Cellular xenotransplantation of animal cells into people: benefits and risk.

The main benefit of xenotransplantation is its potential to overcome the worldwide organ shortage experienced in allotransplantation. Allogeneic transplantation is the only successful therapy for several life-threatening diseases, with cell, tissue or organ donation only partially meeting the demand and many patients dying while waiting for treatment. With supply falling short of demand, it is foreseen that the use of porcine material may at some stage overcome the existing gap between organ availability and clinical need. Recently, pig islet cells have been utilised in clinical trials, with safety being demonstrated. Indeed, pig-derived cells present several advantages: i) porcine cells have a stable function and differentiation pattern and are not tumorigenic; ii) pig cells have been shown to meet the physiological needs in large animal models; iii) the source of pig cells can be scaled up to meet demands in a highly standardised manner, and with respect to animal welfare regulations; iv) 'designated-pathogen-free' (DPF) pig lines can be produced, which could result in a higher safety profile than allotransplantation itself; v) the risk of zoonosis, which was raised years ago as the major hurdle, has been recently circumvented and is actually viewed as a controlled risk; and vi) immune risks are being circumvented via the use of genetically modified donor animals and encapsulation of porcine cells, particularly for the treatment of diabetes. Overall, the benefit appears to outweigh potential risks with respect to cellular xenotransplantation and this is discussed further in this review.

La xénotransplantation (ou hétérogreffe) a pour principal avantage de contourner le problème de la pénurie d'organes disponibles dans le monde pour réaliser des allogreffes. En effet, la transplantation allogénique est la seule thérapie qui permet de traiter avec succès certaines maladies potentiellement mortelles, mais les dons de cellules, de tissus et d'organes ne satisfont qu'une partie de la demande, de sorte que nombre de patients meurent dans l'attente d'un traitement. L'offre étant inférieure à la demande, on peut prévoir que le recours à des organes porcins puisse s'imposer dans un avenir plus ou moins proche afin de réduire l'écart entre les organes disponibles et les besoins cliniques. Récemment, des cellules d'îlots pancréatiques porcins ont été utilisées dans le cadre d'essais cliniques et leur innocuité a été démontrée. En effet, les cellules d'origine porcine présentent plusieurs avantages : i) les cellules porcines ont un fonctionnement et une différenciation cellulaires stables et ne sont pas tumorigènes ; ii) il a été démontré que les cellules porcines sont physiologiquement compatibles avec celles de modèles de grands animaux ; iii) le recours aux cellules porcines peut être échelonné en suivant des normes précises, en fonction de la demande et dans le respect de la réglementation applicable au bien-être animal ; iv) il est possible de produire des lignées cellulaires exemptes de microorganismes pathogènes spécifiques, ce qui offre encore plus de garanties de sécurité qu'une allogreffe ; v) le risque de zoonose, qui constituait le principal obstacle il y a quelques années a été récemment surmonté et on le considère aujourd'hui comme maîtrisé ; vi) les risques pour le système immunitaire du receveur ont été surmontés grâce à l'utilisation d'animaux génétiquement modifiés en tant que donneurs et à l'encapsulation des cellules porcines, en particulier pour les greffes destinées à des patients diabétiques. Les auteurs approfondissent l'examen des avantages de la xénotransplantation, qui l'emportent largement sur ses risques potentiels.

La principal ventaja del xenotrasplante reside en las posibilidades que ofrece para poner remedio a la penuria mundial de órganos destinados a alotrasplantes. El trasplante alogénico es la única terapia eficaz para muchas enfermedades potencialmente mortales, pero las donaciones de células, tejidos y órganos cubren solo una parte de la demanda y muchos pacientes mueren en espera de recibir tratamiento. Ante una oferta que no alcanza a cubrir la demanda, es previsible que en algún momento se recurra a material porcino como medio de subsanar el déficit de órganos disponibles para atender las necesidades clínicas existentes. En fechas recientes se han realizado ensayos clínicos con células de islote pancreático de cerdo y se ha demostrado que resultan seguras. De hecho, el uso de células de origen porcino presenta varias ventajas: i) las células porcinas tienen un patrón estable de funcionamiento y diferenciación y no son tumorígenas; ii) en modelos de animales de gran tamaño está demostrado que las células de cerdo responden a las necesidades fisiológicas; iii) es posible multiplicar las fuentes de células porcinas para responder a la demanda de modo sumamente normalizado y respetando las reglamentaciones de bienestar animal; iv) es posible generar linajes porcinos certificados como «exentos de patógenos¼, lo que podría ofrecer niveles de seguridad incluso mayores que los del propio alotrasplante; v) últimamente se ha podido conjurar el riesgo de zoonosis, que hace unos años parecía constituir el principal obstáculo y actualmente se considera un riesgo controlado; y vi) actualmente ya se evita el riesgo inmunitario gracias al uso de animales donantes genéticamente modificados y al encapsulamiento de las células porcinas, en especial para tratar la diabetes. Globalmente, por lo que respecta al xenotrasplante celular, los beneficios parecen pesar más que los eventuales riesgos, como indican los autores en su examen detallado.

Posttransplant Lymphoproliferative Disorders in Neuronal Xenotransplanted Macaques.

Posttransplant lymphoproliferative disorders (PTLDs) are a heterogeneous group of lymphoid proliferations that occur in the setting of depressed T-cell function due to immunosuppressive therapy used following solid organ transplantation, hematopoietic stem cell transplantation, and also xenotransplantation. In the present study, 28 immunosuppressed parkinsonian Macaca fascicularis were intracerebrally injected with wild-type or CTLA4-Ig transgenic porcine xenografts to identify a suitable strategy to enable long-term cell survival, maturation, and differentiation. Nine of 28 (32%) immunosuppressed primates developed masses compatible with PTLD, located mainly in the gastrointestinal tract and/or nasal cavity. The masses were classified as monomorphic PTLD according to the World Health Organization classification. Immunohistochemistry and polymerase chain reaction (PCR) analyses revealed that the PTLDs were associated with macaca lymphocryptovirus as confirmed by double-labeling immunohistochemistry for CD20 and Epstein-Barr nuclear antigen 2 (EBNA-2), where the viral protein was located within the CD20+ neoplastic B cells. In sera from 3 distinct phases of the experimental life of the primates, testing by quantitative PCR revealed a progression of the viral load that paralleled the PTLD progression and no evidence of zoonotic transmission of porcine lymphotropic herpesvirus through xenoneuronal grafts. These data suggest that monitoring the variation of macaca lymphocryptovirus DNA in primates could be used as a possible early diagnostic tool for PTLD progression, allowing preemptive treatment such as immunosuppression therapy reduction.

Bortezomib, C1-inhibitor and plasma exchange do not prolong the survival of multi-transgenic GalT-KO pig kidney xenografts in baboons.

Galactosyl-transferase KO (GalT-KO) pigs represent a potential solution to xenograft rejection, particularly in the context of additional genetic modifications. We have performed life supporting kidney xenotransplantation into baboons utilizing GalT-KO pigs transgenic for human CD55/CD59/CD39/HT. Baboons received tacrolimus, mycophenolate mofetil, corticosteroids and recombinant human C1 inhibitor combined with cyclophosphamide or bortezomib with or without 2-3 plasma exchanges. One baboon received a control GalT-KO xenograft with the latter immunosuppression. All immunosuppressed baboons rejected the xenografts between days 9 and 15 with signs of acute humoral rejection, in contrast to untreated controls (n = 2) that lost their grafts on days 3 and 4. Immunofluorescence analyses showed deposition of IgM, C3, C5b-9 in rejected grafts, without C4d staining, indicating classical complement pathway blockade but alternate pathway activation. Moreover, rejected organs exhibited predominantly monocyte/macrophage infiltration with minimal lymphocyte representation. None of the recipients showed any signs of porcine endogenous retrovirus transmission but some showed evidence of porcine cytomegalovirus (PCMV) replication within the xenografts. Our work indicates that the addition of bortezomib and plasma exchange to the immunosuppressive regimen did not significantly prolong the survival of multi-transgenic GalT-KO renal xenografts. Non-Gal antibodies, the alternative complement pathway, innate mechanisms with monocyte activation and PCMV replication may have contributed to rejection.

Unusual serological response to hepatitis E virus in plasma donors consistent with re-infection.

Hepatitis E virus (HEV)-positive plasma donations, identified by a plasma mini-pool screening approach, were analysed using serological methods for the presence of anti-HEV IgM and IgG. Avidity testing was performed on the IgG-reactive donations. Anti-HEV IgG with high avidity was observed in two donors together with high viral loads, but with the absence of anti-HEV IgM. These data are suggestive of re-infection in a small proportion of plasma donors, which has not previously been reported.

Prevalence of hepatitis E virus in slaughter-age pigs in Scotland.

The prevalence of anti-HEV isotype-specific antibodies and viraemia were investigated in serum samples collected from slaughter-age pigs (aged 22-24 weeks) from 23 farms in Scotland. Of 176 serum samples tested, 29·0% (n = 51) were anti-HEV IgG positive, 36·9% (n = 65) anti-HEV IgA positive and 29·0% (n = 51) anti-HEV IgM positive. Overall seroprevalence (anti-HEV IgG+ and/or IgA+ and/or IgM+) was 61·4% (n = 108). HEV RNA was detected in 72/162 serum samples (44·4%). Partial sequence of ORF2 (98 nt) was obtained from eight HEV RNA-positive samples and phylogenetic analysis confirmed that they were all of genotype 3. This is the first report on the prevalence of HEV in pigs in Scotland. Given the increasing incidence of locally acquired HEV infection in the UK, evidence that HEV is a foodborne zoonosis emphasizes the need for surveillance in pigs.

Hepatitis E: source and route of infection, clinical manifestations and new developments.

Hepatitis E was previously thought to be a disease of developing countries causing significant morbidity and mortality in young adults, particularly among pregnant women and patients with pre-existing chronic liver disease. Recent studies have shown that hepatitis E is also an issue in developed countries. In this setting, hepatitis E is a zoonotic infection and causes acute infection mainly in middle-aged and elderly men; and chronic infection in the immunosuppressed. The scope and burden of disease are still emerging. The diagnosis of hepatitis E should be considered in any patient with hepatitis, irrespective of their age or travel history.

Hepatitis E virus in Scottish blood donors.

BACKGROUND AND OBJECTIVES: Published prevalence figures for hepatitis E virus (HEV) reveal significant regional differences. Several studies have reported virus transmission via blood transfusion. The aim of this study was to establish HEV seroprevalence and investigate a potential HEV RNA presence in Scottish blood donors. MATERIALS AND METHODS: IgG and IgM were determined in individual serum samples. HEV RNA was investigated in plasma mini-pools corresponding to 43 560 individual donations using nested PCR. Samples amenable to reamplification with primers from a different region were considered confirmed positives, sequenced and analysed. RESULTS: A total of 73 of 1559 tested individual sera (4·7%) were IgG positive, none tested positive for IgM. Plasma mini-pool testing revealed an HEV RNA frequency of 1 in 14 520 donations. Three confirmed positives belonged, as expected to genotype 3. CONCLUSIONS: HEV IgG and RNA figures in Scottish blood donors are lower than those published for the rest of the UK, but sufficiently high to prompt further studies on potential transmission rates and effects of HEV infection, especially for immunosuppressed individuals.

Porcine endogenous retrovirus (PERV) and its transmission characteristics: a study of the New Zealand designated pathogen-free herd.

Previously a strategy for monitoring of pigs intended for cell transplantation was developed and successfully applied to several representative herds in New Zealand. A designated pathogen-free (DPF) herd has been chosen as a good candidate for xenotransplantation. This herd has previously tested free of infectious agents relevant to xenotransplantation and we present here an in depth study of porcine endogenous retrovirus (PERV) transmission. A panel of assays that describes the constraints for the transmission of PERV has been suggested. It includes a) infectivity test in coculture of DPF pig primary cells with both human and pig target cell lines; b) RT activity in supernatant of stimulated primary cells from DPF pigs; c) viral load in donor's blood plasma; d) PERV proviral copy number in DPF pig genome; e) PERV class C prevalence in the herd and its recombination potential. There was no evidence of PERV transmission from DPF pig tissue to either pig or human cells. Additionally, there was no evidence of PERV RNA present in pig blood plasma. PERV copy number differs in individual pigs from as low as 3 copies to 30 copies and the presence of PERV-C varied between animals and breeds. In all DPF pigs tested, a specific locus for PERV-C potentially associated with the recombination of PERV in miniature swine was absent. Presented data on the PERV transmission allows us to classify the DPF potential donors as "null" or noninfectious pigs.

Porcine endogenous retrovirus transmission characteristics from a designated pathogen-free herd.

Previously, a strategy for monitoring pigs intended for cell transplantation was developed and successfully applied to several representative herds in New Zealand. A better understanding of porcine viruses' epidemiology in New Zealand has been achieved, and, as a result, a designated pathogen-free (DPF) herd has been chosen as a good candidate for xenotransplantation. This herd is free of all infectious agents relevant to xenotransplantation. The presented study of pig endogenous retrovirus (PERV) transmission with cocultures in vitro has shown no evidence of PERV transmission from DPF pig tissue. Additionally, in PERV-C-positive DPF donor pigs tested, a specific locus for PERV-C present in miniature swine possibly associated with the transmission of PERV was absent. The data on PERV transmission allowed classifying the DPF potential donors as "null" or noninfectious pigs.

Review article: hepatitis E-a concise review of virology, epidemiology, clinical presentation and therapy.

BACKGROUND: Hepatitis E virus (HEV) is a leading cause of acute icteric hepatitis and acute liver failure in the developing world. During the last decade, there has been increasing recognition of autochthonous (locally acquired) HEV infection in developed countries. Chronic HEV infection is now recognised, and in transplant recipients this may lead to cirrhosis and organ failure. AIM: To detail current understanding of the molecular biology of HEV, diagnostic and therapeutic strategies and propose future directions for basic science and clinical research. METHODS: PubMed was searched for English language articles using the key words "hepatitis E", "viral hepatitis", "autochthonous infection", "antiviral therapy", "liver transplantation", "acute", "chronic", "HEV", "genotype", "transmission" "food-borne", "transfusion". Additional relevant publications were identified from article reference lists. RESULTS: There has been increasing recognition of autochthonous HEV infection in Western countries, mainly associated with genotype 3. Chronic HEV infection has been recognised since 2008, and in transplant recipients this may lead to cirrhosis and organ failure. Modes of transmission include food-borne transmission, transfusion of blood products and solid organ transplantation. Ribavirin therapy is used to treat patients with chronic HEV infection, but new therapies are required as there have been reports of treatment failure with ribavirin. CONCLUSIONS: Autochthonous HEV infection is a clinical issue with increasing burden. Future work should focus on increasing awareness of HEV infection in the developed world, emphasising the need for clinicians to have a low threshold for HEV testing, particularly in immunosuppressed patients. Patients at potential risk of chronic HEV infection must also be educated and given advice regarding prevention of infection.

Hepatitis E virus in patients with decompensated chronic liver disease: a prospective UK/French study.

BACKGROUND: In developed countries, hepatitis E is a porcine zoonosis caused by hepatitis E virus (HEV) genotype 3. In developing countries, hepatitis E is mainly caused by genotype 1, and causes increased mortality in patients with pre-existing chronic liver disease (CLD). AIM: To determine the role of HEV in patients with decompensated CLD. METHODS: Prospective HEV testing of 343 patients with decompensated CLD at three UK centres and Toulouse France, with follow-up for 6 months or death. IgG seroprevalence was compared with 911 controls. RESULTS: 11/343 patients (3.2%) had acute hepatitis E infection, and three died. There were no differences in mortality (27% vs. 26%, OR 1.1, 95% CI 0.28-4.1), age (P = 0.9), bilirubin (P = 0.5), alanine aminotransferase (P = 0.06) albumin (P = 0.5) or international normalised ratio (P = 0.6) in patients with and without hepatitis E infection. Five cases were polymerase chain reaction (PCR) positive (genotype 3). Hepatitis E was more common in Toulouse (7.9%) compared to the UK cohort (1.2%, P = 0.003). HEV IgG seroprevalence was higher in Toulouse (OR 17, 95% CI 9.2-30) and Truro (OR 2.5, 95% CI 1.4-4.6) than in Glasgow, but lower in cases, compared to controls (OR 0.59, 95% CI 0.41-0.86). CONCLUSIONS: Hepatitis E occurs in a minority of patients with decompensated chronic liver disease. The mortality is no different to the mortality in patients without hepatitis E infection. The diagnosis can only be established by a combination of serology and PCR, the yield and utility of which vary by geographical location.

Viral proteins of bovine papillomavirus type 4 during the development of alimentary canal tumours.

In cattle infection of the upper alimentary canal mucosa by bovine papillomavirus type 4 (BPV-4) results in the development of papillomas which can progress to cancer in animals fed on bracken fern. This paper describes a study of the cellular and subcellular distribution of a number of different BPV-4 products in experimentally-induced BPV-4 tumours. E8 and E4 proteins were detected solely as cytoplasmic antigens in the undifferentiated and differentiated layers of the papilloma, respectively; L2 was detected solely as a nuclear antigen in the differentiated layers, whereas E7 was present in either the nucleus or the cytoplasm depending on the differentiation stage of the keratinocyte. Replicative forms of viral DNA were detected from the spinous to the squamous layers. Viral antigens were not detected during papilloma regression or in carcinomas. E8 was most prominent in early developmental stages, while E4 and L2 were most abundant in mature papillomas. E7 was present in large amounts in both early and mature stages, declining at later stages. These results suggest a temporal and spatial requirement for the expression and function of the viral proteins.

Detection of porcine endogenous retrovirus (PERV) viremia in diseased versus healthy US pigs by qualitative and quantitative real-time RT-PCR.

Previous studies have linked levels of porcine endogenous retroviruses (PERV) with poor health and disease in pigs. To determine the levels of expression of PERVs and their potential association with disease expression, real-time reverse transcriptase (RT) PCR assays were used to assess PERV-ABC, PERV-C and PERV-A/C levels in three commercial swine operations in the United States. Pigs (n = 204) aged 3-25 weeks were screened, and all 369 serum samples collected were found to be positive for PERV-ABC RNA as expected. PERV-C and PERV-A/C RNA were detected in 24.1% (89/369) and 18.7% (69/369) of the samples, respectively. When divided into age groups, PERV-A/C RNA was identified in 20.0% (43/215) of the nursery pig samples (3-9 weeks of age) compared to 16.9% (26/154) finisher pig samples (12-25 weeks of age). On two of the farms, serum was collected from healthy pigs (n = 60) and from pen-mates with various clinical conditions including diarrhoea, wasting and respiratory disease (n = 60). Overall, 25% (15/60) of the samples from clinically affected pigs were found to be positive for PERV-A/C RNA, whereas in clinically healthy pigs, only 8.3% (5/60) of the samples were found to be PERV-A/C positive (P = 0.026). It was possible to identify PERV-A/C in the same pigs on more than one consecutive bleeding, indicating variable length of PERV-A/C viremia. The results indicate that there is an increased incidence of PERV-A/C viremia in diseased pigs and that PERV-A/C can be detected over time in selected pigs within commercial pig production systems in the United States.

Xenotransplantation of galactosyl-transferase knockout, CD55, CD59, CD39, and fucosyl-transferase transgenic pig kidneys into baboons.

Galactosyl-transferase knockout (GT-KO) pigs represent the latest major progress to reduce immune reactions in xenotransplantation. However, their organs are still subject to rapid humoral rejection involving complement activation requiring the ongoing development of further genetic modifications in the pig. In a pig-to-baboon renal transplantation setting, we have used donor pigs that are not only GT-KO, but also transgenic for human CD55 (hCD55), hCD59, hCD39, and fucosyl-transferase (hHT). We studied kidney xenograft survival, physiological and immunologic parameters, xenogeneic rejection characteristics, as well as viral transmission aspects among two groups of baboons: control animals (n = 2), versus those (n = 4) treated with a cocktail of cyclophosphamide, tacrolimus, mycophenolate mofetil, steroids, and a recombinant human C1 inhibitor. Whereas control animals showed clear acute humoral rejection at around day 4, the treated animals showed moderately improved graft survival with rejection at around 2 weeks posttransplantation. Biopsies showed signs of acute vascular rejection (interstitial hemorrhage, glomerular thrombi, and acute tubular necrosis) as well as immunoglobulin (Ig)M and complement deposition in the glomerular and peritubular capillaries. The low level of preformed non-Gal-α1.3Gal IgM detected prior to transplantation increased at 6 days posttransplantation, whereas induced IgG appeared after day 6. No porcine endogenous retrovirus (PERV) transmission was detected in any transplanted baboon. Thus, surprisingly, organs from the GT-KO, hCD55, hCD59, hCD39, and hHT transgenic donors did not appear to convey significant protection against baboon anti-pig antibodies and complement activation, which obviously continue to be significant factors under a suboptimal immunosuppression regimen. The association, timing, and doses of immunosuppressive drugs remain critical. They will have to be optimized to achieve longer graft survivals.

The role of exogenous p53 and E6 oncoproteins in in vitro transformation by bovine papillomavirus type 4 (BPV-4): significance of the absence of an E6 ORF in the BPV-4 genome.

Bovine papillomavirus type 4 (BPV-4) does not possess an E6 ORF. The E6 oncoprotein of human papillomavirus (HPV) binds and degrades the tumour suppressor protein p53, thus contributing to tumour progression. Since BPV-4 lacks E6, it is unknown how the virus evades the tumour suppressor properties of p53 in the induction of tumours of the gastrointestinal tract. Mutations in the p53 gene have been detected both in papillomas and carcinomas, suggesting that p53 dysfunction plays a part in these neoplasias. BPV-4 can transform primary foetal bovine cells (PalFs) in cooperation with an activated ras gene, but the transformed cells are neither immortal nor tumorigenic. Co-transfection with the HPV-16 E6 (16E6) ORF confers immortality but not tumorigenicity. To investigate the role of p53 in BPV-4 cell transformation in vitro, we transfected PalFs and p53-null mouse fibroblasts with BPV-4 DNA in combinations with ras, 16E6 ORF and mutant (V143A) p53 cDNA. Transfection of PalFs with BPV-4 DNA, ras and mutant p53 led to cell immortalization, indicating that 16E6 and mutant p53 are functionally equivalent in conferring immortality. However, co-transfection of PalFs with BPV-4 DNA, ras, and both mutant p53 cDNA and 16E6 ORF resulted in cells which were fully transformed to tumorigenicity. In p53-null mouse fibroblasts, BPV-4 DNA induced transformation by itself, but the transformed cells were incapable of suspension growth. The co-transfection of BPV-4 DNA with 16E6 ORF produced many more transformed colonies and the cells were capable of growing in suspension. In this system, therefore, 16E6 confers anchorage-independence to BPV-4-transformed cells in a p53-independent fashion.

The antibody response of cattle infected with bovine immunodeficiency virus to peptides of the viral transmembrane protein.

The development of the antibody response to peptides of the transmembrane glycoprotein of bovine immunodeficiency virus (BIV) was followed over a period of 50 weeks in six cattle experimentally infected with the BIV(FL112) isolate. Antibody was detected by an enzyme immunoassay using either a linear or a cyclized peptide with structural features common to an immunodominant region of other lentiviruses. The assay was specific for BIV, detecting antibody in bovine sera to BIV(FL112) or BIV(R29) but not to six other common viruses of cattle. Antibody was present in the sera of all cattle inoculated with BIV(FL112) within 4 weeks of infection, peaked between 10 and 30 weeks and persisted in most cattle during the 50 weeks of observation. These features indicate that this assay may be useful in identifying cattle infected with other strains of BIV in the field.

Expression and sequence analysis of cDNAs induced during the early stages of tuberisation in different organs of the potato plant (Solanum tuberosum L.).

cDNA clones of two genes (TUB8 and TUB13) which show a 25-30-fold increase in transcript in the stolon tip during the early stages of tuberisation, have been isolated by differential screening. These genes are also expressed in leaves, stems and roots and the expression pattern in these organs changes on tuberisation. Southern analysis shows homologous sequences in the non-tuberising wild type potato species Solanum brevidens and in Lycopersicon esculentum (tomato). Sequence analysis reveals a high degree of similarity between the TUB13 cDNA, and a human S-adenosylmethionine decarboxylase gene. The predicted TUB8 peptide sequence shows several repeats of alanine, glutamate and proline which suggests a structural role for the encoded protein.