The detection of PCMV in cells in all of the organs ofbaboonrecipients 57 and 64, as shown by IHC, is one of the most impressive results of this study. Although it is still unclear whether PCMV is able to infect baboon or human cells, most data indicate that herpes viruses are species-specific [ 47 ]. This was also shown, for example, for mouse cytomegalovirus (CMV) [ 48 ]. In the case of PCMV and human cells, one publication reported the infection of human fibroblasts [ 49 ], while another reported the failure of infection of human cells, showing that the co-cultivation of PCMV-infectedpig macrophages with two human cell lines (293 and Raji) did not facilitate virus transmission [ 13 ]. Therefore, the question of whether PCMV can infect human cells and replicate in humans is still unanswered. Our data here suggest that porcine cells expressing PCMV antigens are found disseminated in the organism of the recipient baboon. Inclusion bodies, a characteristic of infection, were not detected in the positive cells; however, inclusion bodies were also occasionally not observed in previous investigations ofinfected animals [ 40 ]. Quantifying the amount of positive cells in different organs ofbaboon 57 using IHC analysis showed a very high amount of positive cells in the baboon lymph node and testis, a lower amount in the pigheart, and a low amount in the other tested baboon organs (liver, spleen, kidney, and lung) (Table 3 ). Similar results were obtained by IHC analysis for baboon 64. Interestingly, in baboon 64, an extremely high amount of PCMV DNA was detected in the spleen using real-time PCR. It remains unclear whether the difference in the findings obtained by PCR and IHC was due to the presence of viral DNA not expressing protein, or whether different parts of the organ with different expression profiles were used for PCR or IHC. Nevertheless, the highest amount of PCMV DNA was found in the spleen, while lower amounts were found in the blood and liver, and nearly no PCMV was found in the kidney (Figure 4 ).
Six candidate pigs (Sus scrofa) were available for transplantation. They were all triple genetically modified, all crossbreds of German Landrace and Large White, with a GalT-KO, expressing human CD46 and human thrombomodulin (Revivicor, Blacksburg, VA, USA and Institute for Molecular Animal Breeding and Biotechnology, Faculty of Veterinary Medicine, LMU, Munich, Germany). They were analysed for virus infections. The transplant recipient, a 4 years old male baboon, was treated with anti-CD20mAb (19 mg/kg, rituximab, Roche, Basle, Switzerland, days -7, 0, +7, +14), anti-CD40mAb (50 mg/kg , 2C10R4, Mass Biologics, Boston, MA, USA, days -1, 0, +3, +7, +10, +14, +19, later weekly), and anti-thymocyte globulin (ATG, 5 mg/kg, Fresenius/ Neovii Biotech, Gräfelfing, Germany, days -2 and -1). Continuous immunosuppressive treatment included mycophenolate mofetil (40mg/kg/d, CellCept, Roche Pharma AG, Basle, Switzerland) and methylprednisolone (10 mg/kg/d, urbason, Sanofi- Aventis, Paris, France). Immunosuppression was performed as described by Mohiuddin et al. [14,15], with slight modifications. Prophylactic anti-herpes virus and anti-bacterial treatment was performed using ganciclovir (5mg/kg/d, given intravenously, Cymeven, Roche Pharma AG, Basel, Switzerland) and cefuroxim
This is the first report showing trans-species transmission of PCV3 to baboons by the transplantationof a heart from a PCV3-positive donorpig. The correlation between the survival time of the transplant in the recipient and the virus load in the organs of the transplanted baboon suggests that the virus was replicating in the animals. PCV3 was found in the removed pigheart after the end of the study as well as in all organs of the baboons. The highest virus load was found in the liver or spleen of the baboons (Figure 1). It remains still unclear whether PCV3 infects baboon cells or whether the replication is only ongoing in the pigheartand the virus is distributed in the baboon with virus-producing pig cells or as free virus in the blood stream. Incubating mitogen-stimulated pig PBMCs from PCV3-positive pigs with human 293 cells as target cells did not result in infection of the 293 cells. Mitogen stimulation was used, because in several publications it has been shown that the related PCV2 was stimulated in mitogen-treated PBMCs [4,25–27]. The T-cell mitogen concanavalin A (ConA) enhanced PCV2 replication not only in vitro, but also in lymphoid tissues in vivo . Treatment with IL-2, ConA, and D-glycosamine increased the PCV2 yield more effectively than other treatments . Furthermore, ConA together with methyl-beta-cyclodextrin (MβCD) and D-glucosamine also increased the PCV2 replication in pig kidney cells (PK15) . Here we used another T-cell mitogen, PHA, which also simulates an immune response of the T lymphocytes, for the stimulation of PBMCs. An increased replication after mitogen-stimulation ofpig PBMCs was shown not only for PCV2 [4,25–27], but also for PCMV  and PERV . The fact that human 293 cells were not infected with PCV3 in this first experiment does not mean that human cell cannot be infected with PCV3. There may be several reasons for the
While most of these pathogens are sufficiently controlled by housing animals within barrier facilities and thus protecting them from exogenous infections, porcine endogenous retroviruses (PERVs) are a more precarious issue as they can be transmitted via the germline. Consequently, PERVs have caused considerable concern for clinical xenotransplantation. But to date there has been no report of PERVs being actually transmitted to the recipient of a xenograft or having caused detectable adverse reactions within a transplant 10 . Recent successes in eliminating PERVs from the genome ofpig lines have further diminished this potential hazard 11,12 . Other pathogens, however, provoke lifelong, latent, transmittable infections, and are prevalent in most, if not all, pig populations and are thus difficult to control. These include the porcinecytomegalovirus (PCMV), a β-herpesvirus related to the human cytomegaloviruses that causes systemic disease and potentially leads to graft failure in human allotransplantation 13 . In xenotransplantation, the PCMV has also been associated with transplant injury. This has been largely attributed to virus activation within the graft following transplantation but initially was not thought to cause invasive disease in the recipient 14,15 . It is still unclear whether PCMV can infect human cells, with one in vitro study suggesting the possibility 16 and a different study presenting evidence for the opposite 17 . There are indications that while in vitro PCMV appears susceptible to standard antiviral medication comparable to that employed in allotransplantation 18 , in vivo data from pig-to-baboon xenotransplantation suggest that the commonly used ganciclovir has no therapeutic efficacy against PCMV at standard doses 19 . Agents that do prevent or treat PCMV infection effectively, such as foscarnet or cidofovir, carry significant toxic potential for the transplant recipient and are thus of limited usefulness. Consequently, there is consensus that potential donor animals for xenotransplantation should be free of PCMV.
4 However, despite the advances in pharmacologic and device based treatment ofheart failure, morbidity and mortality remains to be very high in this patient cohort, so that surgical treatment represents an option consisting of treatment with left ventricular assist devices (LVAD) or hearttransplantation [5,4]. Hearttransplantation is a reliable therapeutic option for patients with end-stage heart failure . During the last years, 1 year survival remained high after transplantation for ischemic (84%) and non-ischemic (86%) reasons . In addition, progression in immunosuppresive therapy resulted in a decrease of acute rejection to 22% after 1 year and 36% after 3 years . Although the current results show that heart transplant is a feasible option, organ shortage is still a problem to be solved in the background of a declining trend of subjects to donate organs [8,7] and future strategies of allocating the organ to the recipient are needed . The International Society ofHeartand Lung Transplantation outlined indications and criteria for listing patients with heart failure  the criteria were especially focusing on cardiopulmonary stress testing, hemodynamic data from right heart catheter, co-morbidities and cardiovascular risk factors. In a more condensed fashion, characteristics to consider for hearttransplantation are outlined in table 1.
Not only in risk prediction studies, but in research, gen- erally one needs to find a balance between the need of long-term follow-up and the need to use contemporary data to address potential era effects. We therefore decided to include donors from as early as 1980 for prediction ofdonor death, because we felt that there was only little change in practice over time and a long enough follow-up after kidney donation is needed to facilitate observation of the rare event of death in these healthy individuals. Under similar considerations, we decided to include recipients for the models to predict recipient mortality and graft loss only after 1995, because more modern immunosuppres- sion with tacrolimus and mycophenolate mofetil were not used before this date, and thus, this change in practice could have had a larger influence, while at the same time, a ten-year observation period for recipient death and graft loss are reasonable and do constitute a longer follow-up than in most transplant studies.
The amount of unlabeled 60S BAV R N A re quired for fifty percent inhibition of hybridization of labeled viral R N A to different baboon DN As reflects the concentration of BAV proviruses in the baboon tissues or infected cell lines. The concentration of unlabeled 60S BAV required for fifty percent competition of 3H HL23V-BAB 30S R N A was 3 —4 times greater for baboon tissue D N A than for BAV infected dog thymus cell D N A (i.e. 5 x l 0 -2 ng/250 /A for baboon tissues D N A and 1.5 X10-2 ^wg/250 /u\ for BAV infected cell line DNA). Therefore, the normal baboon tissues appear to contain 3 —4 times more BAV pro viral D N A sequences than the infected dog thymus cells, AS/HOS/A7573 or M-7 (8155) , Our data are in agreement with the results of Benveniste and Todaro  who have detected 5 — 15 copies of BAV proviral D N A per haploid cell genome in D N A from baboon tissues and 1 copy per haploid cell genome in D N A from BAV infected cell lines.
(NH 3 ), methane (CH 4 ) and nitrous oxide (N 2 O) are considerable sources for emissions from pig faeces and contribute to a larger extent to the environmental eutrophication and disturbances of sensitive ecosystems (45) . Further, losses of soil borne N decrease its fertility and productivity ( 46) . Indeed, N which is incorporated into the protein of bacteria is less mobile and gradually available for the crops, since it first has to be mineralised (45,47) . Towards the slower breakdown of bacterial protein, taking in dependence on the temperature weeks or even months, the degradation of urinary urea to ammonia and carbon dioxide (CO 2 ) needs only several hours (23,47) . Thus, the proportion of bacteria in faeces is a well known size for N emissions from pig facilities. The potential of increasing the amount of faecal bacteria by the enlarged inclusion of fibre in pig’s diet has been approved as an appropriate tool to shift N excretion from urea in the urine to bacterial protein in the faeces in several studies (22,23,26,48-50) . Thereby, the dietary fibre serves as an energy source on the fermentation process of the gut bacteria and stimulates their growth (3) . The increased de novo synthesis of bacterial protein enhances utilisation of intestinal available NH 3 and the secretion of urea from the blood into the colon lumen (23,26,45) . In consequence, urinary N decreases ( 46) . The potential savings of N emission to the environment has been investigated by Canh et al. (26) , Z ervas & Zijlstra (24) and Bindelle et al. (23) . The inclusion of fermentable dietary fibre from soybean hulls and sugar beet pulp in an investigation of Z ervas & Zijlstra (24) increased faecal N output from 5·1 to 7·7 g/day and decreased urinary to faecal N excretion ratio from 2·0 to 1·3 and 1·0. Canh et al. (26) and Bindelle et al. (23) showed in their studies, that the inclusion of 30% sugar beet pulp reduced the urinary to faecal N ratio from 3·8 to 1·2 resp. 2·1 to 1·2, which corresponds to a decrease in urinary N excretion of the total excreted N of up to 23%. Also from slurry ammonium (NH 4 ) emissions could have been lowered up to 53% with including 30% of sugar beet pulp into the diet ofpigs (48) .
Charlotte U. Brey 1 , Julia Proff 1 , Natascha Teufert 2 , Benjamin Salzer 1 , Johannes Brozy 3 , Markus Münz 3 , Jochen Pendzialek 3 , Armin Ensser 2 , Wolfgang Holter 1,4 & Manfred Lehner 1,4 Bispecific T cell engager (BiTE) antibody constructs are successfully used as cancer therapeutics. We hypothesized that this treatment strategy could also be applicable for therapy of human cytomegalovirus (HCMV) infection, since HCMV-encoded proteins are abundantly expressed on the surface ofinfected cells. Here we show that a BiTE antibody construct directed against HCMV glycoprotein B (gB) and CD3 efficiently triggers T cells to secrete IFN-γ and TNF upon co-culture with fibroblasts infected with HCMV strain AD169, Towne or Toledo. Titration of gB expression levels in non- infected cells confirmed that already low levels of gB are sufficient for efficient triggering of T cells in presence of the BiTE antibody construct. Comparison of redirecting T cells with the bispecific antibody versus a chimeric antigen receptor (CAR) based on the same scFv showed a similar sensitivity for gB expression. Although lysis ofinfected target cells was absent, the BiTE antibody construct inhibited HCMV replication by triggering cytokine production. Notably, even strongly diluted supernatants of the activated T cells efficiently blocked the replication of HCMV in infected primary fibroblasts. In summary, our data prove the functionality of the first BiTE antibody construct targeting an HCMV-encoded glycoprotein for inhibiting HCMV replication in infected cells.
Background- — We evaluated depression and social isolation assessed at time of waitlisting as predictors of survival in heart transplant (HTx) recipients.
Methods and Results- — Between 2005 and 2006, 318 adult HTx candidates were enrolled in the Waiting for a New Heart Study, and 164 received transplantation. Patients were followed until February 2013. Psychosocial characteristics were assessed by questionnaires. Eurotransplant provided medical data at waitlisting, transplantation dates, anddonor characteristics; hospitals reported medical data at HTx and date of death after HTx. During a median follow-up of 70 months ( <1–93 months post-HTx), 56 (38%) of 148 transplanted patients with complete data died. Depression scores were unrelated to social isolation, and neither correlated with disease severity. Higher depression scores increased the risk of dying (hazard ratio =1.07, 95% conﬁdence interval, 1.01, 1.15, P =0.032), which was moderated by social isolation scores (signiﬁcant interaction term; hazard ratio = 0.985, 95% con ﬁdence interval, 0.973, 0.998; P=0.022). These ﬁndings were maintained in multivariate models controlling for covariates (P values 0.020 –0.039). Actuarial 1-year/5-year survival was best for patients with low depression who were not socially isolated at waitlisting (86% after 1 year, 79% after 5 years). Survival of those who were either depressed, or socially isolated or both, was lower, especially 5 years posttransplant (56%, 60%, and 62%, respectively).
to 12.9 patients needed by supra-sternal doppler ultra sound) to get enough experience required for satisfactory results (59). The disadvantages of pulmonary artery catheter (PAC) in hemodynamic monitoring include invasiveness, difficulty in insertion, high cost and the fact that data are not always understood or well responded to (59). The ESCAPE study investigators (60) established that therapy to reduce volume overload during hospitalization for heart failure led to improvement of signs and symptoms of elevated filling pressures both with and without PAC and concluded that PAC was associated with anticipated adverse effects. They suggested research on non-invasive assessment possibilities which could be used to better tailor therapy. Methods other than bioimpedance for estimating cardiac output (CO) include echocardiography, pulse contour analysis, ventriculography, thermodilution, and the direct and indirect Fick methods (61). The indirect Fick method utilizes carbon dioxide (CO2) production after re-breathing to estimate CO. This is only applicable in ventilated patients. Thermodilution in PAC using single measurements is quite inaccurate (22% error) which can be reduced to 13% by averaging triple measurements. Although thermodilution has established itself well in the measurement of CO, it still largely relies on the user technique (injecting technique, timing of injection in dependence to respiratory circle, number of CO values taken and injectate temperature). Pulse contour analysis is becoming increasingly popular in the ICU but it requires femoral artery access and can therefore not be readily used. Esophageal Doppler is invasive and requires sedation of the patient whereas echocardiography is operator dependent and usable only intermittently. Intense monitoring is of benefit to sick patients in early stages of illness. A less user dependent and less invasive measurement technique is therefore necessary.
10 seconds after changing the membrane potential from 0 mV to a given value. Relative frequency of events was calculated by dividing the conductance range of 5 nSi into intervals of 50 pSi and counting the number of events in a given time period (total record length, 60s, was 512 points) divided by the number of selected records. A: Um: — 10 mV, first 10 s after voltage jump. Number of records («) = 17. B: Um: — 10 mV, steady state conductance (s 30 to s 60 are taken for calculation). « = 1 7 . A second conducting state with a conductivity of 2.0 nSi is indicated. C: Um: — 25 mV, first 10 s after voltage jump. « = 1 6 . At least 5 different states of conductivity can be discerned (1.4 nSi; 2.0 nSi; 2.2 nSi and 2.4 nSi for the lower conduct ing states). D: Um: - 2 5 mV, steady state conductance (s 30 to s 60). « = 16. Number of counted open states with lower conductivity far exceed the number of counted total open states. Membrane lipid: 1% Asolectin in «-decane; elec trolyte: 1 M KC1.
Reperfusion injury has a deleterious effect on endothelial cells, since the endothelium is the first barrier contacting the ROS produced by reperfusion injury. The cardioprotective effect of IPreC was demonstrated in both endothelial cells and cardiomyocytes. Endothelium of blood vessels is directly exposed to IPreC stimuli reflecting changes in production of ROS and expression of endothelial nitric oxide synthase (eNOS), a regulator of vascular function. Yang et al. demonstrated in the knockout mouse model that eNOS is crucial for IPreC-elicited cardioprotection and its expression increases in the early phase of cardioprotection (40). Similarly, eNOS signalling persists in SWOP and it is targeted to phosphorylation by protein kinase B (Akt), a key interaction partner of phosphatidylinositol-4, 5-bisphosphate 3-kinase (PI3K) signalling pathway. Activation of PI3K/Akt pathway abrogates the formation of ROS, which is a consequence of hypoxic insult (8, 49). Unbalanced and high levels of ROS released after injurious infarction is a major determinant of myocardial cell death (55). He et al. demonstrated significantly improved endothelial function by IPreC in preconditioned rats and the IPreC abolished production of ROS also in delayed preconditioning (39). However, ROS might exhibit dual function in IPreC-elicited cardioprotection, since ROS contribute to intracellular signalling, and thereby activate endogenous cardioprotection (42, 55). The beneficial effect of IPreC-induced ROS takes place in mitochondria by mediating redox signalling. Dost et al. demonstrated on isolated preconditioned rabbit hearts that an intravenous injection of radical scavengers leads to a diminished effect of IPreC (10). Similarly, in vivo experiments performed on mouse hearts demonstrated that inhibited activity of manganese superoxide dismutase (Mn-SOD), intrinsic ROS scavenger, induces the entrance into the IPreC phase (50).
The identification of a M94 DN mutant leads to the conclusion of at least two essential functions inherited by M94 as one function probably binding in a complex needs to remain like wt, whereas a second function must be destroyed to generate the inhibitory pheno- type. One of these functions is located in the N-terminus of M94 as mutations in this region specifically inhibited a protein function causing the DN phenotype. The second essential function might well be located in the C-terminus of M94 as this was identified as an essential region in the loss-of-function screen. Although other reasons as incorrect protein folding might have resulted in the lethal phenotype of the C-terminal mutants, the assumption of an essential region related to a distinct protein function is reasonable. A publication on the α-Herpesvirinae homologue UL16 demonstrated a transient binding of UL16 to the viral capsid by the conserved cysteines of UL16 family members, which are located in the C-terminus of the homologues . The strict conservation of these cysteines implies a function related to these cysteines, which is also conserved and shared between α-Herpesvirinae and β-Herpesvirinae. So from the two essential functions of M94 one is located in the N-terminus and the second one probably in the C-terminus.
Liver fibrogenesis in NEMO Dhepa recipients is reduced after hepatocyte transplantation
Since our previous findings indicated that HT exerts a beneficial effect on NEMO-dependent liver pathogenesis, we next investi- gated the development of liver fibrosis, which is typically detectable in NEMO Dhepa mice already at the age of 8–13 weeks. Sirius red staining of collagen deposition and immunohistochem- ical analysis of Collagen-1a fibres displayed a strong signal in age- matched naı¨ve and BMT NEMO Dhepa mice. In contrast, mice which underwent HT showed significantly less collagen accumu- lation (Fig. 3A+B). Quantitative analysis of Collagen-1a mRNA expression by real time PCR displayed a significant reduction in mice subjected to HT independent on the genotype ofdonor mice (Fig. 3C). The onset of liver fibrosis was further graded according to an adapted METAVIR score , which revealed an improvement of liver histology in mice subjected to HT as compared to control mice (Table 1). Altogether these results suggest that HT attenuates the progression of fibrosis in NEMO Dhepa mice.
The mixed chimerism approach has been successfully translated into the clinical setting; however, widespread clinical application in transplant recipients has been hindered by the toxicity of current BMT protocols. We previously reported a noncytotoxic protocol by combining therapeutic Treg treatment and a clinically feasible number of stem cells. Notably, Treg treatment not only facilitates BM engraftment and the induction of hematopoietic chimerism, but was also shown to prevent chronic rejection ofheart allografts, making it superior over protocols based on irradiation or cytostatic drugs [ 12 ]. Here, we could show that nTregs are the preferable Treg population for therapeutic application, in both theoretical and quantitative terms. Although generation of iTregs in vitro has practical advantages in murine models, as they are easy to obtain in large numbers, the risk of reconversion is not negligible and induction of stable Tregs in human is more complex than in rodents [ 30 ]. In our previous reports, all tested Treg populations were effective in promoting the induction of chimerism and tolerance at high
In summary, within the present study we demonstrate that the NEMO Dhepa mouse is a versatile model to study technical and biological aspects related to HT. Indeed there are several more studies showing effective and in part higher levels ofdonor cell repopulation. The fumaryl acetoacetat hydrolase mouse (Fah -/- ) - representing an ideal model of metabolic disorders - was shown to be cured from hereditery tyrosinemia by transplantationof only very few hepatocytes . Chemical preconditiong via retrorsine - a cell cycle inhibiting pyrrolizidin alkaloid thus inducing chronic liver injury - resulted in strong liver mass replacement as well . However, a genetic model triggering liver fibrogenesis was not applied to HT up-to-date. Therefore the use of NEMO Dhepa mice as recipients for HT represents a promising novel experimental model. Donor cell engraftment after HT may here function as a modulator of the spontaneous progression of liver fibrosis. This seems to represent a special feature of the NEMO Dhepa mouse, which cannot be investigated in other HT-models to the same extent. Notwithstanding, the expansion of liver repopulation by transplanted hepatocytes is rather modest (up to 13%) which clearly suggests that other factors (growth factors e.g. HGF, cytokines like IL-10,) might help to ameliorate the progression of chronic liver injury in NEMO Dhepa - transplanted mice. This could explain the observed improvement in the NEMO Dhepa phenotype which became also apparent in recipientsof WT-donors cells, displaying a less repopulation efficacy. Similar effects are described already in experiments involving transplation of stem cells in
TR is the most common valvular heart disease after HTx. 40 Indication for surgery is present in symptomatic patients with severe TR due to primary valvular pathologies such as endo- carditis, valve perforation, or rupture of chordae or flail leaflet combined with signs of right-sided heart failure refractory to medical treatment. 40 Risk-benefit ratio for operating on func- tional TR due to severe graft vasculopathy with reduced right ventricular function or combined with pulmonary hyperten- sion is of uttermost importance. There exist several patholo- gies of TR that are typical for HTx patients: biopsy-induced injury of the chordae or leaflet, ischemic damage of papillary muscle, or structural distortion of atrial geometry due to im- plantation technique. 4,7,41
Active control of the growth of broiler chickens andpigs has potential ben- efits for farmers in terms of improved production efficiency, as well as for animal welfare in terms of improved leg health in broiler chickens. In this work, a differential recurrent neural network (DRNN) was identified from experimental data to represent animal growth using a nonlinear system iden- tification algorithm. The DRNN model was then used as the internal model for nonlinear model predicative control (NMPC) to achieve a group of de- sired growth curves. The experimental results demonstrated that the DRNN model captured the underlying dynamics of the broiler andpig growth pro- cess reasonably well. The DRNN based NMPC was able to specify feed intakes in real time so that the broiler andpig weights accurately followed the desired growth curves ranging from −12% to +12% and −20% to +20% of the standard curve for broiler chickens andpigs, respectively. The overall mean relative error between the desired and achieved broiler or pig weight was 1.8% for the period from day 12 to day 51 and 10.5% for the period from week 5 to week 21, respectively.