The chronic shortage of human transplants to treat tissue and organ failure has led to the development of xeno- transplantation, the transplantation of cells, tissues and organs from another species to human recipients. For a number of reasons, pigs are best suited as donor animals. Successful, routine xenotransplantation would have an enormous impact on the health of the human population, including the young, who sometimes require a re- placement organ or islet cells, but especially the elderly, who more often suffer the consequences of organ failure. The ﬁrst form of xenotransplantation applied to humans is the use of pig islet cells to treat insulin-dependent di- abetes, a procedure that will have a signiﬁcant economic impact. However, although xenotransplantation using pig cells, tissues and organs may save and prolong the lives of patients, it may also be associated with the trans- mission of porcine microorganisms to the recipient, eventually resulting in emerging infectious diseases. For this reason, the health of both the donor animals andthe human recipients represents a special and sensitive case of the One Health concept. Basic research leading to strategies how to prevent transmission of porcine microorgan- isms by selection of virus-free animals, treatment of donor pigs by antiviral drugs, vaccines, colostrum depriva- tion, early weaning, Caesarean delivery, embryo transfer and/or gene editing should be undertaken to supply an increasing number of potential recipients with urgently required transplants. The methods developed for the detection and elimination of porcine microorganisms in the context of xenotransplantation will also contrib- ute to an improvement in the health of pig populations in general and an increase in the quality of meat products. At present, there is evidence for transmission of porcine viruses to humans eating pork and having contact with pigs, however the impact of these viruses on public health is still unknown.
PCV3 is considered a putative cause of reproductive failure, encephalitis and myocarditis in perinatal piglets, porcine dermatitis and nephropathy syndrome, and periarteritis in swine in the United States [ 32 ], andporcine dermatitis and nephropathy in China [ 33 ]. Furthermore, PCV3 has a potential association with swine respiratory disease and diarrhea [ 34 ]. However, since PCV3 was found in also in healthy pigs as well as in pigs with numerous severe diseases [ 13 ], it seems likely that co-factors such as a second virus infection, an infection with other microorganisms, or genetic factors are required for a pathogenic effect. In the case of PCV2 additional factors also seem to be required for the induction of the overt clinical symptoms. Under experimental conditions, clinical PCVAD is often difficult to reproduce in pigs infected with PCV2 alone [ 35 – 38 ]. In addition, only a portion of PCV2-infected pigs actually develop the full spectrum of clinical PCVAD, even in the presence of some known co-factors [ 38 ]. PCV2 is an immunosuppressive virus, it preferentially targets the lymphoid tissues, which leads to lymphoid depletion and immunosuppression in pigs. PCV2 significantly alters the cytokine responses in infected animals, with IL-10 upregulated, and IL-2 and IL-4 downregulated [ 39 ]. Most likely the immunosuppression induced by PCV2 infection predisposes pigs to co-infecting agents.
The Commission’s general rationale for funding health related research is to improve both the health of Europeans as well as the economic position of Europe; it was described by an interviewee as intended "to improve quality of life and health of the citizens and to improve the competitiveness of the European industry" (g: 257-258). Xenotransplantation was funded under the heading of "new therapies" (g: 16), which includes "non-pharmaceutical cell based therapies, tissue engineering, gene therapy, regenerative medicine (...) human embryonic stem cells, stem cells" (ibid. 17-19). Public funding in this area is considered justified if several conditions are met; these include cases where new therapies (a) would have a high potential to cure life threatening, currently incurable diseases; (b) they would be at a very early stage of development, and, (c) although they would be risky there would be "proof of principle", mostly in animal models, that research therein was "worth trying" (c.f. g: 16-26). Within the "wider program" of new therapies, xenotransplantation was described by an interviewee as one of several "routes" to alleviate organ shortage, amongst treatment of diseases leading to organ failure, regenerative medicine, and artificial organs (g: 36-41; 48- 56; 99-102). After a "hiatus" (g: 64) of funding, stimulated by a concern about porcine endogenous retroviruses (PERV) and potential cross-species infection, in 2006 the European Commission started funding the integrated project XENOME, which looks at solid organ and cellular xenotransplantation. The fact that the risk of cross infection was no longer regarded as an issue necessarily blocking research encouraged this renewed interest. (g: 68-70). XENOME is “the principle European effort in the field of xenotransplantation” (Stein 2010: 13). Commissioner Geoghegan-Quiinn referred to the project as “the reference for
XMRV (xenotropic murine leukaemia virus-related virus) is a gammaretrovirus that has been detected in human patients with prostate carcinoma, chronic fatigue syndrome (CFS) and also in a small percentage of clinically healthy individuals. It is not yet clear whether the distribution of this virus is primarily limited to the USA or whether it is causally associated with human disease. If future investigations confirm a broad distribution of XMRV and its association with disease, this would have an impact on xenotransplantation of porcine tissues and organs. Xenotransplantation is currently being developed to compensate for the increasing shortage of human material for the treatment of tissue and organ failure but could result in the transmission of porcine pathogens. Maintenance of pathogen-free donor animals will dramatically reduce this risk, but some of theporcine endogenous retroviruses (PERVs) found in the genome of all pigs, can produce infectious virus and infect cultured human cells. PERVs are closely related to XMRV so it is critical to develop tests that discriminate between them. Since recombination can occur between viruses, and recombinants can exhibit synergism, recipients should be tested for XMRV before xenotransplantation.
Hyperacute rejection is the first immunologic barrier for transplantation between human or NHP and pig. It starts immediately when the pig coronary arteries are perfused by primate blood. The graft is destroyed within 24 hours, but often even within the first hour: primate blood contains "natural" anti-pig antibodies that bind the vascular endothelial cells of the pig heart and activate the complement cascade. This leads to immediate injury of the endothelium, which causes thrombosis in vessels and edema that disrupts the function of the endothelium and heart within minutes . The rejection is caused by the interaction between a carbohydrate epitope, galactose-α1,3-galactose (Gal), from porcine endothelial cells and antibodies present in the primate blood. During neonatal life of all primates, these antibodies develop and are probably a reaction to micro-organisms that colonize the gastrointestinal tract [12, 13]. This response is similar to that in ABO-incompatible allotransplantation rejections .
In this study, we aimed at establishing a PCMV-free breeding herd of gm pigs for xenotransplantation. Pregnant F0 sows were purchased from an external designated pathogen-free barrier facility and introduced into CiMM (Figure 1). CiMM was opened in December 2016 as a newly built pig barrier facility. Quarantine requirements of 48 hours without outside pig contact before entry are in place. Cleanroom showers are used, and a full change of clothes is performed upon entering the facility. Schönhammer ventilation system was installed (Schönhammer, Mengkofen, Germany) to discharge pollutants and odors from the stable, target temperature stability and control humidity levels to prevent precipitation. F0 sows were the first pigs to enter this facility. In addition to PCMV screening, all animals were examined for the presence of the following pathogens on a serological and/or antigen basis: Serological testing was performed for Actinobacillus pleuropneumoniae, Haemophilus parasuis, Lawsonia intracellularis, Leptospira spp., Mycoplasma hyopneumoniae, Pasteurella multocida, porcine reproductive and respiratory syndrome virus, swine influenza virus, transmissible gastroenteritis, and hepatitis E virus. In addition, antigen testing took place for Brachyspira hyodysenteriae, Lawsonia intracellularis, salmonella and swine influenza virus, and fecal swabs were examined for bacteriological content and endoparasites. All testing is repeated continuously every 6 months on a representative proportion of the current pig population within CiMM to ensure adequate hygiene monitoring. To date, antigen detection of Brachyspira hyodysenteriae, Lawsonia intracellularis, and salmonella and swine influenza virus has remained negative. Serologically, the cohort is positive only for Actinobacillus pleuropneumoniae and Lawsonia intracellularis.
is the risk to transmit porcine microorganisms to the recipi- ent which may induce severe disease (zoonosis or xenosis). Microorganisms include bacteria, protozoa, fungi and vi- ruses. In this context viruses are certainly the most harmful microorganisms due to the lack of effective antivirals and vaccines. Among theporcine viruses of interest are DNA viruses such as PCMV, theporcine circoviruses 1, 2 and 3 (PCV1, PCV2, PCV3), andtheporcine lymphotropic herpesviruses (PLHV-1, PLHV-2, PLHV-3) as well as RNA viruses such as the hepatitis E virus (HEV), theporcine reproductive and respiratory syndrome virus (PRRSV) andthe Nipah virus (NIV) (for reviews discussing each virus type in the context of xenotransplantation see [ 18 – 25 ]). Some of these viruses, e.g. PCV2, PRRSV and NIV, cause severe disease in the infected pigs and can easily be detected. For the detection of the viruses not causing diseases in the infected pigs sensitive screening methods
As the initial focus, to modify livestock for agricultural purpose, such as growth performance, feed efficiency and body composition (Pursel, 1998), lactation performance (Zuelke, 1998), reproduction, disease resistance and immune responsiveness (Muller et al., 1998) does not meet the expectations, large animals came in a tighter focus for alternative areas. Especially the pig, due to indications like physique, the ability to standardize the environmental situation (housing, feeding, and sanitation standard), the well established reproductive technology and advanced techniques of genetic modification of theporcine genome, represents an ideal model organism for both, human diseases andxenotransplantation (Aigner et al., 2010). Several different technologies to produce transgenic animals, primarily developed in the mouse, such as pronuclear microinjection (PMI) (Gordon et al., 1980), sperm-mediated gene transfer (SMGT) (Lavitrano et al., 1989) or viral gene transfer (Jaenisch et al., 1976), were later on adapted to livestock (Brem et al., 1985; Hofmann et al., 2003; Kurome et al., 2006). In mice the main disadvantages arising from those methods, reported as random, partially multicopy integration of the transgene, insertional mutagenesis, positional effects, oncogene activation, low integration efficiencies or offspring mosaicism (Wheeler, 2003) have been partially bypassed with the establishment of murine embryonic stem cells (ESCs) (Evans et al., 1981; Martin, 1981) andthe development of strategies to genetically modify them (Kuehn et al., 1987). In order to circumvent the lack of porcine ESCs, an alternative method, termed somatic cell nuclear transfer (SCNT), has become an indispensable tool to generate large animal models from genetically modified somatic cells (Campbell
As described , Gag protein could be measured for PERV-A(42)-transfected cells in vitro several days after transfection was performed. Those findings was confirmed as part of this study. The effect is however fleeting and vanishes overtime, as cells were split andthe viral DNA became more dilute. Finally, no more Gag was detected anymore in the harvested supernatant (Figure 3.4). There was reason to believe that the PERV-A(42) therefore has a defect in it’s env gene. The possibility of frameshift mutations, random deletions, and nonsense mutations had to be investigated. Possible causes for such events could be subcloning into a sequencing vector or re-transformation and random recombi- nation due to bacterial recombinases. Also, preceeding PCR amplifications, may have caused point mutations leading to a non-infectious clone. As sequencing data revealed, the clone used for transfection of 293T cells shows a sequence identical to its published reference (accession №: AJ133817). Since Western blot analysis did reveal that Gag pro- tein is produced, so called Gag-particles were formed which do not posses the ability to infect 293T cells in vitro. To elucidate if any virus particles were formed after transfec- tion, transmission electron microscopy was employed. Photographs show a virus-particle without visible Env protrusions sticking out from the viral envelope.
Abstract: Allotransplantation andxenotransplantation may be associated with the transmission of pathogens from the donor to the recipient. Whereas in the case of allotransplantation the transmitted microorganisms and their pathogenic effect are well characterized, the possible influence of porcine microorganisms on humans is mostly unknown. Porcine circoviruses (PCVs) are common in pig breeds and they belong to porcine microorganisms that still have not been fully addressed in terms of evaluating the potential risk of xenotransplantation using pig cells, tissues, and organs. Two types of PCVs are known: porcine circovirus (PCV) 1 and PCV2. Whereas PCV1 is apathogenic in pigs, PCV2 may induce severe pig diseases. Although most pigs are subclinically infected, we do not know whether this infection impairs pig transplant functionality, particularly because PCV2 is immunosuppressive. In addition, vaccination against PCV2 is able to prevent diseases, but in most cases not transmission of the virus. Therefore, PCV2 has to be eliminated to obtain xenotransplants from uninfected healthy animals. Although there is evidence that PCV2 does not infect—at least immunocompetent—humans, animals should be screened using sensitive methods to ensure virus elimination by selection, Cesarean delivery, vaccination, or embryo transfer.
Non-human primates (NHPs: chimpanzee, gorilla, baboon) are phylogenetically the closest to human and show anatomical, physiological and immunological similarities and thus present the obvious choice as donor animals for xenotransplantation. Despite the fact that the only successful organ transplantation was a primat-to-human transplantation of kidney , they were ruled out from xenotransplantation for several reasons: (i) Ethical approaches were raised because of their superior intelligence degree andthe physiological and behavioral features similar to humans and which implies that NHPs can suffer like humans [28, 29]. (ii) Most of NHPs species are threatened with extinction and have long gestation periods and low number of offspring. (iii) Concerns were also raised regarding the safety of the process since most of NHPs harbor several pathogens which may be infectious for human considering the similarity of the immune systems among the primates. The most striking example presents the evolutionary studies that suggested a cross-species transmission of the simian immunodeficiency virus (SIV) from chimpanzee (SIVcpz) and sooty mangabey (SIVsm) which generated the human immunodeficiency virus types 1 and 2 (HIV-1 and -2) . Furthermore, baboons harbor several exogenous and endogenous retroviruses such as simian foamy viruses (SFV), simian T-cell lyphotropic virus (STLV), baboon endogenous virus (BaEV) and simian endogenous retrovirus (SERV) which may be transmitted to humans .
Frontiers in Immunology | www.frontiersin.org March 2018 | Volume 9 | Article 435 increase in the percentage of CD4 + CD25 + Treg out of all CD4 +
cells directly after trauma in humans, but a significant increase of their share on day 7 after trauma ( 19 ). Despite no changes in the percentage of Treg, the authors found a progressive increase in Treg suppressive activity regarding T cell proliferation in patients after severe trauma. Compared to Treg from healthy controls and patients 1 day after injury, CD4 + CD25 high Treg from 7 days after injury had a significantly higher suppressive potency ( 19 ). In a mouse burn injury model, the suppressive potential of Treg from peripheral lymphoid tissues was assessed by examining the proliferation rate of lymph node cells ( 67 ). On day 1 after trauma, the Treg suppressive potential did not significantly differ in injured animals, while on day 7 after trauma, CD4 + CD25 + Treg from injured mice exhibited a significantly increased activity compared to sham ( 67 ). In a porcine burn injury-induced sepsis model Zu et al. showed significantly decreased CD4 + CD25 + Treg apoptosis rate in intestinal lymph nodes, demonstrating that the intestinal Treg may play an important role in the intestinal immune barrier system after severe burn injuries ( 68 ). In the last two studies, only the positive expression of CD4 and CD25 was used to characterize Treg, and due to this study design, it is not assured that specifically pure Treg were evaluated. In our study, we did not evaluate the immunosuppressive activity and functionality of Treg, and this remains to be elucidated in further studies. Gupta et al. found no significant change in the percentage of CD4 + CD25 + FoxP3 + Treg in blood samples collected instantly after admittance to ED from patients suffering from trauma (ISS = 18.71 ± 8.48) and hemorrhagic shock ( 23 ). However, we have included only severe polytrauma in both TP and pigs
The boar testes secrete large amounts of C-16 unsaturated androgens. These steroids act as pheromones when they are excreted (Billen et al. 2009). Attempts to identify chemical compounds responsible for boar taint in pork were initiated by Lerche (1936), who described the parotid gland as processing the bad odor (Lerche 1936). The androst-16-ene (16-androstene) steroids are the most abundant steroids produced by the pig testes, reaching total levels of approximately 0.6 mg/g of testicular tissue. The 16-androstenes are synthesized primarily in Leydig cells of boar testes along with other androgens and estrogens, with lesser contributions from the adrenals (Gower 1984). CYP17A1 catalyzes the key regulatory step in the formation of the 16-androstene steroids from pregnenolone by the andien-beta synthase reaction. It has been shown that the two forms of cytochrome b5 (CYB5A and CYB5B) exert different effects on the three activities of porcine CYP17A1 and that CYB5B does not stimulate the andien-beta synthase activity of CYP17A1 (Billen et al. 2009). The 16-androstenes produced in the testes, 5α-androstenone, 3α-androstenol and 3β-androstenol, are released into the systemic circulation via the spermatic vein (Gower et al. 1970, Saat et al. 1972). Due to their hydrophobic property, circulating 16-androstenes are then transported to fat tissue where they are stored (Bonneau 1982, Brooks & Pearson 1986). Androstenone storage in fat is reversible. Castrating mature boars results in a progressive decline in serum and loin fat concentrations of androstenone (Claus 1976, Cliplef et al. 1985, Grinwich et al. 1988). The apparent half-life of fat androstenone ranges from 4–14 days in boars of 100 kg (220 lb) of live weight (Claus 1976, Bonneau et al. 1982). Androstenone and other 16-androstene steroids are probably catabolized in the liver (Claus 1979, Fish et al. 1980). In young boars (100 kg, 220 lb), androstenone is eliminated mainly through the urine, in the form of 5β-androstenol, and in trace amounts through feces (Bonneau & Terqui 1983). In adult boars, 5β-androstenol and, to a lesser extent, 5α-androstenol are the only 16-androstenes that are eliminated in urine (Saat et al. 1972, Gower et al. 1970, Gower et al. 1972).
A variety of applications in biomedicine and biotechnology, including the generation of transgenic animals depend on the capability to alter DNA sequences stably and in a site-specific manner. Until recently, gene targeting has been conventionally achieved by homologous recombination of host genome with DNA-based vectors [55, 67]. Despite several pig models having been established by different gene targeting strategies [5, 55, 67], modifications of theporcine genome remain a time consuming and complex procedure. Hence, alternative targeting tools providing rapid and reliable genetic modification are required. The novel technology of designed nucleases offers a powerful tool and enlarges the options to modify theporcine genome in a specific manner. However, the overall suitability of these technologies still needs to be proven in porcine primary cells. This study aimed at the evaluation of designed nucleases for modification of theporcine genome and presents strategies to improve the targeting efficiency. Nucleases were tested for NHEJ- or HR-introduced modification on autosomes and sex chromosomes. In addition to establishment of reproducible targeting and screening protocols, a lacZ- reporter for the CFTR gene was generated. Moreover, the gained data suggest that the usage of nucleases in combination with DNA-based vectors provides an efficient tool to examine the cellular repair machinery in mammalian cells.
Bei den CD44 positiven Zelllinien ist aufgefallen, dass die disseminierten Zellen in den Pulmonalarterien und dem angrenzenden perivaskulären Raum verblieben sind und nicht in das Lungenstroma einwandern konnten. Das perivaskuläre Lungenkomparti- ment ist von Pabst (2004) erstmalig beschrieben worden und hat eine Bedeutung für die Lungenödembildung und Leukozytenwanderung bei entzündlichen and allergischen Prozessen (Pabst, 2004). Das isolierte Auftreten von Metastasen in dieser Region ist bisher nicht beschrieben worden und bestätigt die speziellen strukturellen und funktio- nellen Eigenschaften dieses perivaskulären Raums. Die genauen Mechanismen, die da- zu führen, dass die disseminierten CD44 positiven Neuroblastomzellen in diesem Raum verbleiben und sich dort vermehren sind jedoch unklar.
Zu den längerfristig denkbaren Kostenersparnissen durch economies of scale ist zumin- dest anzumerken, daß bei medizinischen Innovationen häufig kein klassischer Innovati- onszyklus durchlaufen wird (F EENY 1985). Vielmehr wird ein Produkt normalerweise anfänglich zu einem hohen Preis angeboten, damit Entwicklungskosten amortisiert wer- den können. Auch bei der Xenotransplantation ist wegen der bereits erteilten Patente davon auszugehen, daß die Organpreise zunächst so festgelegt werden, daß sie die Inve- stitionen der Unternehmen ausgleichen. Von den Experten wurde in den Interviews des- halb auch auf das Problem hingewiesen, daß monopolistische Positionen auf der An- bieterseite der Xeno-Organe dringend vermieden werden müßten (z.B. Z7/33) 157 . Darüber hinaus werden zwar andere technologische Innovationen oder Verfahren später von anderen Anbietern und zu niedrigeren Preisen auf den Markt gebracht, in der Medi- zin existiert jedoch aufgrund der Kostenübernahme durch die Krankenversicherungen wenig Anreiz zur Kostensenkung. Teure und günstige Anbieter können deshalb häufig koexistieren, so daß möglicherweise auch dann nur mit geringen Einsparungen zu rech- nen ist, wenn die Patente bei der Xenotransplantation auslaufen. Diese Sorge wird auch durch die Überlegung L AINGS (1996;93) bestätigt, daß die Preise für Organe zunächst gesenkt und nach erfolgreicher Implementation wieder erhöht werden könnten. Zu be- rücksichtigen ist auch, daß einige der genannten Kostenersparnisse gegenüber der Al- lotransplantation, z.B. bei der Beschaffung menschlicher Organe, erst dann wirksam werden können, wenn die Allotransplantation vollkommen durch die Xenotransplantati- on ersetzt wird. Mit einer Kostenreduktion ist deshalb auch aus dieser Perspektive höch- stens langfristig zu rechnen. Bis dahin ist u.a. aufgrund der Parallelexistenz und - finanzierung beider Systeme eher von einer deutlichen Steigerung auszugehen.
Durch die Möglichkeit der Produktion so genannter Mini-Pig-Linien sowie langsam wachsender Rassen könnte sich die klinische Xenotransplantation variabler auf die Bedürfnisse der Empfänger einstellen, z.B. durch die Anpassung der Organgröße (20). Bestehende Unterschiede hinsichtlich der Anatomie und der Physiologie zwischen dem Hausschwein und dem Menschen wurden bereits von mehreren Autoren detailliert beschrieben (20, 24-26). Trotz einiger physiologischer Unterschiede bezüglich der Körpertemperatur, der Blutviskosität, des Hormonsystems und des enzymatischen Haushaltes überwiegen dennoch die Gemeinsamkeiten zwischen Menschen und Schweinen. Gerade im Hinblick auf das Herz ist z.B. die koronare Durchblutung des Herzens identisch zum Menschen sowie stimmen Größe des Herzens und der Gefäße von ausgewachsenen Mini-Pigs mit der humanen Herzanatomie überein (25). Des Weiteren ist eine ausreichende Organfunktion eines porcinen Spenderorgans in einem aufrechtgehenden Empfängerorganismus erwiesen (27). Dennoch gilt es neben all den Vorteilen auch noch die Hürden zu überwinden, die eine diskordante Transplantation von Schwein zu Menschen mit sich bringt. Zu den Hürden zählen vor allem die immunologischen Barrieren sowie mögliche Übertragung von mikrobiologischen Keimen (19).
While liberation of enzymes by plant injury is rather well known, reports of similar reactions of mammalian cells are rare: Tappel reported in 1960 that lipid peroxides are readily formed whenever tissue hom ogenates were exposed to air (Zalkin and Tappel, 1960). A few years later, Wills investi gated the LPO of organ homogenates. He ob served in agreem ent with the results of Tappel a fast production of peroxides by hom ogenation of tissue especially derived from liver and kidney (Wills, 1966). Bergers et al. investigated LPO pro duction in homogenized skin samples under vari ous conditions (Bergers and Verhagen, 1986). They found enhanced am ounts of hydroperoxides, but nearly none after therm al treatm ent of the samples explaining this fact by postulating an inhi bition of phospholipase A 2 responsible for libera tion of fatty acids. We have been able to support these results by subjecting a porcine liver to hom o genation before and after therm al treatm ent. The identification of reaction products by GC/MS proved the involvement of hydroperoxy fatty acids (H erold and Spiteller, 1996). In this paper we re port on kinetic studies of hydroperoxide form ation in porcine kidney tissue.
engage in systematic reflection of the ethics of science and technology. But we learned from NSD2 that certain preconditions must be fulfilled to make NSD work. NSD is certainly not a method useful for every topic and for everybody. The requirements for successful NSD have to do with the participating individuals and with the particular debate. NSD participants must have certain individual qualities and skills. NSD requires what one could call "open- mindedness" in the participants, i.e. willingness to show and to evaluate their own standpoints and values on the basis of their own experiences and not according to "textbook" theories. This is totally different from the usual conception of professional expertise and, as NSD2 proved, not a natural process. On the level of debate, the intensity of conflict might be an important criterion for the successfulness of NSD. If the intensity of conflict and/or of the participants’ unwillingness to show and question their own values make NSD impossible, other forms of debate or decision-making such as mediation, bargaining or decision-making by law courts may be more appropriate. Further research is needed on the preconditions for successful NSD, both with respect to the individuals involved and to the debate itself.