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The blockade of the CD40/CD40L immune checkpoint is considered essential for cardiac xenotransplantation. However, it is still unclear which single antibody directed against CD40 or CD40L (CD154), or which combination of antibodies, is better at preventing organ rejection. For example, the high doses of antibody administered in previous experiments might not be feasible for the treatment of humans, while thrombotic side effects were described for first-generation anti-CD40L antibodies. To address these issues, we conducted six orthotopic pig-to-baboon cardiac xenotransplantation experiments, combining a chimeric anti-CD40 antibody with an investigational long-acting PASylated anti-CD40L Fab fragment. The combination therapy effectively resulted in animal survival with a rate comparable to a previous study that utilized anti-CD40 monotherapy. Importantly, no incidence of thromboembolic events associated with the administration of the anti-CD40L PAS-Fab was observed. Two experiments failed early because of technical reasons, two were terminated deliberately after 90 days with the baboons in excellent condition and two were extended to 120 and 170 days, respectively. Unexpectedly, and despite the absence of any clinical signs, histopathology revealed fungal infections in all four recipients. This study provides, for the first time, insights into a combination therapy with anti-CD40/anti-CD40L antibodies to block this immune checkpoint.

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INTRODUCTION: Inflammatory responses and coagulation disorders are a relevant challenge for successful cardiac xenotransplantation on its way to the clinic. To cope with this, an effective and clinically practicable anti-inflammatory and anti-coagulatory regimen is needed. The inflammatory and coagulatory response can be reduced by genetic engineering of the organ-source pigs. Furthermore, there are several therapeutic strategies to prevent or reduce inflammatory responses and coagulation disorders following xenotransplantation. However, it is still unclear, which combination of drugs should be used in the clinical setting. To elucidate this, we present data from pig-to-baboon orthotopic cardiac xenotransplantation experiments using a combination of several anti-inflammatory drugs. METHODS: Genetically modified piglets (GGTA1-KO, hCD46/hTBM transgenic) were used for orthotopic cardiac xenotransplantation into captive-bred baboons (n = 14). All animals received an anti-inflammatory drug therapy including a C1 esterase inhibitor, an IL-6 receptor antagonist, a TNF-α inhibitor, and an IL-1 receptor antagonist. As an additive medication, acetylsalicylic acid and unfractionated heparin were administered. The immunosuppressive regimen was based on CD40/CD40L co-stimulation blockade. During the experiments, leukocyte counts, levels of C-reactive protein (CRP) as well as systemic cytokine and chemokine levels and coagulation parameters were assessed at multiple timepoints. Four animals were excluded from further data analyses due to porcine cytomegalovirus/porcine roseolovirus (PCMV/PRV) infections (n = 2) or technical failures (n = 2). RESULTS: Leukocyte counts showed a relevant perioperative decrease, CRP levels an increase. In the postoperative period, leukocyte counts remained consistently within normal ranges, CRP levels showed three further peaks after about 35, 50, and 80 postoperative days. Analyses of cytokines and chemokines revealed different patterns. Some cytokines, like IL-8, increased about 2-fold in the perioperative period, but then decreased to levels comparable to the preoperative values or even lower. Other cytokines, such as IL-12/IL-23, decreased in the perioperative period and stayed at these levels. Besides perioperative decreases, there were no relevant alterations observed in coagulation parameters. In summary, all parameters showed an unremarkable course with regard to inflammatory responses and coagulation disorders following cardiac xenotransplantation and thus showed the effectiveness of our approach. CONCLUSION: Our preclinical experience with the anti-inflammatory drug therapy proved that controlling of inflammation and coagulation disorders in xenotransplantation is possible and well-practicable under the condition that transmission of pathogens, especially of PCMV/PRV to the recipient is prevented because PCMV/PRV also induces inflammation and coagulation disorders. Our anti-inflammatory regimen should also be applicable and effective in the clinical setting of cardiac xenotransplantation.

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Cardiac xenotransplantation has seen remarkable success in recent years and is emerging as the most promising alternative to human cardiac allotransplantation. Despite these achievements, acute vascular rejection still presents a challenge for long-term xenograft acceptance and new insights into innate and adaptive immune responses as well as detailed characterizations of signaling pathways are necessary. In allotransplantation, endothelial cells and their sugar-rich surface-the endothelial glycocalyx-are known to influence organ rejection. In xenotransplantation, however, only in vitro data exist on the role of the endothelial glycocalyx so far. Thus, in the current study, we analyzed the changes of the endothelial glycocalyx components hyaluronan, heparan sulfate and syndecan-1 after pig-to-baboon cardiac xenotransplantations in the perioperative (n = 4) and postoperative (n = 5) periods. These analyses provide first insights into changes of the endothelial glycocalyx after pig-to-baboon cardiac xenotransplantation and show that damage to the endothelial glycocalyx seems to be comparable or even less pronounced than in similar human settings when current strategies of cardiac xenotransplantation are applied. At the same time, data from the experiments where current strategies, like non-ischemic preservation, growth inhibition or porcine cytomegalovirus (a porcine roseolovirus (PCMV/PRV)) elimination could not be applied indicate that damage of the endothelial glycocalyx also plays an important role in cardiac xenotransplantation.

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This report comprises the contents of the presentations and following discussions of a workshop of the German Heart Transplant Centers in Martinsried, Germany on cardiac xenotransplantation. The production and current availability of genetically modified donor pigs, preservation techniques during organ harvesting, and immunosuppressive regimens in the recipient are described. Selection criteria for suitable patients and possible solutions to the problem of overgrowth of the xenotransplant are discussed. Obviously microbiological safety for the recipient and close contacts is essential, and ethical considerations to gain public acceptance for clinical applications are addressed. The first clinical trial will be regulated and supervised by the Paul-Ehrlich-Institute as the National Competent Authority for Germany, and the German Heart Transplant Centers agreed to cooperatively select the first patients for cardiac xenotransplantation.

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BACKGROUND: Using pigs as organ donors has advanced xenotransplantation to the point that it is almost ready for clinical use. However, there is still a zoonotic risk associated with xenotransplantation, and the potential transmission of porcine endogenous retroviruses needs to be surveyed. Despite significant attempts to eliminate this risk, by the selection of PERV-C free pigs with low expression of PERV-A, -B, and by the genome-wide inactivation of PERV using CRISPR/Cas9, the impact of superinfection resistance (SIR) was not investigated. SIR is a viral trait that prevents reinfection (superinfection). For PERV, the underlying mechanism is unclear, whether and how cells, that harbor functional PERV, are protected. Using PERV-C(5683) as a reference virus, we investigated SIR in a newly developed in vitro model to pursue the mechanism and confirm its protective effect. RESULTS: We developed three PERV-C constructs on the basis of PERV-C(5683), each of which carries a hemagglutinin tag (HA-tag) at a different position of the envelope gene (SP-HA, HA-VRA, and RPep-HA), to distinguish between primary infection and superinfection. The newly generated PERV-C(5683)-HA viruses were characterized while quantifying the viral RNA, reverse transcriptase activity, protein expression analysis, and infection studies. It was demonstrated that SP-HA and RPep-HA were comparable to PERV-C(5683), whereas HA-VRA was not replication competent. SP-HA and RPep-HA were chosen to challenge PERV-C(5683)-positive ST-IOWA cells demonstrating that PERV-C-HA viruses are not able to superinfect those cells. They do not integrate into the genome and are not expressed. CONCLUSIONS: The mechanism of SIR applies to PERV-C. The production of PERV-C particles serves as a defense mechanism from superinfection with exogenous PERV-C. It was demonstrated by newly generated PERV-C(5683)-HA clones that might be used as a cutting-edge tool. The HA-tagging of PERV-C is novel, providing a blueprint for the tagging of other human tropic PERV viruses. The tagged viruses are suitable for additional in vitro and in vivo infection studies and will contribute, to basic research on viral invasion and pathogenesis. It will maintain the virus safety of XTx.

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We study the transition to synchronization in large, dense networks of chaotic circle maps, where an exact solution of the mean-field dynamics in the infinite network and all-to-all coupling limit is known. In dense networks of finite size and link probability of smaller than one, the incoherent state is meta-stable for coupling strengths that are larger than the mean-field critical coupling. We observe chaotic transients with exponentially distributed escape times and study the scaling behavior of the mean time to synchronization.

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Porcine cytomegalovirus (PCMV) is widely distributed in pigs and difficult to detect due to latency. PCMV infection of source pigs was associated with early graft failure after cardiac and renal xenotransplantation into nonhuman primates. Importantly, PCMV infection of the first genetically modified pig heart into a human may have contributed to the reduced survival of the patient. Sensitive and reliable assays for detection of latent PCMV infection are thus indispensable. Here, we report the development of five peptide-induced rabbit antisera specific for PCMV glycoprotein B (gB) and their validation for detection of PCMV in infected pig fallopian tube (PFT) cells by immunofluorescence and electron microscopy (EM). The anti-gB antibodies were also used for detection by Western blot analysis of PCMV purified from the supernatant of infected PFT cells. Sera of infected versus non-infected pigs have been compared. In parallel, PCMV viral load in blood samples of the animals was quantified by a novel highly sensitive nested-PCR and qPCR assay. A combination of four partly overlapping peptides from the gB C-terminus was used to establish a diagnostic ELISA for PCMV gB specific pig antibodies which is able to differentiate infected from non-infected animals and to quantify maternal antibodies in neonates. The combination of a highly sensitive nested PCR for direct virus detection with a sensitive peptide-based ELISA detecting anti-PCMV gB-antibodies, supplemented by Western blot analysis and/or immunohistochemistry for virus detection will reliably differentiate pigs with active infection, latently infected pigs, and non-infected pigs. It may significantly improve the virologic safety of xenotransplantation.

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Xenotransplantation may compensate the limited number of human allografts for transplantation using pigs as organ donors. Porcine endogenous retroviruses inherit infectious potential if pig cells, tissues, or organs were transplanted to immunosuppressed human recipients. Particularly, ecotropic PERV-C that could recombine with PERV-A to highly replication-competent human-tropic PERV-A/C should be excluded from pig breeds designed for xenotransplantation. Because of their low proviral background, SLAD/D (SLA, swine leukocyte antigen) haplotype pigs are potential candidates as organ donors as they do not bear replication-competent PERV-A and -B, even if they carry PERV-C. In this work, we characterized their PERV-C background isolating a full-length PERV-C proviral clone number 561 from a SLAD/D haplotype pig genome displayed in a bacteriophage lambda library. The provirus truncated in env due to cloning in lambda was complemented by PCR, and the recombinants were functionally characterized, confirming an increased infectivity in vitro compared to other PERV-C. Recombinant clone PERV-C(561) was chromosomally mapped by its 5'-proviral flanking sequences. Full-length PCR using 5'-and 3'-flanking primers specific to the PERV-C(561) locus verified that this specific SLAD/D haplotype pig harbors at least one full-length PERV-C provirus. The chromosomal location is different from that of the previously described PERV-C(1312) provirus, which was derived from the porcine cell-line MAX-T. The sequence data presented here provide further knowledge about PERV-C infectivity and contribute to targeted knockout in order to generate PERV-C-free founder animals. IMPORTANCE Yucatan SLAD/D haplotype miniature swine are candidates as organ donors for xenotransplantation. A full-length replication-competent PERV-C provirus was characterized. The provirus was chromosomally mapped in the pig genome. In vitro, the virus showed increased infectivity compared to other functional PERV-C isolates. Data may be used for targeted knockout to generate PERV-C free founder animals.

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For many patients with terminal/advanced cardiac failure, heart transplantation is the most effective, durable treatment option, and offers the best prospects for a high quality of life. The number of potentially life-saving donated human organs is far fewer than the population who could benefit from a new heart, resulting in increasing numbers of patients awaiting replacement of their failing heart, high waitlist mortality, and frequent reliance on interim mechanical support for many of those deemed among the best candidates but who are deteriorating as they wait. Currently, mechanical assist devices supporting left ventricular or biventricular heart function are the only alternative to heart transplant that is in clinical use. Unfortunately, the complication rate with mechanical assistance remains high despite advances in device design and patient selection and management, and the quality of life of the patients even with good outcomes is only moderately improved. Cardiac xenotransplantation from genetically multi-modified (GM) organ-source pigs is an emerging new option as demonstrated by the consistent long-term success of heterotopic (non-life-supporting) abdominal and life-supporting orthotopic porcine heart transplantation in baboons, and by a recent 'compassionate use' transplant of the heart from a GM pig with 10 modifications into a terminally ill patient who survived for 2 months. In this review, we discuss pig heart xenotransplantation as a concept, including pathobiological aspects related to immune rejection, coagulation dysregulation, and detrimental overgrowth of the heart, as well as GM strategies in pigs to prevent or minimize these problems. Additional topics discussed include relevant results of heterotopic and orthotopic heart transplantation experiments in the pig-to-baboon model, microbiological and virologic safety concepts, and efficacy requirements for initiating formal clinical trials. An adequate regulatory and ethical framework as well as stringent criteria for the selection of patients will be critical for the safe clinical development of cardiac xenotransplantation, which we expect will be clinically tested during the next few years.

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We formulate a linear phase and frequency response theory for hyperbolic flows, which generalizes phase response theory for autonomous limit cycle oscillators to hyperbolic chaotic dynamics. The theory is based on a shadowing conjecture, stating the existence of a perturbed trajectory shadowing every unperturbed trajectory on the system attractor for any small enough perturbation of arbitrary duration and a corresponding unique time isomorphism, which we identify as phase such that phase shifts between the unperturbed trajectory and its perturbed shadow are well defined. The phase sensitivity function is the solution of an adjoint linear equation and can be used to estimate the average change of phase velocity to small time dependent or independent perturbations. These changes in frequency are experimentally accessible, giving a convenient way to define and measure phase response curves for chaotic oscillators. The shadowing trajectory and the phase can be constructed explicitly in the tangent space of an unperturbed trajectory using co-variant Lyapunov vectors. It can also be used to identify the limits of the regime of linear response.

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INTRODUCTION: Porcine endogenous retroviruses (PERVs) are an integral part of the pig genome with infectious potential, as shown in vitro. HYPOTHESIS/GAP STATEMENT: In view of nonclinical and clinical xenotransplantation, data are essential that give an insight into viral pathogenicity. This includes PERV's environmental stability and environmental risk. AIM: We analyzed two ecotropic PERV-C (PERV-C[1312] and -[5683]), monitoring cell-free culture supernatants of infected ST-IOWA cells at various time intervals at room temperature (22°C +/-1°C). The virus was stored in the presence or absence of sterile wood litter, as used for large animal husbandry. This approach was set to determine the environmental stability of exogenous PERV-C at defined conditions for the first time. METHODOLOGY: Reverse transcriptase (RT) activity and viral RNA were monitored for up to 57 days and remaining infectivity of supernatant without wood litter was tested from day 7 onwards on naïve ST-IOWA cells. RESULTS: Results show that viral RNA decreases but remains detectable over the whole observation period, whereas RT activity showed 83%-96% reduction from day 7 on. This effect was stronger in the presence of wood litter and fresh harvested virus was more stable than frozen virus stocks. Even under these optimal conditions, no infectivity was shown for viral RNA-positive and RT-reduced supernatant harvested at day 7. CONCLUSION: The results confirm that PERV-C is less stable and the reduction of RT activity is accompanied by reduced infectivity, independently of existing viral RNA. The combination of both RT and viral RNA measurement is a suitable method to differentiate infectious PERV-C.

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We report on the effect of spatially correlated noise on the velocities of self-propelled particles. Correlations in the random forces acting on self-propelled particles can induce directed collective motion, i.e., swarming. Even with repulsive coupling in the velocity directions, which favors a disordered state, strong correlations in the fluctuations can align the velocities locally leading to a macroscopic, turbulent velocity field. On the other hand, while spatially correlated noise is aligning the velocities locally, the swarming transition to globally directed motion is inhibited when the correlation length of the noise is nonzero, but smaller than the system size. We analyze the swarming transition in d-dimensional space in a mean field model of globally coupled velocity vectors.

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Due to the rapid development of the Internet of Things (IoT) and consequently, the availability of more and more IoT data sources, mechanisms for searching and integrating IoT data sources become essential to leverage all relevant data for improving processes and services. This paper presents the IoT search framework IoTCrawler. The IoTCrawler framework is not only another IoT framework, it is a system of systems which connects existing solutions to offer interoperability and to overcome data fragmentation. In addition to its domain-independent design, IoTCrawler features a layered approach, offering solutions for crawling, indexing and searching IoT data sources, while ensuring privacy and security, adaptivity and reliability. The concept is proven by addressing a list of requirements defined for searching the IoT and an extensive evaluation. In addition, real world use cases showcase the applicability of the framework and provide examples of how it can be instantiated for new scenarios.

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Complex networks are abundant in nature and many share an important structural property: they contain a few nodes that are abnormally highly connected (hubs). Some of these hubs are called influencers because they couple strongly to the network and play fundamental dynamical and structural roles. Strikingly, despite the abundance of networks with influencers, little is known about their response to stochastic forcing. Here, for oscillatory dynamics on influencer networks, we show that subjecting influencers to an optimal intensity of noise can result in enhanced network synchronization. This new network dynamical effect, which we call coherence resonance in influencer networks, emerges from a synergy between network structure and stochasticity and is highly nonlinear, vanishing when the noise is too weak or too strong. Our results reveal that the influencer backbone can sharply increase the dynamical response in complex systems of coupled oscillators.

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We study ensembles of globally coupled or forced identical phase oscillators subject to independent white Cauchy noise. We demonstrate that if the oscillators are forced in several harmonics, stationary synchronous regimes can be exactly described with a finite number of complex order parameters. The corresponding distribution of phases is a product of wrapped Cauchy distributions. For sinusoidal forcing, the Ott-Antonsen low-dimensional reduction is recovered.

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BACKGROUND: The demand for donated human hearts far exceeds the number available. Xenotransplantation of genetically modified porcine organs provides an alternative. In 2000, an Advisory Board of the International Society for Heart and Lung Transplantation set the benchmark for commencing clinical cardiac xenotransplantation as consistent 60% survival of non-human primates after life-supporting porcine heart transplantations. Recently, we reported the stepwise optimization of pig-to-baboon orthotopic cardiac xenotransplantation finally resulting in consistent success, with 4 recipients surviving 90 (n = 2), 182, and 195 days. Here, we report on 4 additional recipients, supporting the efficacy of our procedure. RESULTS: The first 2 additional recipients succumbed to porcine cytomegalovirus (PCMV) infections on Days 15 and 27, respectively. In 2 further experiments, PCMV infections were successfully avoided, and 3-months survival was achieved. Throughout all the long-term experiments, heart, liver, and renal functions remained within normal ranges. Post-mortem cardiac diameters were slightly increased when compared with that at the time of transplantation but with no detrimental effect. There were no signs of thrombotic microangiopathy. The current regimen enabled the prolonged survival and function of orthotopic cardiac xenografts in altogether 6 of 8 baboons, of which 4 were now added. These results exceed the threshold set by the Advisory Board of the International Society for Heart and Lung Transplantation. CONCLUSIONS: The results of our current and previous experimental cardiac xenotransplantations together fulfill for the first time the pre-clinical efficacy suggestions. PCMV-positive donor animals must be avoided.

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BACKGROUND: The lack of human donors for allotransplantation forces the development of other strategies to circumvent the existing organ shortage documented on the waiting lists. Here, xenotransplantation offers a suitable option since the genetic modification of animals has become an established method that allows the generation of animals as donors of cells, tissues, and organs with reduced antigenicity. METHODS: Focus is given on the generation of decellularized matrix scaffolds, for example, for valve transplantation and/or repair, that have the potential being fully assimilated by the recipient as they are no longer a mechanical implant with risk of calcification and related failure. RESULTS: This new class of products is transplants that will be regulated either as medical devices or as cell-based medicinal products, that is, advanced therapy medicinal products, according to the regulations in the European Union. CONCLUSIONS: In this review, we compile relevant regulatory aspects and point out the possibilities of how these products for human use may be regulated in the future.

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BACKGROUND: Decellularized human pulmonary heart valve (dhHV) scaffolds have been shown to be the gold standard especially for younger, adolescent patients. However, human heart valves are limited in availability. Xenogeneic decellularized pig heart valves (dpHV) may serve as alternative. METHODS: The efficacy of DNA reduction processes upon decellularization of heart valves from German Landrace pigs was analyzed by measurements of remaining nucleic acids including proviral porcine endogenous retrovirus (PERV) sequences. Porcine pulmonary heart valves (pPHV) were decellularized by three different protocols and further treated with DNaseI or Benzonase, at varying incubation times. DNA isolated from valve associated muscle (m), valve cusp (c), and pulmonary artery (pa) was monitored by PCR and qRT-PCR using GAPDH and the PERV polymerase (pol) for read-out. RESULTS: Decellularization of pPHV led to a significant reduction of DNA (>99%) which could be further significantly increased for (m) and (pa) by nuclease treatment, reducing proviral PERV pol from approximately 5 × 107 to 5 × 103  copies/mg in nuclease treated tissues. CONCLUSIONS: Both nucleases demonstrated comparable activities. But DNaseI revealed to be less consistent for PERV, especially at muscular tissue. Noteworthy, remaining proviral sequences are still detectable by PCR; however, due to the absence of the cellular replication machinery the production of infectious particles is not expected. Decellularization and nuclease treatment of pPHV is an efficient procedure to reduce the DNA content including PERV, thus represents a valuable option to increase virus safety independently from the source animal background.

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The infection of human transplant recipients by porcine endogenous retrovirus (PERV) is a safety issue for xenotransplantation (XTx). CRISPR/Cas9 technology has enabled the generation of pigs free of functional PERVs, and the susceptibility of these animals to reinfection by PERVs remains unclear. To assess virological safety, we characterized a cell line in which PERVs have been inactivated by CRISPR/Cas9 (PK15 clone 15) for its susceptibility to infectious PERV. First, basal expression of PERV pol, the porcine PERV-A receptor (POPAR), and reverse transcriptase (RT) activity of PERV were determined. PK15 clone 15 cells were inoculated with PERV and monitored post infection for virus expression and RT activity. Particles were visualized by electron microscopy. Our data show that PK15 clone 15 cells still produce viral proteins that assemble to produce impaired viral particles. These virions have an irregular morphology that diverges from that of mature wild type. The particles are no longer infectious when tested in a downstream infection assay using supernatants of PK15 clone 15 cells to infect susceptible swine testis-IOWA (ST-IOWA) cells. The expression of POPAR was quantified to exclude the possibility that lack of susceptibility to reinfection, for PERV-A, is caused by absence of viral host receptor(s). PK15 and PK15 clone 15 cells do, in fact, express POPAR equally. PERV RT inactivation mediated by CRISPR/Cas9 does not compromise virus assembly but affects virion structure and proviral integration. The constitutive virion production seems to maintain cellular resistance to superinfection and possibly indicates a protective side effect of this specific CRISPR/Cas9 mediated RT inactivation.

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