perfusion model.

Species incompatibilities in the pig-to-macaque islet xenotransplant model affect transplant outcome: a comparison with allotransplantation.

BACKGROUND: Porcine islet transplantation into diabetic non-human primates is considered most relevant in translational research supporting a clinical application. Most studies have focused on immunosuppressive protocols, while metabolic aspects have mainly been utilized in graft monitoring. We evaluated data from our group regarding human and non-human primate (NHP) allotransplantation and pig-to-NHP xenotransplantation to identify incompatibilities in metabolic factors and their consequences for transplant outcomes. METHODS: Basic gluco-metabolic parameters (fasting blood glucose, C-peptide, and response to stimulation with arginine or glucose) were derived from literature (humans), 72 macaques, and 47 adult Landrace pigs. Islet preparations from 15 human deceased donors, 61 macaques, and 23 adult pigs were compared with respect to yield, fractional viability assessed by oxygen consumption normalized for DNA, and in vitro glucose-induced insulin release. Metabolic parameters at day 75 after a single islet transplantation in the liver were compared for 19 patients and 9 macaques receiving an allotransplant and 11 macaques receiving a porcine xenotransplant: recipients received chronic immunosuppression. RESULTS: Pigs differ from NHPs and humans by a much lower C-peptide level (0.42 vs. 1.3 to 2.0 ng/ml, respectively) and a 2- to 7-fold lower C-peptide response to arginine stimulation. In contrast, NHPs have the highest metabolic demand as evidenced by a high C-peptide and high C-peptide response to arginine stimulation; values are about twice higher than in humans. For manufactured islet preparations, these differences are reflected by glucose-stimulated insulin release (the stimulation index for pigs is 1.5, for humans 3.8, and for macaques 7.7), but not by fractional viability, which was in the same range. The day 75 outcome after transplantation assessed by C-peptide was similar for allotransplanted humans and NHPs (80 to 90% good graft function) and lower in xenografted NHPs (36% good graft function); gluco-metabolic parameters were in accordance with graft function, albeit different between species because normoglycemia under exogenous insulin is maintained more aggressively in patients than in NHPs. In xenografted NHPs, the shift in glycemic control with respect to normal values, combined with low values of circulating porcine C-peptide, resembled more the normal condition in a pig than that in a macaque. CONCLUSIONS: The substantially lower glucose-induced insulin response in adult porcine islet preparations as opposed to islets manufactured from humans or macaques combined with the much higher need for insulin in macaques than in humans creates an imbalance between the metabolic demand and the engrafted islet mass in the pig-to-NHP xenograft recipient. Engrafted islet mass is affected by dose, suggesting that a much higher dose level of islets is necessary in the xenogeneic setting than in human or NHP allotransplantation, and pig islets need to be given at a higher dose in macaques than the anticipated effective dose in humans. To cope with differences in metabolic demand and presumably also metabolic dynamics, a liberal regime in supportive exogenous insulin might be essential to achieve long-term survival. These intrinsic characteristics of the NHP model deserve consideration to optimally design experimental studies with the perspective of translational value of results.

Islet isolation from adult designated pathogen-free pigs: use of the newer bovine nervous tissue-free enzymes and a revised donor selection strategy would improve the islet graft function.

BACKGROUND: In clinical trials using adult porcine islet products, islets should be isolated from the designated pathogen-free (DPF) pigs under the current good manufacturing practice (GMP) regulations. Our previous studies suggested that male DPF pigs are better donors than retired breeder pigs and histomorphometrical parameters of donor pancreas predict the porcine islet quality. We aimed to investigate whether the use of the newer bovine nervous tissue-free enzymes and a revised donor selection strategy could improve the islet graft function in the context of islet isolation with DPF pigs. METHODS: Using 30 DPF pigs within a closed herd, we compared the islet yield of porcine islets isolated with Liberase PI (n = 11, as a historical control group), Liberase MTF C/T, which is a GMP-grade enzyme (n = 12), and CIzyme collagenase MA/BP protease (n = 7). We analyzed the relationship between the diabetes reversal rate of recipient NOD/SCID mice (n = 75) and histomorphometric parameters of each donor pancreas as well as donor characteristics. RESULTS: Proportion of islets larger than 200 µm from the biopsied donor pancreas (P = 0.006) better predicted islet yield than age (P = 0.760) or body weight (P = 0.371) of donor. The proportion of islets larger than 200 µm from the biopsied donor pancreas was not related to the sex of the donor miniature pig (P = 0.358). The islet yield obtained with the three enzymes did not differ, even after stratification of the donor with the histomorphometric parameters of the biopsied donor pancreas and the sex of donor. The use of the newer bovine nervous tissue-free enzymes (P < 0.001), a higher proportion of large islets in donor pancreas (P = 0.006), and a male sex of the donor (P = 0.025) were independent predictors of earlier diabetes reversal. CONCLUSIONS: Use of the newer bovine nervous tissue-free enzymes including a GMP-grade enzyme resulted in better islet quality than that of islet isolated using Liberase PI. To obtain high-quality islet from DPF pigs, the donor should be male pig and histomorphometrical parameters from donor pancreas should be considered.

Absence of Gal epitope prolongs survival of swine lungs in an ex vivo model of hyperacute rejection.

BACKGROUND: Galactosyl transferase gene knock-out (GalTKO) swine offer a unique tool to evaluate the role of the Gal antigen in xenogenic lung hyperacute rejection. METHODS: We perfused GalTKO miniature swine lungs with human blood. Results were compared with those from previous studies using wild-type and human decay-accelerating factor-transgenic (hDAF(+/+) ) pig lungs. RESULTS: GalTKO lungs survived 132 ± 52 min compared to 10 ± 9 min for wild-type lungs (P = 0.001) and 45 ± 60 min for hDAF(+/+) lungs (P = 0.18). GalTKO lungs displayed stable physiologic flow and pulmonary vascular resistance (PVR) until shortly before graft demise, similar to autologous perfusion, and unlike wild-type or hDAF(+/+) lungs. Early (15 and 60 min) complement (C3a) and platelet activation and intrapulmonary platelet deposition were significantly diminished in GalTKO lungs relative to wild-type or hDAF(+/+) lungs. However, GalTKO lungs adsorbed cytotoxic anti-non-Gal antibody and elaborated high levels of thrombin; their demise was associated with increased PVR, capillary congestion, intravascular thrombi and strong CD41 deposition not seen at earlier time points. CONCLUSIONS: In summary, GalTKO lungs are substantially protected from injury but, in addition to anti-non-Gal antibody and complement, platelet adhesion and non-physiologic intravascular coagulation contribute to Gal-independent lung injury mechanisms.

Identification of soluble and membrane-bound isoforms of porcine tumor necrosis factor receptor 2.

BACKGROUND: TNF and its receptors TNF-Receptor 1 (TNFR1, CD120a) and TNF-Receptor 2 (TNFR2, CD120b) have been implicated in the rejection of transplanted cells and organs. Although pig TNFR1 (pTNFR1) is known to mediate the effects of human TNF in a xenogeneic setting, it is unclear whether pig TNFR2 (pTNFR2) could contribute to xenograft rejection. METHODS: We have cloned the cDNA of various pTNFR2 variants by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends. We have characterized the predicted proteins with bioinformatic tools and conducted expression, affinity, and functional studies to investigate their roles. RESULTS: We have identified four isoforms of pTNFR2: one comprising the four cysteine-rich domains (CRD) conserved between species, a shorter variant (pTNFR2ΔE7-10) encoding for a soluble isoform, another with only three CRD due to the lack of exon 4 (pTNFR2ΔE4), and a fourth variant containing both modifications. Accordingly, multiple mRNA transcripts were observed by northern blotting. Quantitative RT-PCR determined high pTNFR2 expression in lung and immune cells and detected the two alternative splicings in all cells/tissues examined. The full receptor was moderately expressed on the surface of pig cells such as porcine aortic endothelial cells and PK-15 and was regulated by TNF. On the contrary, the membrane-bound pTNFR2ΔE4 was located only intracellularly. Plasmon resonance studies showed that pTNFR2 binds pig and human TNFα with high affinity, but pTNFR2ΔE4 interacts poorly with pig TNFα and does not bind to the human cytokine. Moreover, pull-down experiments with the two recombinant soluble isoforms consistently demonstrated that the two bound together and soluble pTNFR2ΔE4 was able to modulate the TNF inhibitory activity of pTNFR2-GST in a cell-based assay. CONCLUSION: The pTNFR2 may participate in the process of xenograft rejection and other related events, as well as be used in soluble form to block TNF in this setting. In addition, we have discovered other pTNFR2 isoforms that may affect the pig immune responses and have an impact on rejection of xenografts.

Proteomic analysis of human epithelial ovarian cancer xenografts in immunodeficient mice exposed to chronic psychological stress.

This work aims at comparing alterations in the proteomes of human epithelial ovarian cancer xenografts between stressed and non-stressed immunodeficient mice as well as exploring the molecular mechanisms linking chronic psychological stress to ovarian cancer oncogenesis and progression. SK-OV-3 cells were injected subcutaneously into nude mice. The stress group was subjected to a chronic physical restraint protocol for 6 h on 35 consecutive days, while the control group was unrestrained. All mice were sacrificed on day 36 after SK-OV-3 cell injection, and tumors were excised. Tumor tissues were processed for 2D gel electrophoresis, mass spectrometry (nanoUPLC-ESI-MS/MS) and Western blotting. The expression of 20 proteins was found to be significantly altered between the stress and control groups, of which 14 were up-regulated, five were down-regulated, and one protein was found only in the stress group. All proteins were identified by UPLC-ESI-MS/MS, and Western blotting results were consistent with those of proteomic methods. The present results provide new evidence relating to the molecular mechanism underlying the relationship between psychological stress and tumor progression.

The immense potential of xenotransplantation in surgery.

There is a limited availability of deceased human organs and cells for the purposes of clinical transplantation. Genetically-engineered pigs may provide an alternative source. Although several immune barriers need to be overcome, considerable progress has been made in experimental models in recent years, largely through the increasing availability of pigs with new genetic modifications. Pig heterotopic heart graft survival in nonhuman primates has extended for 8 months, with orthotopic grafts supporting life for almost 2 months. Life-supporting kidney transplants have functioned for almost 3 months. The current barriers are related to coagulation dysfunction between pig and primate that results in thrombotic microangiopathy and/or a consumptive coagulopathy, which may in part be related to molecular incompatibilities in the coagulation systems of pigs and primates. Current efforts are concentrated on genetically-modifying the organ- or islet-source pigs by the introduction of 'anticoagulant' or 'anti-thrombotic' genes to provide protection from the recipient coagulation cascade and platelet activation. Progress with pig islet xenotransplantation has been particularly encouraging with complete control of glycemia in diabetic monkeys extending in one case for >12 months. Other areas where experimental data suggest the possibility of early clinical trials are corneal xenotransplantation and pig neuronal cell xenotransplantation, for example, in patients with Parkinson's disease. With the speed of advances in genetic engineering increasing steadily, it is almost certain that the remaining problems will be overcome within the foreseeable future, and clinical allotransplantation will eventually become of historical interest only.

An in vitro model of pig liver xenotransplantation--pig complement is associated with reduced lysis of wild-type and genetically modified pig cells.

BACKGROUND: After pig liver transplantation in humans, the graft will produce pig complement (C). We investigated in vitro the lysis of wild-type (WT), α1,3-galactosyltransferase gene-knockout (GTKO), and CD46 transgenic (CD46) pig peripheral blood mononuclear cells (PBMC) caused by human anti-pig antibodies (Abs) + pig C. METHODS: Human serum IgM/IgG binding to WT and GTKO PBMC was determined by flow cytometry, and lysis of pig PBMC by a C-dependent cytotoxicity assay using (i) human serum (human Abs + C), (ii) GTKO pig serum (anti-Gal Abs + pig C), (iii) heat-inactivated human serum (human Abs) + rabbit C, or (iv) human Abs + pig C (serum). RESULTS: Binding of human IgM and IgG to GTKO PBMC was less than to WT PBMC (P < 0.05). In the presence of human Abs, lysis of WT and GTKO PBMC by rabbit C was 87 and 13%, respectively (WT vs. GTKO, P < 0.01), but was only 37 and 0.4% in the presence of pig C (WT vs. GTKO, P < 0.05). Human/rabbit C-induced lysis was greater than pig C-induced lysis for both WT and GTKO PBMC. CD46 pig PBMC reduced rabbit/human C- and pig C-mediated lysis (P < 0.05). CONCLUSIONS: Pig livers, particularly from GTKO and CD46 pigs, are likely to have an immunologic advantage over other organs after transplantation into humans. In the absence of pig antibodies directed to human tissues, pig complement is unlikely to cause problems after liver xenotransplantation, especially if GTKO/CD46 pigs are used as the source of the livers.

Hepatic function after genetically engineered pig liver transplantation in baboons.

BACKGROUND: If "bridging" to allo-transplantation (Tx) is to be achieved by a pig liver xenograft, adequate hepatic function needs to be assured. METHODS: We have studied hepatic function in baboons after Tx of livers from alpha1,3-galactosyltransferase gene-knockout (GTKO, n=1) or GTKO pigs transgenic for CD46 (GTKO/CD46, n=5). Monitoring was by liver function tests and coagulation parameters. Pig-specific proteins in the baboon serum/plasma were identified by Western blot. In four baboons, coagulation factors were measured. The results were compared with values from healthy humans, baboons, and pigs. RESULTS: Recipient baboons died or were euthanized after 4 to 7 days after internal bleeding associated with profound thrombocytopenia. However, parameters of liver function, including coagulation, remained in the near-normal range, except for some cholestasis. Western blot demonstrated that pig proteins (albumin, fibrinogen, haptoglobin, and plasminogen) were produced by the liver from day 1. Production of several pig coagulation factors was confirmed. CONCLUSIONS: After the Tx of genetically engineered pig livers into baboons (1) many parameters of hepatic function, including coagulation, were normal or near normal; (2) there was evidence for production of pig proteins, including coagulation factors; and (3) these appeared to function adequately in baboons although interspecies compatibility of such proteins remains to be confirmed.