quantification-circulating-pig-specific-dna-blood-xenotransplantation

Quantification of Circulating Pig-Specific DNA in the Blood of a Xenotransplantation Model

Xenotransplantation is a feasible method to treat organ failure. However, how to effectively monitor the immune rejection of xenotransplantation is a ...

Shenzhen University School of Medicine, First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital

Idiopathic azoospermia (IA) is a severe form of male infertility due to unknown causes. The HSF2 gene, encoding the heat shock transcription factor 2, had been suggested to play a significant role in the spermatogenesis process since the Hsf2-knockout male mice showed spermatogenesis defects. To examine whether HSF2 is involved in the pathogenesis of IA in human, we sequenced all the exons of HSF2 in 766 patients diagnosed with IA and 521 proven fertile men. A number of coding mutations private to the patient group, which include three synonymous mutations and five missense mutations, were identified. Of the missense mutations, our functional assay demonstrated that one heterozygous mutation, R502H, caused a complete loss of HSF2 function and that the mutant suppressed the normal function of the wild-type (WT) allele through a dominant-negative effect, thus leading to the dominant penetrance of the mutant allele. These results support a role for HSF2 in the pathogenesis of IA and further implicate this transcription factor as a potential therapeutic target.

A dominant-negative mutation of HSF2 associated with idiopathic azoospermia.

Clinical organ allotransplantation is limited by the availability of deceased human donors. However, the transplantation of human organs produced in other species would provide an unlimited number of organs. The pig has been identified as the most suitable source of organs for humans as organs of any size would be available. Genome editing by RNA-guided endonucleases, also known as clustered regularly interspaced short palindromic repeat (CRISPR/Cas9), in combination with induced pluripotent stem cells (iPSC), may have the potential to enable the creation of human organs from genetically-modified chimaeric pigs. These could potentially provide an unlimited supply of organs that would not be rejected by the recipient's immune system. However, substantial research is needed to prove that this approach will work. Genetic modification of chimaeric pigs could also provide useful models for developing therapies for various human diseases, especially in relation to drug development. 

The potential of the combination of CRISPR/Cas9 and pluripotent stem cells to provide human organs from chimaeric pigs.

There is an increasing worldwide shortage of organs and cells for transplantation in patients with end-stage organ failure or cellular dysfunction. This shortage could be resolved by the transplantation of organs or cells from pigs into humans. What competing approaches might provide support for the patient with end-stage organ or cell failure? Four main approaches are receiving increasing attention - (i) implantable mechanical devices, although these are currently limited almost entirely to devices aimed at supporting or replacing the heart, (ii) stem cell technology, at present directed mainly to replace absent or failing cells, but which is also fundamental to progress in (iii) tissue engineering and regenerative medicine, in which the ultimate aim is to replace an entire organ. A final novel potential approach is (iv) blastocyst complementation. These potential alternative approaches are briefly reviewed, and comments added on their current status and whether they are now (or will soon become) realistic alternative therapies to xenotransplantation. 

Potential alternative approaches to xenotransplantation.

A variety of genetic variants lead to abnormal human spermatogenesis. The ubiquitin-conjugating enzyme E2B (UBE2B) plays a significant role in spermatogenesis as Ube2b-knockout male mice are infertile.

A functional variant in the UBE2B gene promoter is associated with idiopathic azoospermia.

Different cell types possess different miRNA expression profiles, and cell/tissue/organ-specific miRNAs (or profiles) indicate different diseases. Circulating miRNA is either actively secreted by living cells or passively released during cell death. Circulating cell/tissue/organ-specific miRNA may serve as a non-invasive biomarker for allo- or xeno-transplantation to monitor organ survival and immune rejection. In this review, we summarize the proof of concept that circulating organ-specific miRNAs serve as non-invasive biomarkers for a wide spectrum of clinical organ-specific manifestations such as liver-related disease, heart-related disease, kidney-related disease, and lung-related disease. Furthermore, we summarize how circulating organ-specific miRNAs may have advantages over conventional methods for monitoring immune rejection in organ transplantation. Finally, we discuss the implications and challenges of applying miRNA to monitor organ survival and immune rejection in allo- or xeno-transplantation. 

Circulating Organ-Specific MicroRNAs Serve as Biomarkers in Organ-Specific Diseases: Implications for Organ Allo- and Xeno-Transplantation.

When hyperacute rejection is avoided by deletion of Gal expression in the pig, delayed xenograft rejection (DXR) becomes a major immunologic barrier to successful xenotransplantation. This study was to investigate the potential antigens involved in DXR. We isolated primary renal microvascular endothelial cells (RMEC) and aortic endothelial cells (AEC) from a GGTA1/CMAH double-knockout (DKO) pig (and a GGTA1-KO pig) and immunized cynomolgus monkeys with both of these cells. After sensitization, monkey serum antibody binding and cytotoxicity to RMEC was significantly higher than to AEC(p < 0.05), suggesting that RMEC are more immunogenic than AEC. Transcriptome sequencing of GGTA1/CMAH DKO pigs indicated that the expression of 1,500 genes was higher in RMEC than in AEC, while expression of 896 genes was lower. Next, we selected 101 candidate genes expressed only in pig RMEC, but not in pig AEC or in monkey or human RMEC. When these genes were knocked out individually in GGTA1/CMAH DKO RMEC, 32 genes were associated with reduced antibody binding, indicating that these genes might be primary immunologic targets involved in DXR. These genes may be important candidates for deletion in producing pigs against which there is a reduced primate immune response in pig kidney xenograft.

Potential Antigens Involved in Delayed Xenograft Rejection in a Ggta1/Cmah Dko Pig-to-Monkey Model.

Chemokine (C-C motif) receptor 6 (CCR6) is present in sperm and plays a significant role in sperm motility and chemotaxis acting in the reproductive tracts. However, the expression and functional significance of CCR6 in testis are still poorly understood, especially in the process of spermatogenesis.

In vitro chemokine (C-C motif) receptor 6-dependent non-inflammatory chemotaxis during spermatogenesis.

Xenotransplantation is an attractive solution to the problem of allograft shortage. However, transplants across discordant species barriers are subject to vigorous immunologic and pathobiologic hurdles, some of which might be overcome with the induction of immunologic tolerance. Several strategies have been designed to induce tolerance to a xenograft at both the central (including induction of mixed chimerism and thymic transplantation) and peripheral (including adoptive transfer of regulatory cells and blocking T cell costimulation) levels. Currently, xenograft tolerance has been well-established in rodent models, but these protocols have not yet achieved similar success in nonhuman primates. This review will discuss the major barriers that impede the establishment of immunological tolerance across xenogeneic barriers and the potential solution to these challenges, and provide a perspective on the future of the development of novel tolerance-inducing strategies.

Transplant Tolerance: Current Insights and Strategies for Long-Term Survival of Xenografts.

Single-field non-mydriatic fundus photography (NMFP) has been used to detect diabetic retinopathy (DR) in many studies; however, its value in a general clinical setting has not been established. Here we performed a meta-analysis to evaluate its diagnostic effectiveness.

Single-Field Non-Mydriatic Fundus Photography for Diabetic Retinopathy Screening: A Systematic Review and Meta-Analysis.

The major limitation of organ transplantation is the shortage of available organs. Xenotransplantation is considered to be an effective way to resolve the problem. Immune rejection is a major hurdle for the successful survival of pig xenografts in primate recipients. Cytokines play important roles in inflammation and many diseases including allotransplantation, however, their roles in xenotransplantation have been less well investigated.

TNF-α promotes human antibody-mediated complement-dependent cytotoxicity of porcine endothelial cells through downregulating P38-mediated Occludin expression.

Controversial results regarding the associations between aldose reductase (AR) genetic polymorphisms and diabetic retinopathy (DR) have been reported for many years. The present meta-analysis was performed to clarify the effects of the AR gene C(-106)T polymorphism on DR risk. The PubMed, Web of Sciences, Cochrane library, EMBASE, Chinese National Knowledge Infrastructure, and Wan Fang databases were extensively searched in Chinese to select relevant studies with an updated date of April 25, 2018. The Newcastle-Ottawa Scale (NOS) was applied to assess quality. The random-effects model was applied to calculate the pooled OR and 95% CI. This meta-analysis identified 23 studies with an average score of 7.52 for NOS analysis, including 4,313 DR cases and 5,128 diabetes mellitus (DM) control cases. In the overall analysis, a significant association between the AR gene C(-106)T polymorphism and DR susceptibility was found. In subgroups stratified by DM type and ethnicity, significantly increased risks for DR were found in DM type 1, East Asian populations, and Middle Eastern populations. Compared with DR control cases, the following associations were found: T vs. C: OR 0.91, 95% CI 0.85-0.97, I2 = 72.9%; CT + TT vs. CC: OR 0.75, 95% CI 0.68-0.81, I2 = 86.7%; and CT vs. CC: OR 0.86, 95% CI 0.78-0.94, I2 = 70.5%. The results of this meta-analysis showed a significant association between the AR gene C(-106)T polymorphism and susceptibility to DR in DM patients. DM patients with allele T and CT+TT genotype of the AR gene may have a lower risk of DR.

Article	Year
A dominant-negative mutation of HSF2 associated with idiopathic azoospermia. Human genetics\| PubMed ID: 23064888	2013
The potential of the combination of CRISPR/Cas9 and pluripotent stem cells to provide human organs from chimaeric pigs. International journal of molecular sciences\| PubMed ID: 25807262	2015
Potential alternative approaches to xenotransplantation. International journal of surgery (London, England)\| PubMed ID: 26209781	2015
A functional variant in the UBE2B gene promoter is associated with idiopathic azoospermia. Reproductive biology and endocrinology : RB&E\| PubMed ID: 26223869	2015
Circulating Organ-Specific MicroRNAs Serve as Biomarkers in Organ-Specific Diseases: Implications for Organ Allo- and Xeno-Transplantation. International journal of molecular sciences\| PubMed ID: 27490531	2016
Potential Antigens Involved in Delayed Xenograft Rejection in a Ggta1/Cmah Dko Pig-to-Monkey Model. Scientific reports\| PubMed ID: 28855711	2017
In vitro chemokine (C-C motif) receptor 6-dependent non-inflammatory chemotaxis during spermatogenesis. Biological research\| PubMed ID: 29788995	2018
Transplant Tolerance: Current Insights and Strategies for Long-Term Survival of Xenografts. Archivum immunologiae et therapiae experimentalis\| PubMed ID: 29992337	2018
Single-Field Non-Mydriatic Fundus Photography for Diabetic Retinopathy Screening: A Systematic Review and Meta-Analysis. Ophthalmic research\| PubMed ID: 31067550	2019
TNF-α promotes human antibody-mediated complement-dependent cytotoxicity of porcine endothelial cells through downregulating P38-mediated Occludin expression. Cell communication and signaling : CCS\| PubMed ID: 31307477	2019
Association between Aldose Reductase Gene C(-106)T Polymorphism and Diabetic Retinopathy: A Systematic Review and Meta-Analysis. Ophthalmic research\| PubMed ID: 31962334	2020

Lisha Mou

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