Porcine Organs Research Articles

Construction of the Porcine Endogenous Retrovirus Envelope Glycoprotein A and B Specific Antibody

Xenotransplantation using porcine organs could potentially associate with the risk of pathogenic infections, because human tropic porcine endogenous retrovirus (PERV) particles could be released from pig cells or organs. While there is no evidence of PERV transmission to human, safety issues become a paramount concern. For the prevention of this transmission, specific immunological tools must be provided for PERV transmission detection. In this study we described the expression of PERV envelope proteins and the production of a specific antibody against PERV envelope (Env) glycoprotein. The nucleotide sequence harboring the partial region of glycoprotein 70 was cloned into the pET vector and envelope protein was expressed in E. coli. Approximately 42 kDa recombinant Env protein (PERV Env-aa357) was purified by the Ni-affinity column. For antibody production, mice were immunized with the recombinant PERV Env-aa357. The generated anti-serum was tested using Western blot and immunocytochemical assay. We found that anti-PERV Env serum displayed the specificity against the PERV Envs (PERV-A and PERV-B) expressed not only in E. coli but also in mammalian cells, and PERV particles within the porcine cell lines (PK 15 and PK-1). Taken together, PERV antibody could be useful for detecting PERV infection or xenotransplantation transmission.

Anticarbohydrate Antibody Repertoires in Patients Transplanted with Fetal Pig Islets Revealed by Glycan Arrays

Ten patients with type I diabetes were transplanted with porcine fetal islet-like cell clusters (ICC) between 1990 and 1993. A significant rise in the anti-α-Gal antibody titers was seen posttransplant, but also non-α-Gal-specific antibodies were detected in some patients. We have reanalyzed the carbohydrate specificity of antibodies in the sera from seven of these patients taken before transplantation, 1, 6 and 12 months posttransplantation using a glycan array with 200 structurally defined glycans. The main findings were: (i) prepig ICC transplantation patients had antibodies reactive with terminal α-GalNAc (e.g. the Forssman antigen, but not the blood group A determinant in blood group A patients), α-Gal (except blood group B determinants in B individuals), β3-linked Gal especially Galβ1,3GlcNAc even if terminally sulfated or sialylated, β-GlcNAc except if β1,3-linked and oligomannosyl compounds; (ii) the titers of all carbohydrate-specific antibodies detected before transplantation rose after transplantation; (iii) the kinetics of the antibody responses differed between patients; (iv) in some patients antibodies reacting with Galα1,3Lex and several structures terminated with Neu5Gc appeared after transplantation. In conclusion, anti-α-Gal antibodies are the predominant anticarbohydrate antibodies detected after porcine ICC transplantation, with some patients also developing Neu5Gc-specific antibodies. Their clinical significance needs to be established.

Xenotransplantation as a model of integrated, multidisciplinary research

Xenotransplantation was proposed a long time ago as a possible solution to the world-wide shortage of human organs. For years, researchers in this field have almost exclusively directed their efforts towards combating the immunological barrier that precluded the long-term xenograft survival. Studies have been conducted in both small and large animal models and the most relevant results have been obtained in pre-clincal studies, specifically those utilising the pig-to-nonhuman primate combination. In this context, a better understanding of the immunological mechanisms underlying the rejection of a xenograft have allowed the identification of specific targets of intervention that have resulted in considerable improvements in survival of porcine organs or cells in nonhuman primates. However it has also become apparent that if xenotransplantation has to enter the clinical arena, a multidisciplinary approach will be needed to comprehensively tackle the different issues related to the use of a xenograft to cure human disease.In this regard, the safety, ethics and regulatory aspects of xenotransplantation are currently being aggressively addressed to enable the initiation of xenotransplantation with a favourable risk/benefit ratio.

Xenotransplantation: Role of natural immunity

Hyperacute rejection, mediated by natural anti-Galα1,3Galβ1,4GlcNAc (αGal) antibodies and the classically activated complement pathway, was identified as the first major barrier to the survival of porcine organs in humans. Subsequently, discordant pig-to-nonhuman primate and concordant rodent models revealed key roles for T and B lymphocytes in the second form of rejection, acute vascular rejection (AVR) or delayed xenograft rejection (DXR). As significant progress was made in strategies to circumvent or suppress xenoreactivity of the adaptive immune system, it became clear that, apart from natural antibodies, other innate immune system elements actively participate in AVR/DXR and represent a barrier to xenograft acceptance that may be particularly difficult to overcome. Observations in pig-to-primate and semi-discordant and concordant rodent models indicate that Natural Killer (NK) cells play a more prominent role in xenograft than in allograft rejection. Several mechanisms through which human NK cells recognize porcine endothelial cells have been elucidated and these appear to be more diverse than those involved in NK cell alloreactivity. Further, it has been demonstrated that human macrophages and neutrophils can directly recognize pig derived cells and can mediate direct xenograft damage. Here, we review the recent progress in the understanding of the xenoreactivity of the natural immune system, focussing on preclinical pig-to-(non)human primate systems, and discuss the proposed strategies to overcome these barriers.

Coagulation in xenotransplantation

Following either discordant transplantation of porcine organs into primate recipients or after ex vivo perfusion of porcine organs with human blood, a profound activation of the primate/human blood coagulation can be observed. This activation may result in thrombotic microangiopathy and changes of coagulation resembling disseminated intravascular coagulation with subsequent death of the recipient animals. There are several factors contributing to this pathology including endothelial cell activation, activation of human blood cells, thrombocyte activation and incompatibilities of molecules regarding to control coagulation. Several potentially important molecular incompatibilities between the porcine and the primate coagulation system have been noted: The inability of porcine tissue factor pathway inhibitor (TFPI) to adequately neutralize human factor Xa (FXa), the aberrant activation of both human prothrombin and FX by porcine endothelial cells and the failure of the porcine natural anticoagulant thrombomodulin to activate the anti‐coagulant human protein C. Normal hemostasis is a complex balance between pro‐ and antithrombotic pathways. These pathways are balanced by inhibitory systems including the heparin‐antithrombin interaction, TFPI, the generation of activated protein C by a proper thrombomodulin/thrombin interaction and the fibrinolytic system. To achieve normal haemostasis after xenotransplantation it is important to overcome the known molecular incompatibilities (i.e. by transgenic organs) and to reliably prevent the derangement of coagulation.

EXISTENCE OF PROVIRAL PORCINE ENDOGENOUS RETROVIRUS IN FRESH AND DECELLULARISED PORCINE TISSUES

Purpose: Swine are expected to be utilized as xenograft donors for both whole organ and cellular transplantation. A major concern in using porcine organs for transplantation is the potential of transmission of porcine endogenous retrovirus (PERV). Tissue-engineered or decellularised heart valves have already been implanted in humans and have been marketed by certain companies after Food and Drug Administration (FDA) approval. The aim of this study was to examine the existence of porcine endogenous retrovirus (PERV) in fresh and decellularised porcine tissues. Methods: Porcine tissues (both fresh and decellularised) were analysed using validated assays specific for PERV: polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR). Results: PERV specific GAG sequences were found in the porcine heart tissue samples using PCR for DNA and RT-PCR for RNA. All tissue samples (both fresh and treated tissues) like aortic valve, pulmonary valve and heart muscle showed the presence of PERV DNA. RT PCR for PERV was positive in all fresh tissues and was found to be negative in decellularised treated tissues. Conclusions: PCR is a rapid, specific test for the detection of PERV virus in xenografts. These findings have demonstrated that the presence of proviral DNA form of PERV in porcine tissues needs to be carefully considered when the infectious disease potential of xenotransplantation is being assessed.

REACTION OF COMPLEMENT WITH ENDOTHEUAL CELLS IN A MODEL OF XEN0TRANSPLANTATION

We review our studies on the role of complement (C) as mediator of xenograft hyperacute rejection using an in vitro model consisting of porcine endothelial cells as target and human serum as source of natural antibodies and C. Cytotoxicity of endothelial cells required IgM antibodies to porcine endothelial cells, and the classical pathway and membrane attack complex of C. These findings correlated with in vivo results of porcine organs transplanted into rhesus monkeys, which showed a) co-deposition of IgM, C3, C4 and C9, along blood vessels of rejecting organs, with trace deposits of factors B or P, and b) minimal deposition of IgM and C components in transplants with prolonged survival that were performed in rhesus monkeys depleted of natural antibodies but with normal C levels. Human serum causes activation of porcine endothelial cells manifested by release of heparan sulfate proteoglycan. Heparan sulfate release was induced by C5a alone. A new approach to avert xenograft hyperacute rejection was tested. To inhibit cytotoxicity of porcine endothelial cells by human C, the membrane-associated C inhibitor decay-accelerating factor (DAF) of human origin was incorporated into endothelial cells. Human DAF was able to efficiently inhibit C-mediated killing of porcine endothelial cells, suggesting that the use of DAF and other C inhibitors could be used to interfere with C-mediated xenograft hyperacute rejection.

Effects of Modifiers of Glycosaminoglycan Biosynthesis on Outflow Facility in Perfusion Culture

Glycosaminoglycans (GAGs) have been implicated in the regulation of outflow resistance of aqueous humor flow through the trabecular meshwork (TM). Their role was further investigated by assessment of the effects of chlorate, an inhibitor of sulfation, and beta-xyloside, which provides a competitive nucleation point for addition of disaccharide units, in anterior segment perfusion culture. Outflow facility was measured in perfused porcine and human anterior organ cultures treated with 20 or 50 mM sodium chlorate, or 1 mM beta-xyloside. Perturbation of extracellular matrix (ECM) components was assessed in paraffin-embedded sections by immunofluorescence and confocal microscopy. Parallel experiments were conducted on cultured TM cells. Outflow facility increased in porcine eyes with chlorate (3-fold) and beta-xyloside (3.5-fold) treatments. In human eyes, outflow increased approximately 1.5-fold and took longer (>48 hours) to occur. By confocal microscopy, immunostaining for chondroitin and heparan sulfates was observed on edges of human TM beams in nontreated eyes, with intense staining in the juxtacanalicular tissue (JCT) region. In treated eyes, staining of beam edges was severely reduced and was instead found in plaques. Chlorate treatment resulted in a striated pattern of GAG staining in the human JCT region. Fibronectin immunostaining was altered in beta-xyloside-treated eyes, whereas in cell culture, chlorate induced formation of thick fibronectin fibrils, to which tenascin C colocalized. Disrupting GAG chain biosynthesis increased outflow facility in perfusion culture and induced atypical ECM molecule interactions in cell culture. This study provides direct evidence of the critical role of GAG chains in regulating outflow resistance in human TM.

APOBEC3 Proteins and Porcine Endogenous Retroviruses

Xenotransplantation of porcine cells, tissues, and organs offers a solution to overcome the shortage of human donor materials. In addition to the immunological and physiological barriers, the existence of numerous porcine microorganisms including viruses poses a risk for xenozoonosis. Three classes of functional gamma-type porcine endogenous retroviruses (PERV) have been identified, whereby functional polytropic PERV-A and PERV-B infect human embryonic kidney (HEK 293) and other cell lines in vitro. In the course of risk assessment for xenotransplantation the capacity of human cells to counteract PERV infections should be analyzed. Primates and other mammals display different means of protection against viral infections. APOBEC3 proteins which are cytidine deaminases and a part of the intrinsic immunity mediate potent activity against a wide range of retroviruses including murine leukemia viruses (MLV). As PERV and MLV belong to the same genus, we raised the question as to whether PERV is affected by APOBEC3 proteins. Initial data indicate that human and porcine cytidine deaminases inhibit PERV replication, thereby possibly reducing the risk for infection of human cells by PERV as a consequence of pig-to-human xenotransplantation.

Is Porcine Endogenous Retrovirus (PERV) Transmission Still Relevant?

Xenotransplantation using porcine cells or organs may be associated with the risk of transmission of zoonotic microorganisms. Porcine endogenous retroviruses (PERVs) pose a potentially high risk because they are integrated into the genome of all pigs and PERV-A and PERV-B at least, which are present in all pigs, can infect human cells. However, PERV transmission could not be demonstrated in the first recipients of clinical xenotransplantation or after numerous experimental pig-to-non-human primate transplantations. In addition, inoculation of immunosuppressed small animals and non-human primates failed to result in demonstrable PERV infection. Nevertheless, strategies to reduce the possible danger of PERV transmission to humans, however low, could be of benefit for the large-scale clinical use of porcine xenotransplants. One strategy is to select pigs free of PERV-C, thereby preventing recombination with PERV-A. A second strategy involves the selection of animals that express only very low levels of PERV-A and PERV-B. To this end, sensitive and specific methods have been developed to allow the distribution and expression of PERV to be analyzed. A third strategy is to develop a vaccine capable of protecting against PERV transmission. Finally, a fourth strategy is based on the inhibition of PERV expression by RNA interference. Using PERV-specific short hairpin RNA (shRNA) and retroviral vectors, inhibition of PERV expression in primary pig cells was demonstrated and transgenic pigs were generated that show reduced PERV expression in all tissues analyzed. Intensive work is required to improve and to combine these strategies to further decrease the putative risk of PERV transmission following xenotransplantation.

Time dependence of electrical bioimpedance on porcine liver and kidney under a 50 Hz ac current

The purpose of this work is to study the changes of the bioimpedance from its ‘in vivo’ value to the values measured in a few hours after the excision from the body. The evolution of electrical impedance with time after surgical extraction has been studied on two porcine organs: the liver and the kidney. Both in vivo and ex vivo measurements of electrical impedance, measuring its real and imaginary components, have been performed. The in vivo measurements have been carried out with the animal anaesthetized. The ex vivo measurements have been made more than 2 h after the extraction of the organ. The latter experiment has been carried out at two different stabilized temperatures: at normal body temperature and at the standard preservation temperature for transplant surgery. The measurements show a correlation between the biological evolution and the electrical bioimpedance of the organs, which increases from its in vivo value immediately after excision, multiplying its value by 2 in a few hours.

The perspectives for porcine-to-human xenografts

The shortage of donated human organs for transplantation continues to be a life threatening problem for patients suffering from complete organ failure. Although this gap is increasing due to the demographic changes in aging Western populations, it is generally accepted that international trading in human organ is not an ethical solution. Alternatives to the use of human organs for transplantation must be developed and these alternatives include stem cell therapy, artificial organs and organs from other species, i.e. xenografts. For practical reasons but most importantly because of its physiological similarity with humans, the pig is generally accepted as the species of choice for xenotransplantation. Nevertheless, before porcine organs can be used in human xenotransplantation, it is necessary to make a series of precise genetic modifications to the porcine genome, including the addition of genes for factors which suppress the rejection of transplanted porcine tissues and the inactivation or removal of undesirable genes which can only be accomplished at this time by targeted recombination and somatic nuclear transfer. This review will give an insight into the advances in transgenic manipulation and cloning in pigs--in the context of porcine-to-human xenotransplantation.

Knockdown of porcine endogenous retrovirus (PERV) expression by PERV‐specific shRNA in transgenic pigs

Xenotransplantation using porcine cells, tissues or organs may be associated with the transmission of porcine endogenous retroviruses (PERVs). More than 50 viral copies have been identified in the pig genome and three different subtypes of PERV were released from pig cells, two of them were able to infect human cells in vitro. RNA interference is a promising option to inhibit PERV transmission. We recently selected an efficient si (small interfering) RNA corresponding to a highly conserved region in the PERV DNA, which is able to inhibit expression of all PERV subtypes in PERV-infected human cells as well as in primary pig cells. Pig fibroblasts were transfected using a lentiviral vector expressing a corresponding sh (short hairpin) RNA and transgenic pigs were produced by somatic nuclear transfer cloning. Integration of the vector was proven by PCR, expression of shRNA and PERV was studied by in-solution hybridization analysis and real-time RT PCR, respectively. All seven born piglets had integrated the transgene. Expression of the shRNA was found in all tissues investigated and PERV expression was significantly inhibited when compared with wild-type control animals. This strategy may lead to animals compatible with PERV safe xenotransplantation.

Existence of proviral porcine endogenous retrovirus in fresh and decellularised porcine tissues

Swine are expected to be utilized as xenograft donors for both whole organ and cellular transplantation. A major concern in using porcine organs for transplantation is the potential of transmission of porcine endogenous retrovirus (PERV). Tissue-engineered or decellularised heart valves have already been implanted in humans and have been marketed by certain companies after Food and Drug Administration (FDA) approval. The aim of this study was to examine the existence of porcine endogenous retrovirus (PERV) in fresh and decellularised porcine tissues. Porcine tissues (both fresh and decellularised) were analysed using validated assays specific for PERV: polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR). PERV specific GAG sequences were found in the porcine heart tissue samples using PCR for DNA and RT- PCR for RNA. All tissue samples (both fresh and treated tissues) like aortic valve, pulmonary valve and heart muscle showed the presence of PERV DNA. RT PCR for PERV was positive in all fresh tissues and was found to be negative in decellularised treated tissues. PCR is a rapid, specific test for the detection of PERV virus in xenografts. These findings have demonstrated that the presence of proviral DNA form of PERV in porcine tissues needs to be carefully considered when the infectious disease potential of xenotransplantation is being assessed.

Proteomic analysis of pancreas derived from adult cloned pig

The potential medical applications of animal cloning include xenotransplantation, but the complex molecular cascades that control porcine organ development are not fully understood. Still, it has become apparent that organs derived from cloned pigs may be suitable for transplantation into humans. In this study, we examined the pancreas of an adult cloned pig developed through somatic cell nuclear transfer (SCNT) using two-dimensional electrophoresis (2-DE) and Western blotting. Proteomic analysis revealed 69 differentially regulated proteins, including such apoptosis-related species as annexins, lamins, and heat shock proteins, which were unanimously upregulated in the SCNT sample. Among the downregulated proteins in SCNT pancreas were peroxiredoxins and catalase. Western blot results indicate that several antioxidant enzymes and the anti-apoptotic protein were downregulated in SCNT pancreas, whereas several caspases were upregulated. Together, these data suggest that the accumulation of reactive oxygen species (ROS) in the pancreas of an adult cloned pig leads to apoptosis.

In vivo mechanical characterization of human liver

The mechanical behavior of human liver has been characterized with aspiration experiments. Measurements have been performed in vivo under sterile conditions during open surgery. Twenty-three measurements on six healthy human livers were performed using the same loading history for each test, so to allow a direct comparison of the measured deformations. The measurement results are reported and the experimental uncertainties evaluated. One of the main objectives of the present paper is to share information on the in vivo mechanical response of human liver with the biomechanics research community: the present data can be used for mechanical model development and validation purposes. The parameters of a quasi-linear viscoelastic model have been determined from the experimental data by means of inverse finite element calculations. The corresponding linear elastic modulus is compared with values from the literature. In particular, a significant discrepancy has been found with respect to the values proposed by Carter et al. [Carter, F.J., Frank, T.G., Davies, P.J., McLean, D., Cuschieri, A., 2001. Measurement and modelling of the compliance of human and porcine organs. Medical Image Analysis 5, 231-236] and the reasons for this difference are discussed. The predictive capabilities of the quasi-linear viscoelastic model and the Rubin Bodner non-linear elastic-viscoplastic model are compared with respect to the tissue response in repeated aspiration cycles. Finally, for demonstration purposes, the constitutive model corresponding to the "average" liver response has been implemented into a finite element whole liver model and used for simulations related to liver surgery.

The Effectiveness of Current Sterility Techniques in Natural Orifice Transluminal Endoscopic Surgery (NOTES)

Background: NOTES represents a potentially less invasive alternative to conventional laparoscopy. However, breach of the gut wall raises the specter of intra-peritoneal infection. Various anti-microbial techniques have been employed in animal survival studies—including sterile saline rinse, intravenous and topical antibiotics, and Betadine—despite a paucity of data regarding their general effectiveness. Aim: To assess the effectiveness of existing sterilization techniques for NOTES by quantifying and speciating colony-forming units (CFU's) before and after treatment in ex vivo porcine organs. Method: Stomachs and distal colons were harvested en bloc from six fasted adult female white pigs following euthanasia. Four of these animals received 1g of Cefazolin intravenously prior to sacrifice. After the organs were filleted open in sterile fashion, they were further sub-divided into 2.0 cm × 2.0 cm full-thickness square samples. Each tissue sample was then assigned to one of five treatment arms: (1) sterile 0.9% saline lavage, (2) Betadine lavage, (3) lavage with a Cefazolin/Metronidazole suspension, (4) 4% Chlorhexidine lavage, or (5) no treatment. Following 10 minutes of contact time with the treatment solution, the mucosal surface of each tissue sample was swabbed with a sterile cotton applicator and inoculated in 5 ml of normal saline for 1 hour, followed by serial dilutions. 100 mL aliquots of each dilution were plated onto Sheep's blood agar plates and incubated in aerobic conditions at 24 degrees Celsius. Colony-forming units (CFU's) were quantified after seven days, and representative samples were sent to a commercial veterinary laboratory for speciation. Results: Initial bacterial density was estimated to be 1.0 × 106 CFU's/cm2 for stomach and 1.2 × 106 CFU's/cm2 for colon. The predominant organisms were Escherichia coli in stomach and both Escherichia coli and Enterococcus sp in colon. Sterile normal saline lavage caused a 1-log reduction to 2.3 × 105 CFU's/cm2. Antibiotic lavage caused a 2-log reduction to 6 × 104 CFU's/cm2. Betadine lavage a 5-log reduction to 4.1 × 101 CFU's/cm2. Finally, Chlorhexidine lavage also caused a 5-log reduction to 6.0 × 101 CFU's/cm2. The use of intravenous antibiotics alone diminished bacterial load to 10 CFU's/cm2. The combined use of intravenous antibiotics and Betadine or Chlorhexidine decreased counts to zero. Data was analyzed using ANOVA 2-way analysis method and found to be statistically significant (P = 0.001). Conclusions: The use of intravenous antibiotics in addition to topical Betadine or Chlorhexidine effectively eradicated bacteria in both gastric and colonic mucosa in this porcine model.

Mechanical Simulators for Training for Laparoscopic Surgery in Urology

The introduction of laparoscopic surgery into urology has led to new training concepts differing significantly from previous concepts of training for open surgery. This paper focuses on the type and importance of mechanical simulators in laparoscopic training. On the basis of our own studies and experience with the development of various concepts of laparoscopic training, including different modules (i.e., Pelvi-trainer, animal models, clinical mentoring) since 1991, we reviewed the current literature concerning all types of simulators. We focused on training for laparoscopic ablative and reconstructive surgery using mechanical simulators. The principle of a mechanical simulator (i.e., a box with the possibility of trocar insertion) has not changed during the last decade. However, the types of Pelvi-trainers and the models used inside have been improved significantly. According to the task of the simulator, various sophisticated models have been developed, including standardized phantoms, animal organs, and even perfused segments of porcine organs. For laparoscopic suturing, various step-by-step training concepts have been presented. These can be used for determination of the ability of a physician with an interest in laparoscopic surgery, but also to classify the training status of a laparosopic surgeon. Training in laparoscopic surgery has become an important topic, not only in learning a procedure, but also in maintaining skills and preparing for the management of complications. For these purposes, mechanical simulators will definitely play an important role in the future.

Histopathological findings in the gastrointestinal tract of primate recipients of porcine renal xenografts following different immunosuppressive regimens

Cyclophosphamide (CYP) and methotrexate (MTX) have been used as immunosuppressants in induction or maintenance protocols in a large variety of xenotransplantation models. Combining the use of transgenic porcine organs expressing human decay-accelerating factor (hDAF) with immunosuppressive therapy that included the use of CYP or MTX, survival of primate recipients of life-supporting renal xenografts has been prolonged. However, both drugs can cause significant systemic toxicity and, in particular, gastrointestinal (GI) toxicity. To date only limited data have been reported on the histopathological features deriving from the use of such agents in non-human primates. Cyclophosphamide or MTX was used as part of the immunosuppressive regimen in 15 bilaterally nephrectomized non-human primate (Macaca fascicularis) recipients of a life-supporting hDAF porcine kidney. At post-mortem, a detailed analysis of the GI tract in animals receiving either CYP or MTX was performed. Paraffin-embedded sections of each portion of the GI tract were prepared and stained with hematoxylin and eosin (H&E). In some animals, additional investigations by immunohistochemistry (CD3, CD5, CD20, CD79 alpha cy, lambda, and kappa light chains) and by in situ hybridization for EBV encoded RNA (EBER) were undertaken. The xenografted animals from the CYP group had a mean survival of 31 days (range: 0 to 90 days); animals from the MTX group survived a median of 14 days (range: 0 to 39 days). GI complications were the most frequent cause of euthanasia after renal failure. In CYP-treated animals GI-tract lesions were primarily characterized by diffuse, severe lymphoplasmocytic mucosal inflammatory infiltrate. Variable degrees of villi atrophy and fusion, gut-associated lymphoid tissue (GALT) and goblet cell hyperplasia were also observed. In MTX-treated primates, findings were consistent with severe villi atrophy associated with mild-to-moderate disseminated lymphoplasmocytic infiltration. In conclusion, GI tract lesions are an early and consistent finding when CYP or MTX are used as induction agents in this model. The two compounds induce different types of GI tract damage, however, in agreement with their different mechanisms of action. Whilst CYP primarily determines inflammatory lesions, MTX leads to a degenerative type of damage. This study indicates that immunosuppressive drugs can cause severe GI tract damage in primate recipients of renal xenografts and may be responsible for life-threatening lesions.

Functional epitopes on porcine endogenous retrovirus envelope protein interacting with neutralizing antibody combining sites

Porcine cell and organ transplantation provides promise for maintaining normal physiological conditions in patients with end-stage organ failure. The approach however poses serious risk of transmitting pig pathogens to humans. Among many potential pathogens, porcine endogenous retroviruses (PERV) are of particular concern due to their ubiquitous nature in pigs and capability of infecting human cells. Major antigenic determinants and receptor binding domains on PERV remain unclear until now. Two monoclonal antibodies (mAb), named 8E10 and 7C4 capable of neutralizing PERV infection in HEK293 cells are isolated at an IC 50 of 3.0 and 2.7 μg/ml, respectively, in this work. Epitope location for mAb 8E10 was mapped to amino acids 427–434, residing at the C-terminal region of the gp70 component of type A PERV Env protein. The mAb 8E10 bound directly to the PERV indicating that the epitope is exposed on the virion surface. The mAb 7C4 epitope was assigned to the region comprising amino acids 517–537 on the p15E component of PERV. In contrast to mAb 8E10, the 7C4 mAb bound native PERV inefficiently suggesting that its epitope is accessible only after the virus interacts with its receptor. Finally, both mAbs variable regions were cloned and nucleotide sequence determined. All together, these results reveal that both mAbs 8E10 and 7C4 effectively neutralize PERV infection and may be used as a mean to prevent PERV infection in patients receiving xenotransplantation.