Browsing by Author "Tector, A. Joseph"

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    A brief history of clinical xenotransplantation
    (Elsevier, 2015-11) Cooper, David K. C.; Ekser, Burcin; Tector, A. Joseph; Department of Surgery, IU School of Medicine
    Between the 17th and 20th centuries, blood was transfused from various animal species into patients with a variety of pathological conditions. Skin grafts were carried out in the 19th century, with grafts from a variety of animals, with frogs being the most popular. In the 1920s, Voronoff advocated the transplantation of slices of chimpanzee testis into elderly men, believing that the hormones produced by the testis would rejuvenate his patients. In 1963-4, when human organs were not available and dialysis was not yet in use, Reemtsma transplanted chimpanzee kidneys into 13 patients, one of whom returned to work for almost 9 months before suddenly dying from what was believed to be an electrolyte disturbance. The first heart transplant in a human ever performed was by Hardy in 1964, using a chimpanzee heart, but the patient died within 2 h. Starzl carried out the first chimpanzee-to-human liver transplantation in 1966; in 1992 he obtained patient survival for 70 days following a baboon liver transplant. The first clinical pig islet transplant was carried out by Groth in 1993. Today, genetically-modified pigs offer hope of a limitless supply of organs and cells for those in need of a transplant.
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    Characterization of antibody binding to swine leukocyte antigen class II
    (2016-05-26) Ladowski, Joseph Matthew; Tector, A. Joseph; Tector, Matthew; Blum, Janice S.
    Though the elimination of carbohydrate xenoantigens has reduced the antibody barrier to clinical xenotransplantation, identification of additional targets of rejection could further increase the immunologic compatibility of pig tissues with humans. Many patients in need of organ transplantation have antibodies to proteins encoded by the human major histocompatibility complex (MHC) which have high similarity to their swine homologs. The goal of this thesis was to determine if the class II genes of the swine MHC can bind human antibodies. To characterize antibody binding effect to class II swine leukocyte antigens (SLA), a constitutively positive SLA class II cell was created through transfection with the human class II transactivator (CIITA). Cells expressing only SLA-DR or SLA-DQ were also created using the CRISPR/Cas9 gene knockout tools. These various lines were incubated with human sera and tested for binding to IgM and IgG in a flow cytometry crossmatch (FCXM). The results demonstrate reliable antibody binding to each of the SLA class II –DR and –DQ derivatives. A two-way paired t-test revealed statistical difference in total sera binding between to the DR(+)DQ(+) and DR(-)DQ(-) clones for IgG (p = 0.0059) but not IgM (p = 0.2460). Looking at the subset of individuals with and without anti-HLA class II sensitization, statistical difference was noted for IgG (p = 0.0229) but not IgM (p = 0.3045). Examining further the role of DR(+) vs DQ(+), statistical analysis revealed difference in the DR(+)DQ(-) vs. the DR(-)DQ(+) FCXM (p = 0.0099), the DR(+)DQ(-) vs. the DR(+)DQ(+) FCXM (p = 0.0192), and the DR(-)DQ(-) parent vs. DR(+)DQ(+) FCXM (p = 0.0329). No difference was found in the DR(-)DQ(+) vs. DR(+)DQ(+) FCXM (p = 0.1601). The results of this project suggest that SLA class II, specifically SLA-DQ, could be a target of antibody binding and cross-reactive anti-HLA class II antibodies may be capable of binding SLA class II.
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    Clinical lung xenotransplantation – what donor genetic modifications may be necessary?
    (Wiley, 2012) Cooper, David K. C.; Ekser, Burcin; Burlak, Christopher; Ezzelarab, Mohamed; Hara, Hidetaka; Paris, Leela; Tector, A. Joseph; Phelps, Carol; Azimzadeh, Agnes M.; Ayares, David; Robson, Simon C.; Pierson, Richard N., III; Surgery, School of Medicine
    Barriers to successful lung xenotransplantation appear to be even greater than for other organs. This difficulty may be related to several macro anatomic factors, such as the uniquely fragile lung parenchyma and associated blood supply that results in heightened vulnerability of graft function to segmental or lobar airway flooding caused by loss of vascular integrity (also applicable to allotransplants). There are also micro-anatomic considerations, such as the presence of large numbers of resident inflammatory cells, such as pulmonary intravascular macrophages and natural killer (NK) T cells, and the high levels of von Willebrand factor (vWF) associated with the microvasculature. We have considered what developments would be necessary to allow successful clinical lung xenotransplantation. We suggest this will only be achieved by multiple genetic modifications of the organ-source pig, in particular to render the vasculature resistant to thrombosis. The major problems that require to be overcome are multiple and include (i) the innate immune response (antibody, complement, donor pulmonary and recipient macrophages, monocytes, neutrophils, and NK cells), (ii) the adaptive immune response (T and B cells), (iii) coagulation dysregulation, and (iv) an inflammatory response (e.g., TNF-α, IL-6, HMGB1, C-reactive protein). We propose that the genetic manipulation required to provide normal thromboregulation alone may include the introduction of genes for human thrombomodulin/endothelial protein C-receptor, and/or tissue factor pathway inhibitor, and/or CD39/CD73; the problem of pig vWF may also need to be addressed. It would appear that exploration of every available therapeutic path will be required if lung xenotransplantation is to be successful. To initiate a clinical trial of lung xenotransplantation, even as a bridge to allotransplantation (with a realistic possibility of survival long enough for a human lung allograft to be obtained), significant advances and much experimental work will be required. Nevertheless, with the steadily increasing developments in techniques of genetic engineering of pigs, we are optimistic that the goal of successful clinical lung xenotransplantation can be achieved within the foreseeable future. The optimistic view would be that if experimental pig lung xenotransplantation could be successfully managed, it is likely that clinical application of this and all other forms of xenotransplantation would become more feasible.
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    Creating class I MHC-null pigs using guide RNA and the Cas9 endonuclease
    (American Association of Immunologists, 2014-12-01) Reyes, Luz M.; Estrada, Jose L; Wang, Zheng Yu; Blosser, Rachel J.; Smith, Rashod F.; Sidner, Richard A.; Paris, Leela L.; Blankenship, Ross L.; Ray, Caitlin N.; Miner, Aaron C.; Tector, Matthew; Tector, A. Joseph; Surgery, School of Medicine
    Pigs are emerging as important large animal models for biomedical research, and they may represent a source of organs for xenotransplantation. The MHC is pivotal to the function of the immune system in health and disease, and it is particularly important in infection and transplant rejection. Pigs deficient in class I MHC could serve as important reagents to study viral immunity as well as allograft and xenograft rejection. In this study, we report the creation and characterization of class I MHC knockout pigs using the Cas9 nuclease and guide RNAs. Pig fetal fibroblasts were genetically engineered using Cas9 and guide RNAs, and class I MHC(-) cells were then used as nuclear donors for somatic cell nuclear transfer. We produced three piglets devoid of all cell surface class I proteins. Although these animals have reduced levels of CD4(-)CD8(+) T cells in peripheral blood, the pigs appear healthy and are developing normally. These pigs are a promising reagent for immunological research.
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    De Novo Malignancy Post Liver Transplantation: A Single Center, Population Controlled Study
    (Wiley, 2013) Chatrath, Hemant; Berman, Kenneth; Vuppalanchi, Raj; Slaven, James; Kwo, Paul; Tector, A. Joseph; Chalasani, Naga; Ghabril, Marwan; Medicine, School of Medicine
    Background: With the growing numbers of liver transplant recipients, it is increasingly important to understand the risks of de novo malignancy after liver transplantation. Aim: To characterize the incidence of de novo malignancy after liver transplantation compared with a control non-transplant population. Methods: We studied 534 Indiana state residents undergoing liver transplantation at our center between 1997 and 2004, followed through August 2010. The incidence and predictors of malignancy were determined. The standardized incidence ratio (SIR) of cancer in our cohort was compared with age-, gender-, and period-matched state population using the Indiana State Cancer Registry. Results: After a mean follow-up of 5.7 ± 3.2 yr, 73 patients (13.7%) developed 80 cancers, with five- and 10-yr incidence rates of 11.7% and 24.8%, respectively. These included 24 (30%) skin, 16 (20%) hematologic, and 40 (50%) solid tumors. The most common solid cancers were aerodigestive. Compared with matched state population, liver transplant recipients had significantly higher incidence of all cancers (SIR: 3.1, 95% CI [Confidence interval]: 2.9-3.2), skin (melanoma) (SIR: 5.8, 95% CI: 4.7-7.0), hematologic (SIR: 7.1, 95% CI: 6.3-8.0), and solid (SIR: 2.7, 95% CI: 2.5-2.8) tumors. Conclusion: There is a significantly increased risk of de novo malignancies after liver transplantation, highlighting the need for surveillance strategies in this population.
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    Erythrocytes from GGTA1/CMAH knockout pigs: implications for xenotransfusion and testing in non-human primates
    (Wiley, 2014-07) Wang, Zheng-Yu; Burlak, Christopher; Estrada, Jose L.; Li, Ping; Tector, Matthew F.; Tector, A. Joseph; Department of Surgery, IU School of Medicine
    Background Pig erythrocytes are potentially useful to solve the worldwide shortage of human blood for transfusion. Domestic pig erythrocytes, however, express antigens that are bound by human preformed antibodies. Advances in genetic engineering have made it possible to rapidly knock out the genes of multiple xenoantigens, namely galactose α1,3 galactose (aGal) and N-glycolylneuraminic acid (Neu5Gc). We have recently targeted the GGTA1 and CMAH genes with zinc finger endonucleases resulting in double knockout pigs that no longer express aGal or Neu5Gc and attract significantly fewer human antibodies. In this study, we characterized erythrocytes from domestic and genetically modified pigs, baboons, chimpanzees, and humans for binding of human and baboon natural antibody, and complement mediated lysis. Methods Distribution of anti Neu5Gc IgG and IgM in pooled human AB serum was analyzed by ELISA. Erythrocytes from domestic pigs (Dom), aGal knockout pigs (GGTA1 KO), aGal and Neu5Gc double knockout pigs (GGTA1/CMAH KO), baboons, chimpanzees, and humans were analyzed by flow cytometry for aGal and Neu5Gc expression. In vitro comparative analysis of erythrocytes was conducted with pooled human AB serum and baboon serum. Total antibody binding was accessed by hemagglutination; complement-dependent lysis was measured by hemolytic assay; IgG or IgM binding to erythrocytes was characterized by flow cytometry. Results The pooled human AB serum contained 0.38 μg/ml anti Neu5Gc IgG and 0.085 μg/ml anti Neu5Gc IgM. Both Gal and Neu5Gc were not detectable on GGTA1/CMAH KO erythrocytes. Hemagglutinaion of GGTA1/CMAH KO erythrocytes with human serum was 3.5-fold lower compared to GGTA1 KO erythrocytes, but 1.6-fold greater when agglutinated with baboon serum. Hemolysis of GGTA1/CMAH KO erythrocytes by human serum (25%) was reduced 9-fold compared to GGTA1 KO erythrocytes, but increased 1.64-fold by baboon serum. Human IgG binding was reduced 27-fold on GGTA1/CMAH KO erythrocytes compared to GGTA1 KO erythrocytes, but markedly increased 3-fold by baboon serum IgG. Human IgM binding was decreased 227-fold on GGTA1/CMAH KO erythrocytes compared to GGTA1 KO erythrocytes, but enhanced 5-fold by baboon serum IgM. Conclusions Removal of aGal and Neu5Gc antigens from pig erythrocytes significantly reduced human preformed antibody-mediated cytotoxicity but may have complicated future in vivo analysis by enhancing reactivity from baboons. The creation of the GGTA1/CMAH KO pig has provided the xenotransplantion researcher with organs and cells that attract fewer human antibodies than baboon and our closest primate relative, chimpanzee. These finding suggest that while GGTA1/CMAH KO erythrocytes may be useful for human transfusions, in vivo testing in the baboon may not provide a direct transplation to the clinic.
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    Expression of NeuGc on Pig Corneas and Its Potential Significance in Pig Corneal Xenotransplantation
    (Wolters Kluwer, 2016-01) Lee, Whayoung; Miyagawa, Yuko; Long, Cassandra; Ekser, Burcin; Walters, Eric; Ramsoondar, Jagdeece; Ayares, David; Tector, A. Joseph; Cooper, David K. C.; Hara, Hidetaka; Department of Surgery, IU School of Medicine
    PURPOSE: Pigs expressing neither galactose-α1,3-galactose (Gal) nor N-glycolylneuraminic acid (NeuGc) take xenotransplantation one step closer to the clinic. Our aims were (1) to document the lack of NeuGc expression on corneas and aortas and cultured endothelial cells [aortic endothelial cells (AECs); corneal (CECs)] of GTKO/NeuGcKO pigs, and (2) to investigate whether the absence of NeuGc reduced human antibody binding to the tissues and cells. METHODS: Wild-type (WT), GTKO, and GTKO/NeuGcKO pigs were used for the study. Human tissues and cultured cells were negative controls. Immunofluorescence staining was performed using anti-Gal and anti-NeuGc antibodies, and human IgM and IgG binding to tissues was determined. Flow cytometric analysis was used to determine Gal and NeuGc expression on cultured CECs and AECs and to measure human IgM/IgG binding to these cells. RESULTS: Both Gal and NeuGc were detected on WT pig corneas and aortas. Although GTKO pigs expressed NeuGc, neither humans nor GTKO/NeuGcKO pigs expressed Gal or NeuGc. Human IgM/IgG binding to corneas and aortas from GTKO and GTKO/NeuGcKO pigs was reduced compared with binding to WT pigs. Human antibody binding to GTKO/NeuGcKO AECs was significantly less than that to GTKO AECs, but there was no significant difference in binding between GTKO and GTKO/NeuGcKO CECs. CONCLUSIONS: The absence of NeuGc on GTKO aortic tissue and AECs is associated with reduced human antibody binding, and possibly will provide a better outcome in clinical xenotransplantation using vascularized organs. For clinical corneal xenotransplantation, the absence of NeuGc expression on GTKO/NeuGcKO pig corneas may not prove an advantage over GTKO corneas.
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    Fatigue as Reported at 12 Time Points during the First Year Post-Liver Transplant
    (2013) Scott, Patricia J.; Krause, Audrey; Tector, A. Joseph; Kwo, Paul
    BACKGROUND: Although liver transplantation has evolved as an effective procedure, fatigue remains a post-transplant complaint [1]. As yet, there are no published accounts of the experience of fatigue at temporal intervals during the first post-transplant year, and this information would benefit LT candidates in recovery planning. Van Ginneken [2] found that time since transplant was not associated with physical fatigue and reduced activity, but was associated with albumin levels less than 25g/l and with lower GFR. METHODS: Data used in this study were collected through an ongoing, longitudinal, prospective design. Results presented here are for fatigue and biometric data at 12 data points starting one week post hospital discharge, continued weekly for the first 8 weeks, then monthly at 3, 6, 9, and 12 months. RESULTS: We sampled 30 subjects: 19 (70.4%) male and 8 (29.6%) female, age 55.4 ± 9.8 years. A mixed models analysis of variance was done to investigate a change in FACIT over time. The initial model included age, MELD, sex, week, albumin, ALT, BILI T, and CREAT. The final model included age, BILI T, and week. Increasing age and BILI T were associated with greater fatigue (p=0.0376 and p=0.0005, respectively). There was significant decrease in fatigue over time (p<0.0001). Pair-wise comparisons were done to determine which weeks significantly differed. Tukey’s adjustment for multiple comparisons was used. Figure 1 indicates which visits significantly differed. DISCUSSION: Our subjects experienced decreased fatigue over time. The data set was rich with prospectively collected longitudinal information helpful for establishing realistic expectations for post-transplant fatigue. Finding include early weeks of recovery (weeks 2-3) differ from weeks 7+ and that there is no significant change after 3 months, up to one year. No association was seen with Albumin levels although total bilirubin and age were associated with greater fatigue.
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    Immunobiological barriers to xenotransplantation
    (Elsevier, 2015-11) Cooper, David K. C.; Ekser, Burcin; Tector, A. Joseph; Department of Surgery, IU School of Medicine
    Binding of natural anti-pig antibodies in humans and nonhuman primates to carbohydrate antigens expressed on the transplanted pig organ, the most important of which is galactose-α1,3-galactose (Gal), activate the complement cascade, which results in destruction of the graft within minutes or hours, known as hyperacute rejection. Even if antibody is removed from the recipient's blood by plasmapheresis, recovery of antibody is associated with acute humoral xenograft rejection. If immunosuppressive therapy is inadequate, the development of high levels of T cell-dependent elicited anti-pig IgG similarly results in graft destruction, though classical acute cellular rejection is rarely seen. Vascular endothelial activation by low levels of anti-nonGal antibody, coupled with dysregulation of the coagulation-anticoagulation systems between pigs and primates, leads to a thrombotic microangiopathy in the graft that may be associated with a consumptive coagulopathy in the recipient. The most successful approach to overcoming these barriers is by genetically-engineering the pig to provide it with resistance to the human humoral and cellular immune responses and to correct the coagulation discrepancies between the two species. Organs and cells from pigs that (i) do not express the important Gal antigen, (ii) express a human complement-regulatory protein, and (iii) express a human coagulation-regulatory protein, when combined with an effective immunosuppressive regimen, have been associated with prolonged pig graft survival in nonhuman primates.
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    Immunobiology of liver xenotransplantation
    (Taylor & Francis, 2012) Ekser, Burcin; Burlak, Christopher; Waldman, Joshua P.; Lutz, Andrew J.; Paris, Leela L.; Veroux, Massimiliano; Robson, Simon C.; Rees, Michael A.; Ayares, David; Gridelli, Bruno; Tector, A. Joseph; Cooper, David K. C.; Surgery, School of Medicine
    Pigs are currently the preferred species for future organ xenotransplantation. With advances in the development of genetically modified pigs, clinical xenotransplantation is becoming closer to reality. In preclinical studies (pig-to-nonhuman primate), the xenotransplantation of livers from pigs transgenic for human CD55 or from α1,3-galactosyltransferase gene-knockout pigs+/- transgenic for human CD46, is associated with survival of approximately 7-9 days. Although hepatic function, including coagulation, has proved to be satisfactory, the immediate development of thrombocytopenia is very limiting for pig liver xenotransplantation even as a 'bridge' to allotransplantation. Current studies are directed to understand the immunobiology of platelet activation, aggregation and phagocytosis, in particular the interaction between platelets and liver sinusoidal endothelial cells, hepatocytes and Kupffer cells, toward identifying interventions that may enable clinical application.