Joseph M Ladowski, Meghan Hu, Janghoon Yoon, Zheng Chen, Stuart Knechtle, Annette M Jackson, Jean Kwun
{"title":"Detection of Anti-Non-α-Gal Xenoreactive Antibodies in Human Blood Products.","authors":"Joseph M Ladowski, Meghan Hu, Janghoon Yoon, Zheng Chen, Stuart Knechtle, Annette M Jackson, Jean Kwun","doi":"10.1111/xen.70034","DOIUrl":"10.1111/xen.70034","url":null,"abstract":"<p><strong>Background: </strong>Surgical bleeding is a risk in any solid organ transplant, and is commonly addressed with the transfusion of human blood products to replace or supplement coagulation factors. It is unknown if these blood products would harm xenotransplanted pig organs in human recipients demonstrating coagulopathy. The aim of this study was to investigate in vitro if blood products such as fresh frozen plasma (FFP) or cryoprecipitate (cryo) contain xenoantibodies capable of cytotoxicity to GTKO pig cells.</p><p><strong>Methods: </strong>We obtained 12 individual single-donor (7 FFP and 5 cryo) blood products from our institution's blood bank for testing. Peripheral blood mononuclear cells (PBMCs) were obtained from a GTKO/hCD55 pig for use as target cells. We performed a series of flow cytometry crossmatch (FCXM) and complement-dependent cytotoxicity (CDC) assays.</p><p><strong>Results: </strong>We found that all the tested blood products contained some degree of IgM and IgG xenoantibody. Tests using a 1:50 dilution revealed a significant decrease in IgM xenoantibody binding, but an increase in the detection of IgG binding. Multiple preparations were capable of GTKO PBMC cytotoxicity but the level of antibody binding and cell death varied by preparation.</p><p><strong>Conclusions: </strong>Both FFP and cryo contain IgM and IgG non-galactose-α-1,3-galactose (αGal) xenoantibodies capable of killing GTKO PBMCs, though the level varies by preparation. Although some centers utilize a genetic background with mutations in the three enzymes responsible for the known xenoantigens, others are investigating the GTKO pig as a potential option. These results suggest that a center pursuing a human xenotransplantation study with a GTKO genetic background should pre-screen blood products prior to administration.</p>","PeriodicalId":23866,"journal":{"name":"Xenotransplantation","volume":"32 2","pages":"e70034"},"PeriodicalIF":3.3,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143664697","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Combinatorial Decellularization as a Better Approach to Porcine Liver Extracellular Matrix Scaffold Fabrication With Preserved Bioactivity: A Comparative Evaluation.","authors":"Jesna Puthiya Veettil, Devika Sasikumar Lolitha, Umashankar Payanam Ramachandra","doi":"10.1111/xen.70031","DOIUrl":"10.1111/xen.70031","url":null,"abstract":"<p><p>Soft tissue repair patches of decellularized extracellular matrices (ECM) with inherently preserved structural components and biomacromolecules are desirable in regenerative applications. This study characterizes three detergent-based decellularization methods to fabricate acellular porcine liver matrices to remove antigenic determinants without compromising the structural integrity, glycosaminoglycans (GAG) content, and bound growth factors within the resulting ECM. Three detergents chosen for decellularization were sodium dodecyl sulfate (SDS), SDS with sodium deoxycholate (SDS+SDC-combinatorial method), and triton X-100 followed by SDS. Combinatorial detergent decellularization effectively removed cellular components and retained intact collagenous structure with minimal residual DNA and protein. It also preserved significantly higher amounts of GAG, HGF, and bFGF. TX100 decellularization was highly destructive with the least preservation of GAG and GFs. The SDS method showed an intermediate level of preservation of biomolecules. The correlation obtained between GAG and GFs revealed quantification of GAG to be an indirect way of estimating the bound GFs preserved within the ECM. In vitro experiments revealed the non-cytotoxic nature of the scaffolds. The study revealed that, among the three methods of decellularization, the ECM scaffold fabricated by combinatorial detergent decellularization is extremely promising to be used as a soft tissue repair patch with inherent bioactive molecules for scaffold-based regenerative therapies.</p>","PeriodicalId":23866,"journal":{"name":"Xenotransplantation","volume":"32 2","pages":"e70031"},"PeriodicalIF":3.3,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143664691","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Joachim Denner, Hina Jhelum, Jinzhao Ban, Ludwig Krabben, Benedikt B Kaufer
{"title":"How to Detect Porcine Endogenous Retrovirus (PERV) Infections in Patients After Transplantation of Pig Organs.","authors":"Joachim Denner, Hina Jhelum, Jinzhao Ban, Ludwig Krabben, Benedikt B Kaufer","doi":"10.1111/xen.70028","DOIUrl":"10.1111/xen.70028","url":null,"abstract":"<p><p>Porcine endogenous retroviruses (PERVs) are integrated into the genome of all pigs and can infect human cells in culture. However, no PERV infections have been reported in recipients following preclinical or clinical xenotransplantation or deliberate infection experiments. Detection of PERV infection in transplanted recipients is challenging due to microchimerism, such as the presence of pig cells containing PERV proviruses in the recipient. Based on our previous publications on PERV detection in xenotransplant recipients, particularly from the first clinical trials, we developed a comprehensive strategy to screen for PERV infections. Recipients can be monitored for increasing levels of viral genomic RNA and mRNA using real-time reverse transcriptase (RT)-PCR, which can indicate PERV expression and replication. To test this strategy, explanted pig hearts and organs from baboons after pig heart transplantation were analyzed. No PERV genomic RNA or mRNA was detected in these tissues, although both were found in PERV-producing human control cells. Screening for antibodies against PERV as indirect evidence of infection is the method of choice. Recombinant viral proteins were prepared for use in Western blot assays. Animal antisera generated through immunization with recombinant PERV proteins served as positive controls. No antibodies against PERV were detected in transplanted baboons, even though microchimerism was observed in many of the animals' organs. For effective antibody screening, at least two PERV proteins should be used as antigens.</p>","PeriodicalId":23866,"journal":{"name":"Xenotransplantation","volume":"32 1","pages":"e70028"},"PeriodicalIF":3.3,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11850954/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143493936","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Elisa J Gordon, Michael K Gusmano, Jessica Gacki-Smith, Hannah L Brooks, Margaret M Matthews, Dahlya Manning, Joseph Leventhal, Karen J Maschke
{"title":"Patients' Information Needs for Informed Consent to Participate in First-in-Human Pig Kidney Xenotransplant Clinical Trials: A Mixed Methods Study.","authors":"Elisa J Gordon, Michael K Gusmano, Jessica Gacki-Smith, Hannah L Brooks, Margaret M Matthews, Dahlya Manning, Joseph Leventhal, Karen J Maschke","doi":"10.1111/xen.70016","DOIUrl":"10.1111/xen.70016","url":null,"abstract":"<p><strong>Background: </strong>Transplant programs preparing to initiate first-in-human pig kidney xenotransplant clinical trials must be especially careful when obtaining participants' informed consent. Little is known about the kind of information patients want for making an informed decision about trial participation.</p><p><strong>Methods: </strong>We conducted semi-structured telephone interviews with waitlisted kidney transplant patients about information needs regarding participating in a first-in-human pig kidney xenotransplant trial, which guided development of a prototype consent form. Subsequent usability testing interviews sought patient feedback on the consent form. We analyzed qualitative data by thematic analysis and quantitative data by descriptive statistics.</p><p><strong>Results: </strong>Twenty-eight patients participated in semi-structured interviews; 16 patients participated in usability testing interviews. Most interview participants were male (68%, 56%), White (54%, 56%), or Black (36%, 31%), respectively. Interview participants identified five types of information needs: (1) the potential for infection contraction and transmission; (2) risks, benefits, and impact of xenotransplant trials; (3) xenotransplant clinical trial and recipient experience; (4) clinical trial logistics; and (5) the pig and its kidney. Usability testing participants suggested adding details to the prototype. Participants' preparedness to make a decision about participating in a xenotransplant trial increased after reviewing the prototype (12.5% vs. 31.3%, n.s.).</p><p><strong>Conclusion: </strong>We identified multiple unique types of information patients desired to make informed decisions about pig kidney xenotransplant trial participation. Transplant programs initiating xenotransplant trials should be prepared to address patients' information needs to optimize informed decision-making for trial participation. The prototype consent form may support a patient-centered approach to informed consent.</p>","PeriodicalId":23866,"journal":{"name":"Xenotransplantation","volume":"32 1","pages":"e70016"},"PeriodicalIF":3.3,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11851052/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143493363","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Kasra Shirini, Joseph M Ladowski, Raphael P H Meier
{"title":"Xenotransplantation Literature Update July-December 2024.","authors":"Kasra Shirini, Joseph M Ladowski, Raphael P H Meier","doi":"10.1111/xen.70027","DOIUrl":"10.1111/xen.70027","url":null,"abstract":"<p><p>This review highlights groundbreaking progress from July to December 2024, including developments in gene-edited pigs, cellular therapies, organ preservation, and transplantation techniques. Recent advancements, particularly in gene-editing technologies and immunosuppressive protocols, have brought the field closer to clinical application. While significant ethical, immunological, and societal challenges remain, this progress underscores the immense potential of xenotransplantation to revolutionize transplantation medicine.</p>","PeriodicalId":23866,"journal":{"name":"Xenotransplantation","volume":"32 1","pages":"e70027"},"PeriodicalIF":3.3,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11856842/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143504637","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Combinatorial Decellularization as a Better Approach to Porcine Liver Extracellular Matrix Scaffold Fabrication With Preserved Bioactivity: A Comparative Evaluation.","authors":"Jesna Puthiya Veettil, Devika Sasikumar Lolitha, Umashankar Payanam Ramachandra","doi":"10.1111/xen.70025","DOIUrl":"10.1111/xen.70025","url":null,"abstract":"<p><p>Soft tissue repair patches of decellularized extracellular matrices (ECM) with inherently preserved structural components and biomacromolecules are desirable in regenerative applications. This study characterizes three detergent-based decellularization methods to fabricate acellular porcine liver matrices to remove antigenic determinants without compromising the structural integrity, glycosaminoglycans (GAG) content, and bound growth factors within the resulting ECM. Three detergents chosen for decellularization were sodium dodecyl sulfate (SDS), SDS with sodium deoxycholate (SDS + SDC-combinatorial method), and Triton X-100 followed by SDS. Combinatorial detergent decellularization effectively removed cellular components and retained intact collagenous structure with minimal residual DNA and protein. It also preserved significantly higher amounts of GAG, HGF, and bFGF. TX100 decellularization was highly destructive with the least preservation of GAG and GFs. The SDS method showed an intermediate level of preservation of biomolecules. The correlation obtained between GAG and GFs revealed quantification of GAG to be an indirect way of estimating the bound GFs preserved within the ECM. In vitro experiments revealed the noncytotoxic nature of the scaffolds. The study revealed that, among the three methods of decellularization, ECM scaffold fabricated by combinatorial detergent decellularization is extremely promising for use as a soft tissue repair patch with inherent bioactive molecules for scaffold-based regenerative therapy.</p>","PeriodicalId":23866,"journal":{"name":"Xenotransplantation","volume":"32 1","pages":"e70025"},"PeriodicalIF":3.3,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143442228","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Hao Feng, Man Zhang, Qiangbing Xia, Jiaxiang Du, Tao Li, Song Chen, Yi Wang, Dengke Pan, Lan Zhu, Gang Chen
{"title":"Evaluation of Complement-Dependent Cytotoxicity Assays for Gene-Edited Pig-to-Human Xenotransplantation.","authors":"Hao Feng, Man Zhang, Qiangbing Xia, Jiaxiang Du, Tao Li, Song Chen, Yi Wang, Dengke Pan, Lan Zhu, Gang Chen","doi":"10.1111/xen.70021","DOIUrl":"10.1111/xen.70021","url":null,"abstract":"<p><strong>Background: </strong>Gene-edited pigs for xenotransplantation usually contain one or more transgenes encoding human complement regulatory proteins (CRPs). Because of species differences, human CRP(s) expressed in gene-edited pigs may have difficulty inhibiting the activation of exogenous rabbit complement added to a complement-dependent cytotoxicity (CDC) assay. The use of human complement instead of rabbit complement in CDC experiments may more accurately reflect the actual regulatory activity of human CRP(s).</p><p><strong>Methods: </strong>Peripheral blood mononuclear cells (PBMCs) were obtained from one GTKO pig and two GTKO/hCD55 pigs with a high or low level of hCD55 expression. After incubation of heat-inactivated normal human sera (HINHS) with porcine PBMCs, CDC levels were measured after the addition of commercial rabbit complement or human complement. In addition, a modified one-step CDC method was established using pooled normal human sera (NHS) without the addition of an exogenous complement.</p><p><strong>Results: </strong>There was no significant difference in the binding of IgM/IgG to PBMCs from the three pigs. Both rabbit and human complement-mediated a significant cytotoxic effect on GTKO pig PBMCs (98.97% vs. 82.73%). Even the high expression of hCD55 only had a very limited inhibitory effect on rabbit complement-mediated cytotoxicity (81.70% vs. 98.97%). However, regardless of whether the expression level was high or low, hCD55 had a very remarkable inhibitory effect on human complement-mediated cytotoxicity (2.94% and 23.83% vs. 82.73%; p < 0.01). Similar results were obtained using the modified one-step CDC method. In addition, the inhibitory effect of hCD55 on C3c and C5b-9 deposition on pig PBMCs was positively correlated with the expression level of hCD55.</p><p><strong>Conclusion: </strong>The use of human complement instead of rabbit complement in CDC assays can better reflect the actual cytotoxic effect of human xenoantibodies against pig PBMCs expressing human CRP(s), and thus may have potential application to gene-edited pig-to-human xenotransplantation.</p>","PeriodicalId":23866,"journal":{"name":"Xenotransplantation","volume":"32 1","pages":"e70021"},"PeriodicalIF":3.3,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143442277","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Zoltan Czigany, Kasra Shirini, Aghnia J Putri, Alban E Longchamp, Subarna Bhusal, Shani Kamberi, Raphael P H Meier
{"title":"Bridging Therapies-Ex Vivo Liver Xenoperfusion and the Role of Machine Perfusion: An Update.","authors":"Zoltan Czigany, Kasra Shirini, Aghnia J Putri, Alban E Longchamp, Subarna Bhusal, Shani Kamberi, Raphael P H Meier","doi":"10.1111/xen.70011","DOIUrl":"10.1111/xen.70011","url":null,"abstract":"<p><p>Advancements in xenotransplantation intersecting with modern machine perfusion technology offer promising solutions to patients with liver failure providing a valuable bridge to transplantation and extending graft viability beyond current limitations. Patients facing acute or acute chronic liver failure, post-hepatectomy liver failure, or fulminant hepatic failure often require urgent liver transplants which are severely limited by organ shortage, emphasizing the importance of effective bridging approaches. Machine perfusion is now increasingly used to test and use genetically engineered porcine livers in translational studies, addressing the limitations and costs of non-human primate models. Current reports about artificial and bioartificial liver support combined with xenografts showcase the potential in ex vivo xenogeneic perfusion. Breakthroughs, such as the perfusion of genetically modified porcine liver with FDA-approved machine perfusion systems connected to human blood circulation, underscore the interest and potential feasibility of a \"liver dialysis\" bridge to allotransplantation or recovery. This review provides an overview of the past and current research in the field of ex vivo pig liver xenoperfusion.</p>","PeriodicalId":23866,"journal":{"name":"Xenotransplantation","volume":"32 1","pages":"e70011"},"PeriodicalIF":3.3,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143012785","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Alessandro Martinino, Timothy J Smith, Zachary C Elmore, Janghoon Yoon, Joseph Ladowski, Davide Schiliro, Joshua A Hull, Allie Schwalb, Meghan Hu, Ryan Spangler, Kyo Won Lee, Min Jung Kim, Kyha Williams, Annette Jackson, Stuart J Knechtle, Aravind Asokan, Jean Kwun
{"title":"An IgM Cleaving Enzyme for Clearance of Anti-Pig Xenoreactive Antibodies in a Nonhuman Primate Model.","authors":"Alessandro Martinino, Timothy J Smith, Zachary C Elmore, Janghoon Yoon, Joseph Ladowski, Davide Schiliro, Joshua A Hull, Allie Schwalb, Meghan Hu, Ryan Spangler, Kyo Won Lee, Min Jung Kim, Kyha Williams, Annette Jackson, Stuart J Knechtle, Aravind Asokan, Jean Kwun","doi":"10.1111/xen.70015","DOIUrl":"10.1111/xen.70015","url":null,"abstract":"<p><strong>Background: </strong>The removal of preformed antibodies with cleaving enzyme like IdeS (Imlifidase) has demonstrated therapeutic potential in organ transplantation for sensitized recipients. However, preformed xenoreactive antibodies (XAbs) against porcine glycans are predominantly IgM and considered detrimental in pig-to-human xenotransplantation.</p><p><strong>Methods: </strong>Recombinant IceM, an endopeptidase cleaving IgM, was generated in Escherichia coli. Four maximally MHC-mismatched rhesus macaques underwent two serial skin transplantations to model allosensitized patients awaiting xenotransplantation. IceM was administered IV in allosensitized animals at 28 and 56 days after the first skin transplantation to assess in vivo IgM cleavage. Total IgG and IgM were quantified with western blot, and anti-pig (xenoreactive) IgG/IgM were evaluated using flowcrossmatch. B cell and its subpopulations were assessed using flow cytometry.</p><p><strong>Results: </strong>IceM selectively cleaved human IgM, while showing no cleavage activity toward other isotypes including IgG, IgA, IgD, and IgE. Additionally, IceM cleaves only human and non-human primate IgM in vitro, but not in sera from other species. At a dose of 0.5 mg/kg, IceM reduced xenoreactive IgM levels to 13.76% ± 4.98% of baseline (B cell flow crossmatch) at 24 h post-administration, with baseline levels restored approximately 2 weeks after treatment. Additionally, animals showed similar kinetics of xenoreative IgM degradation with the repeated dose of IceM.</p><p><strong>Conclusion: </strong>In this study, we report a recombinant bacterial enzyme that selectively cleaves IgM in human and non-human primate sera. Repeat administration of IceM in macaques enables selective, robust clearance of circulating xenoreactive IgM. This approach will be useful in treating preformed natural and rebound IgM in xenotransplantation.</p>","PeriodicalId":23866,"journal":{"name":"Xenotransplantation","volume":"32 1","pages":"e70015"},"PeriodicalIF":3.3,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11897829/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143047753","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Hao Feng, Man Zhang, Qiangbing Xia, Jiaxiang Du, Tao Li, Song Chen, Yi Wang, Dengke Pan, Lan Zhu, Gang Chen
{"title":"Evaluation of Complement-Dependent Cytotoxicity Assays for Gene-Edited Pig-to-Human Xenotransplantation.","authors":"Hao Feng, Man Zhang, Qiangbing Xia, Jiaxiang Du, Tao Li, Song Chen, Yi Wang, Dengke Pan, Lan Zhu, Gang Chen","doi":"10.1111/xen.70012","DOIUrl":"10.1111/xen.70012","url":null,"abstract":"<p><strong>Background: </strong>Gene-edited pigs for xenotransplantation usually contain one or more transgenes encoding human complement regulatory proteins (CRPs). Because of species differences, human CRP(s) expressed in gene-edited pigs may have difficulty inhibiting the activation of exogenous rabbit complement added to a complement-dependent cytotoxicity (CDC) assay. The use of human complement instead of rabbit complement in CDC experiments may more accurately reflect the actual regulatory activity of human CRP(s).</p><p><strong>Methods: </strong>Peripheral blood mononuclear cells (PBMCs) were obtained from one GTKO pig and two GTKO/hCD55 pigs with a high or low level of hCD55 expression. After incubation of heat-inactivated normal human sera (HINHS) with porcine PBMCs, CDC levels were measured after the addition of commercial rabbit complement or human complement. In addition, a modified one-step CDC method was established using pooled normal human sera (NHS) without the addition of exogenous complement.</p><p><strong>Results: </strong>There was no significant difference in the binding of IgM/IgG to PBMCs from the three pigs. Both rabbit and human complement mediated a significant cytotoxic effect on GTKO pig PBMCs (98.97% vs. 82.73%). Even the high expression of hCD55 only had a very limited inhibitory effect on rabbit complement-mediated cytotoxicity (81.70% vs. 98.97%). However, regardless of whether the expression level was high or low, hCD55 had a very remarkable inhibitory effect on human complement-mediated cytotoxicity (2.94% and 23.83% vs. 82.73%; p < 0.01). Similar results were obtained using the modified one-step CDC method. In addition, the inhibitory effect of hCD55 on C3c and C5b-9 deposition on pig PBMCs was positively correlated with the expression level of hCD55.</p><p><strong>Conclusion: </strong>The use of human complement instead of rabbit complement in CDC assays can better reflect the actual cytotoxic effect of human xenoantibodies against pig PBMCs expressing human CRP(s), and thus may have potential application to gene-edited pig-to-human xenotransplantation.</p>","PeriodicalId":23866,"journal":{"name":"Xenotransplantation","volume":"32 1","pages":"e70012"},"PeriodicalIF":3.3,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143012787","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}