Graft dysfunction in compassionate use of genetically engineered pig-to-human cardiac xenotransplantation: a case report.

BACKGROUND: A genetically engineered pig cardiac xenotransplantation was done on Jan 7, 2022, in a non-ambulatory male patient, aged 57 years, with end-stage heart failure, and on veno-arterial extracorporeal membrane oxygenation support, who was ineligible for an allograft. This report details our current understanding of factors important to the xenotransplantation outcome. METHODS: Physiological and biochemical parameters critical for the care of all heart transplant recipients were collected in extensive clinical monitoring in an intensive care unit. To ascertain the cause of xenograft dysfunction, we did extensive immunological and histopathological studies, including electron microscopy and quantification of porcine cytomegalovirus or porcine roseolovirus (PCMV/PRV) in the xenograft, recipient cells, and tissue by DNA PCR and RNA transcription. We performed intravenous immunoglobulin (IVIG) binding to donor cells and single-cell RNA sequencing of peripheral blood mononuclear cells. FINDINGS: After successful xenotransplantation, the graft functioned well on echocardiography and sustained cardiovascular and other organ systems functions until postoperative day 47 when diastolic heart failure occurred. At postoperative day 50, the endomyocardial biopsy revealed damaged capillaries with interstitial oedema, red cell extravasation, rare thrombotic microangiopathy, and complement deposition. Increased anti-pig xenoantibodies, mainly IgG, were detected after IVIG administration for hypogammaglobulinaemia and during the first plasma exchange. Endomyocardial biopsy on postoperative day 56 showed fibrotic changes consistent with progressive myocardial stiffness. Microbial cell-free DNA testing indicated increasing titres of PCMV/PRV cell-free DNA. Post-mortem single-cell RNA sequencing showed overlapping causes. INTERPRETATION: Hyperacute rejection was avoided. We identified potential mediators of the observed endothelial injury. First, widespread endothelial injury indicates antibody-mediated rejection. Second, IVIG bound strongly to donor endothelium, possibly causing immune activation. Finally, reactivation and replication of latent PCMV/PRV in the xenograft possibly initiated a damaging inflammatory response. The findings point to specific measures to improve xenotransplant outcomes in the future. FUNDING: The University of Maryland School of Medicine, and the University of Maryland Medical Center.

Large-gap peripheral nerve repair using xenogeneic transplants in rhesus macaques.

Surgical intervention is required to successfully treat severe, large-gap (≥4 cm) peripheral nerve injuries. However, all existing treatments have shortcomings and an alternative to the use of autologous nerves is needed. Human and porcine nerves are physiologically similar, with comparable dimensions and architecture, presence and distribution of Schwann cells, and conserved features of the extracellular matrix (ECM). We report the repair of fully transected radial nerves in 10 Rhesus Macaques using viable, whole sciatic nerve from genetically engineered (GalT-KO), designated pathogen free (DPF) porcine donors. This resulted in the regeneration of the transected nerve, and importantly, recovery of wrist extension function, distal muscle reinnervation, and recovery of nerve conduction velocities and compound muscle action potentials similar to autologous controls. We also demonstrate the absence of immune rejection, systemic porcine cell migration, and detectable residual porcine material. Our preliminary findings support the safety and efficacy of viable porcine nerve transplants, suggest the interchangeable therapeutic use of cross-species cells, and highlight the broader clinical potential of xenotransplantation.

Organ transplants of the future: planning for innovations including xenotransplantation.

The future clinical application of animal-to-human transplantation (xenotransplantation) is of importance to society as a whole. Favourable preclinical data relevant to cell, tissue and solid organ xenotransplants have been obtained from many animal models utilizing genetic engineering and protocols of pathogen-free husbandry. Findings have reached a tipping point, and xenotransplantation of solid organs is approaching clinical evaluation, the process of which now requires close deliberation. Such discussions include considering when there is sufficient evidence from preclinical animal studies to start first-in-human xenotransplantation trials. The present article is based on evidence and opinions formulated by members of the European Society for Organ Transplantation who are involved in the Transplantation Learning Journey project. The article includes a brief overview of preclinical concepts and biology of solid organ xenotransplantation, discusses the selection of candidates for first-in-human studies and considers requirements for study design and conduct. In addition, the paper emphasizes the need for a regulatory framework for xenotransplantation of solid organs and the essential requirement for input from public and patient stakeholders.

Immunological response in cynomolgus macaques to porcine α-1,3 galactosyltransferase knockout viable skin xenotransplants-A pre-clinical study.

BACKGROUND: Allogeneic skin recovered from human deceased donors (HDD) has been a mainstay interim treatment for severe burns, but unfortunately risk of infectious disease and availability limitations exist. Genetically engineered ɑ-1,3 galactosyltransferase knockout (GalT-KO) porcine source animals for viable skin xenotransplants may provide a promising clinical alternative. METHODS: Four cynomolgus macaque recipients received full-thickness surgical wounds to model the defects arising from excision of full-thickness burn injury and were treated with biologically active skin xenotransplants derived from GalT-KO, Designated Pathogen Free (DPF) miniature swine. Evaluations were conducted for safety, tolerability, and recipient immunological response. RESULTS: All skin xenotransplants demonstrated prolonged survival, vascularity, and persistent dermal adhesion until the study endpoint at post-operative day 30. No adverse outcomes were observed during the study. Varying levels of epidermolysis coincided with histologic detection of CD4+ and CD8+ T cells, and other cellular infiltrates in the epidermis. Recipient sera IgM and IgG demonstrated significant antibody immune response to non-α-1,3-galactose porcine xenoantigens. Separately, specific wound healing mediators were quantified. Neither porcine cell migration nor PERV were detected in circulation or any visceral organs. CONCLUSIONS: These results provide a detailed analysis of vital skin xenotransplants utilizing a non-human primate model to predict the anticipated immunological response of human patients. The lack of adverse rejection even in the presence of elevated Ig indicates this is a prospective therapeutic option. The findings reported here directly supported regulatory clearance for a first-in-man, Phase I xenotransplantation clinical trial.

Examining the potential for porcine-derived islet cells to harbour viral pathogens.

With an onus on safety in the potential use of porcine islet cells as a treatment for diabetes, the use of animals lacking exogenous pathogens is clearly important and multilevel screening strategies have been presented on testing animals and the product. In this study, we wished to investigate whether islet cells indeed harboured the same viral pathogens of concern in the source animal. PMBC and islet cells from both adult and neonatal source animals were directly compared and tested for PCMV, PLHV, PCV2, PPV and HEV using both molecular and serological assays. Adult PBMC were found positive for all viruses with the exception of PCV2 and HEV. Neonatal PBMC were only found positive for PCMV and HEV. All animals were found negative for HEV antibodies. Interestingly, islet cells were negative for all viruses tested regardless of status in the animal-derived PBMC. Given that other laboratories have demonstrated the lack of virus detection during the culture of islets, this study also demonstrates that the hygiene status of the herd may not reflect the status of the product. This is important for establishing guidelines for any risk evaluation and mitigation process utilised during product manufacture.

Assessment of porcine endogenous retrovirus transmission across an alginate barrier used for the encapsulation of porcine islets.

BACKGROUND: Subcutaneous implantation of a macroencapsulated patch containing human allogenic islets has been successfully used to alleviate type 1 diabetes mellitus (T1DM) in a human recipient without the need for immunosuppression. The use of encapsulated porcine islets to treat T1DM has also been reported. Although no evidence of pathogen transfer using this technology has been reported to date, we deemed it appropriate to determine if the encapsulation technology would prevent the release of virus, in particular, the porcine endogenous retrovirus (PERV). METHODS: HEK293 (human epithelial kidney) and swine testis (ST) cells were co-cultured with macroencapsulated pig islets embedded in an alginate patch, macroencapsulated PK15 (swine kidney epithelial) cells embedded in an alginate patch and free PK15 cells. Cells and supernatant were harvested at weekly time points from the cultures for up to 60 days and screened for evidence of PERV release using qRT-PCR to detect PERV RNA and SG-PERT to detect reverse transcriptase (RT). RESULTS: No PERV virus, or evidence of PERV replication, was detected in the culture medium of HEK293 or pig cells cultured with encapsulated porcine islets. Increased PERV activity relative to the background was not detected in ST cells cultured with encapsulated PK15 cells. However, PERV was detected in 1 of the 3 experimental replicates of HEK293 cells cultured with encapsulated PK15 cells. Both HEK293 and ST cells cultured with free PK15 cells showed an increase in RT detection. CONCLUSIONS: With the exception of 1 replicate, there does not appear to be evidence of transmission of replication competent PERV from the encapsulated islet cells or the positive control PK15 cells across the alginate barrier. The detection of PERV would suggest the alginate barrier of this replicate may have become compromised, emphasizing the importance of quality control when producing encapsulated islet patches.

Selective CD28 Antagonist Blunts Memory Immune Responses and Promotes Long-Term Control of Skin Inflammation in Nonhuman Primates.

Novel therapies that specifically target activation and expansion of pathogenic immune cell subsets responsible for autoimmune attacks are needed to confer long-term remission. Pathogenic cells in autoimmunity include memory T lymphocytes that are long-lived and present rapid recall effector functions with reduced activation requirements. Whereas the CD28 costimulation pathway predominantly controls priming of naive T cells and hence generation of adaptive memory cells, the roles of CD28 costimulation on established memory T lymphocytes and the recall of memory responses remain controversial. In contrast to CD80/86 antagonists (CTLA4-Ig), selective CD28 antagonists blunt T cell costimulation while sparing CTLA-4 and PD-L1-dependent coinhibitory signals. Using a new selective CD28 antagonist, we showed that Ag-specific reactivation of human memory T lymphocytes was prevented. Selective CD28 blockade controlled both cellular and humoral memory recall in nonhuman primates and induced long-term Ag-specific unresponsiveness in a memory T cell-mediated inflammatory skin model. No modification of memory T lymphocytes subsets or numbers was observed in the periphery, and importantly no significant reactivation of quiescent viruses was noticed. These findings indicate that pathogenic memory T cell responses are controlled by both CD28 and CTLA-4/PD-L1 cosignals in vivo and that selectively targeting CD28 would help to promote remission of autoimmune diseases and control chronic inflammation.

Hepatitis E virus (HEV) in Scotland: evidence of recent increase in viral circulation in humans.

BackgroundPrevious studies showed low levels of circulating hepatitis E virus (HEV) in Scotland. We aimed to reassess current Scottish HEV epidemiology. Methods: Blood donor samples from five Scottish blood centres, the minipools for routine HEV screening and liver transplant recipients were tested for HEV antibodies and RNA to determine seroprevalence and viraemia. Blood donor data were compared with results from previous studies covering 2004-08. Notified laboratory-confirmed hepatitis E cases (2009-16) were extracted from national surveillance data. Viraemic samples from blood donors (2016) and chronic hepatitis E transplant patients (2014-16) were sequenced. Results: Anti-HEV IgG seroprevalence varied geographically and was highest in Edinburgh where it increased from 4.5% in 2004-08) to 9.3% in 2014-15 (p = 0.001). It was most marked in donors < 35 years. HEV RNA was found in 1:2,481 donors, compared with 1:14,520 in 2011. Notified laboratory-confirmed cases increased by a factor of 15 between 2011 and 2016, from 13 to 206. In 2011-13, 1 of 329 transplant recipients tested positive for acute HEV, compared with six cases of chronic infection during 2014-16. Of 10 sequenced viraemic donors eight and all six patients were infected with genotype 3 clade 1 virus, common in European pigs. Conclusions: The seroprevalence, number of viraemic donors and numbers of notified laboratory-confirmed cases of HEV in Scotland have all recently increased. The causes of this change are unknown, but need further investigation. Clinicians in Scotland, particularly those caring for immunocompromised patients, should have a low threshold for testing for HEV.

Detection of viral hepatitis E in clinical liver biopsies.

AIMS: To determine the relative utility of in-situ testing for hepatitis E virus (HEV) RNA and paraffin-section polymerase chain reaction (PCR) to diagnose HEV infection in paraffin-embedded clinical liver biopsies, and to correlate with clinicopathological characteristics. METHODS AND RESULTS: We evaluated in-situ and quantitative PCR (qPCR)-based approaches to identifying HEV in clinical liver biopsies from infected patients from multiple centres, correlating with clinical setting (immunocompetent, allograft or immunosuppressed native liver) and histological findings. Thirty-six biopsies from 29 patients had histological data, 27 and 23 of which had satisfactory material for in-situ RNA testing and tissue qPCR, respectively. Both approaches specifically identified HEV infection, but tissue qPCR was significantly more sensitive than RNAscope in-situ testing (P = 0.035). In immunocompetent but not immunosuppressed patients the tissue qPCR yield correlated with the severity of lobular hepatitis (rho = 0.94, P < 0.001). qPCR viral yield was comparably high in allografts and immunosuppressed native livers and significantly greater than with native liver infection. Immunosuppressed patients showed reduced severity of hepatitis and cholestatic changes, compared with immunocompetent patients. Indeed, HEV-infected liver allografts could show minimal hepatitis for many months. In individual cases each technique was useful when serum was not available to identify chronic infection retrospectively (in biopsies taken 4-31 months before diagnosis), to identify persistent/residual infection when contemporary serum PCR was negative and to identify cleared infection. CONCLUSIONS: qPCR is more effective than in-situ RNA testing to identify HEV infection in paraffin-embedded liver biopsies and has diagnostic utility in selected settings.

Characterization of porcine endogenous retrovirus expression in neonatal and adult pig pancreatic islets.

BACKGROUND: Pig islets represent an alternative to the current modes of treatment for patients with diabetes. However, the concerns over pathogen transmission including that of PERV limit their immediate, widespread usage in humans. It has been previously demonstrated that PERV copy number and particularly expression levels can vary considerably between individuals and within different tissues of a single animal. In general, expression levels have been found to be particularly low in the pancreas compared to other porcine tissues suggesting a reduced risk associated with the use of this tissue. Data regarding this crucial aspect, however, remain limited and little is known about PERV status of islets themselves, which represent the final product to be transplanted. In addition, comparative analysis of the PERV status of neonatal piglets with adults is important as they are increasingly considered as potential islet donors for xenotransplantation. METHODS: Tissue samples from 51 neonatal piglets (age between 14 and 21 days) and 29 adult pigs were collected from Belgian landrace pigs used for pancreas procurement and islet isolation. Tissue biopsies were used to extract DNA for PERV copy number quantification by qPCR and RNA for PERV expression by qRT-PCR. RESULTS: As expected, PERV expression demonstrated great variation and was significantly lower in pancreas compared to other tissues. More importantly, PERV RNA expression was found to be specifically enriched in pancreatic islets reaching values similar to those found in other tissues such as liver and kidney. Interestingly, this expression was not coupled with the detection of reverse transcriptase in islet cultures or indeed detection of PERV virus. Lung, spleen, and lymph node consistently showed the highest levels of PERV expression. Comparison of PERV in neonatal and adult pigs showed that copy number did not vary significantly from birth to adulthood. PERV expression on the other hand was significantly lower in neonatal pig islets compared to adult islets and did not increase over the period of culture. CONCLUSION: Our study confirms the low level of PERV expression in whole pancreas in a large population of both neonatal and adult pigs (n=80). The level of PERV expression was however higher in the endocrine tissue than in the exocrine cells. There was no correlation between PERV status in donor PBMCs and islet cells, and no evidence of active replication in in vitro regardless of PERV expression in islet cells. Moreover, neonatal pig islets were found to have significantly lower PERV expression compared to adult islets. Neonatal islets have been suggested as the best choice for xenotransplantation in terms of economic and procurement considerations; the PERV status reported here would also potentially support their use.

Selective blockade of CD28-mediated T cell costimulation protects rhesus monkeys against acute fatal experimental autoimmune encephalomyelitis.

Costimulatory and coinhibitory receptor-ligand pairs on T cells and APC control the immune response. We have investigated whether selective blockade of CD28-CD80/86 costimulatory interactions, which preserves the coinhibitory CTLA4-CD80/86 interactions and the function of regulatory T (Treg) cells, abrogates the induction of experimental autoimmune encephalomyelitis (EAE) in rhesus monkeys. EAE was induced by intracutaneous immunization with recombinant human myelin oligodendrocyte glycoprotein (rhMOG) in CFA on day 0. FR104 is a monovalent, PEGylated-humanized Fab' Ab fragment against human CD28, cross-reactive with rhesus monkey CD28. FR104 or placebo was administered on days 0, 7, 14, and 21. FR104 levels remained high until the end of the study (day 42). Placebo-treated animals all developed clinical EAE between days 12 and 27. FR104-treated animals did not develop clinical EAE and were sacrificed at the end of the study resulting in a significantly prolonged survival. FR104 treatment diminished T and B cell responses against rhMOG, significantly reduced CNS inflammation and prevented demyelination. The inflammatory profile in the cerebrospinal fluid and brain material was also strongly reduced. Recrudescence of latent virus was investigated in blood, spleen, and brain. No differences between groups were observed for the ß-herpesvirus CMV and the polyomaviruses SV40 and SA12. Cross-sectional measurement of lymphocryptovirus, the rhesus monkey EBV, demonstrated elevated levels in the blood of FR104-treated animals. Blocking rhesus monkey CD28 with FR104 mitigated autoreactive T and B cell activation and prevented CNS pathology in the rhMOG/CFA EAE model in rhesus monkeys.

First update of the International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes--Chapter 5: recipient monitoring and response plan for preventing disease transmission.

Xenotransplantation of porcine cells, tissues, and organs may be associated with the transmission of porcine microorganisms to the human recipient. A previous, 2009, version of this consensus statement focused on strategies to prevent transmission of porcine endogenous retroviruses (PERVs). This version addresses potential transmission of all porcine microorganisms including monitoring of the recipient and provides suggested approaches to the monitoring and prevention of disease transmission. Prior analyses assumed that most microorganisms other than the endogenous retroviruses could be eliminated from donor animals under appropriate conditions which have been called "designated pathogen-free" (DPF) source animal production. PERVs integrated as proviruses in the genome of all pigs cannot be eliminated in that manner and represent a unique risk. Certain microorganisms are by nature difficult to eliminate even under DPF conditions; any such clinically relevant microorganisms should be included in pig screening programs. With the use of porcine islets in clinical trials, special consideration has to be given to the presence of microorganisms in the isolated islet tissue to be used and also to the potential use of encapsulation. It is proposed that microorganisms absent in the donor animals by sensitive microbiological examination do not need to be monitored in the transplant recipient; this will reduce costs and screening requirements. Valid detection assays for donor and manufacturing-derived microorganisms must be established. Special consideration is needed to preempt potential unknown pathogens which may pose a risk to the recipient. This statement summarizes the main achievements in the field since 2009 and focus on issues and solutions with microorganisms other than PERV.

First update of the International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes--Chapter 2a: source pigs--preventing xenozoonoses.

Chapter 2 of the original consensus statement published in 2009 by IXA represents an excellent basis for the production of safe donor pigs and pig-derived materials for porcine islet xenotransplantation. It was intended that the consensus statement was to be reviewed at interval to remain relevant. Indeed, many of the original salient points remain relevant today, especially when porcine islet xenotransplantation is performed in conjunction with immunosuppressants. However, progress in the field including demonstrated safe clinical porcine xenograft studies, increased understanding of risks including those posed by PERV, and advancement of diagnostic capabilities now allow for further consideration. Agents of known and unknown pathogenic significance continue to be identified and should be considered on a geographic, risk-based, dynamic, and product-specific basis, where appropriate using validated, advanced diagnostic techniques. PERV risk can be sufficiently reduced via multicomponent profiling including subtype expression levels in combination with infectivity assays. Barrier facilities built and operated against the AAALAC Ag Guide or suitable alternative criteria should be considered for source animal production as long as cGMPs and SOPs are followed. Bovine material-free feed for source animals should be considered appropriate instead of mammalian free materials to sufficiently reduce TSE risks. Finally, the sponsor retention period for archival samples of donor materials was deemed sufficient until the death of the recipient if conclusively determined to be of unrelated and non-infectious cause or for a reasonable period, that is, five to 10 yrs. In summary, the safe and economical production of suitable pigs and porcine islet xenograft materials, under appropriate guidance and regulatory control, is believed to be a viable means of addressing the unmet need for clinical islet replacement materials.

Expression of recipient cytomegalovirus in immunosuppressed and xenotransplanted Macaca fascicularis may be related to more severe gastrointestinal lesions.

BACKGROUND: Xenotransplantation is a potential answer to the current organ shortage, but the risk of infections related to overimmunosuppression is an important parameter that may predict the recipient's long-term survival. Cytomegalovirus (CMV) in xenotransplanted and immunosuppressed primates is a well-known cause of disease particularly affecting the gastrointestinal (GI) tract and a zoonotic concern. METHODS: Post-mortem sera and tissues from 45 immunosuppressed and xenografted Macaca fascicularis were evaluated for CMV using antisera specific for the immediate early 1 (IE1), anti-RhCMV, and QPCR for virus. RESULTS: Serological analysis showed 100% positivity for the presence of CMV antibodies following the application of a specific test designed for RhCMV. Five of 45 primates showed typical lesions of CMV infection in the GI tract, including neutrophilic enteritis and inclusion bodies. Molecular analysis confirmed the presence of recipient's CMV in the tissues with CMV histopathology. Porcine CMV from the donor animals was not found in any of the CMV-specific IHC-positive recipients. CONCLUSION: The presence of active CMV infection in animals intended for xenograft experiments can lead to severe gastrointestinal lesions that could impact the overall aims of the study. In such cases, the animals should be investigated using appropriate (non-human primate-specific) diagnostic tools prior to use and treated aggressively with state-of-the-art antiviral therapy.

Long-term IgG response to porcine Neu5Gc antigens without transmission of PERV in burn patients treated with porcine skin xenografts.

Acellular materials of xenogenic origin are used worldwide as xenografts, and phase I trials of viable pig pancreatic islets are currently being performed. However, limited information is available on transmission of porcine endogenous retrovirus (PERV) after xenotransplantation and on the long-term immune response of recipients to xenoantigens. We analyzed the blood of burn patients who had received living pig-skin dressings for up to 8 wk for the presence of PERV as well as for the level and nature of their long term (maximum, 34 y) immune response against pig Ags. Although no evidence of PERV genomic material or anti-PERV Ab response was found, we observed a moderate increase in anti-αGal Abs and a high and sustained anti-non-αGal IgG response in those patients. Abs against the nonhuman sialic acid Neu5Gc constituted the anti-non-αGal response with the recognition pattern on a sialoglycan array differing from that of burn patients treated without pig skin. These data suggest that anti-Neu5Gc Abs represent a barrier for long-term acceptance of porcine xenografts. Because anti-Neu5Gc Abs can promote chronic inflammation, the long-term safety of living and acellular pig tissue implants in recipients warrants further evaluation.

Role of DNA methylation in expression and transmission of porcine endogenous retroviruses.

Porcine endogenous retroviruses (PERV) represent a major safety concern in pig-to-human xenotransplantation. To date, no PERV infection of a xenograft recipient has been recorded; however, PERVs are transmissible to human cells in vitro. Some recombinants of the A and C PERV subgroups are particularly efficient in infection and replication in human cells. Transcription of PERVs has been described in most pig cells, but their sequence and insertion polymorphism in the pig genome impede identification of transcriptionally active or silenced proviral copies. Furthermore, little is known about the epigenetic regulation of PERV transcription. Here, we report on the transcriptional suppression of PERV by DNA methylation in vitro and describe heavy methylation in the majority of PERV 5' long terminal repeats (LTR) in porcine tissues. In contrast, we have detected sparsely methylated or nonmethylated proviruses in the porcine PK15 cells, which express human cell-tropic PERVs. We also demonstrate the resistance of PERV DNA methylation to inhibitors of methylation and deacetylation. Finally, we show that the high permissiveness of various human cell lines to PERV infection coincides with the inability to efficiently silence the PERV proviruses by 5'LTR methylation. In conclusion, we suggest that DNA methylation is involved in PERV regulation, and that only a minor fraction of proviruses are responsible for the PERV RNA expression and porcine cell infectivity.

The International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of xenocorneal transplantation.

To develop an international consensus regarding the appropriate conditions for undertaking clinical trials in xenocorneal transplantation, here we review specific ethical, logistical, scientific, and regulatory issues regarding xenocorneal transplantation, and propose guidelines for conduct of clinical xenocorneal transplantation trials. These proposed guidelines are modeled on the published consensus statement of the International Xenotransplantation Association regarding recommended guidelines for conduct of clinical islet xenotransplantation. It is expected that this initial consensus statement will be revised over time in response to scientific advances in the field, and changes in the regulatory framework based on accumulating clinical experience.