Prolonged xenokidney graft survival in sensitized NHP recipients by expression of multiple human transgenes in a triple knockout pig.

Genetic modification of porcine donors, combined with optimized immunosuppression, has been shown to improve outcomes of experimental xenotransplant. However, little is known about outcomes in sensitized recipients, a population that could potentially benefit the most from the clinical implementation of xenotransplantation. Here, five highly allosensitized rhesus macaques received a porcine kidney from GGTA1 (α1,3-galactosyltransferase) knockout pigs expressing the human CD55 transgene (1KO.1TG) and were maintained on an anti-CD154 monoclonal antibody (mAb)-based immunosuppressive regimen. These recipients developed de novo xenoreactive antibodies and experienced xenograft rejection with evidence of thrombotic microangiopathy and antibody-mediated rejection (AMR). In comparison, three highly allosensitized rhesus macaques receiving a kidney from GGTA1, CMAH (cytidine monophospho-N-acetylneuraminic acid hydroxylase), and b4GNT2/b4GALNT2 (ß-1,4-N-acetyl-galactosaminyltransferase 2) knockout pigs expressing seven human transgenes including human CD46, CD55, CD47, THBD (thrombomodulin), PROCR (protein C receptor), TNFAIP3 (tumor necrosis factor-α-induced protein 3), and HMOX1 (heme oxygenase 1) (3KO.7TG) experienced significantly prolonged graft survival and reduced AMR, associated with dampened post-transplant humoral responses, early monocyte and neutrophil activation, and T cell repopulation. After withdrawal of all immunosuppression, recipients who received kidneys from 3KO.7TG pigs rejected the xenografts via AMR. These data suggest that allosensitized recipients may be suitable candidates for xenografts from genetically modified porcine donors and could benefit from an optimized immunosuppression regimen designed to target the post-transplant humoral response, thereby avoiding AMR.

HLA-G1+ Expression in GGTA1KO Pigs Suppresses Human and Monkey Anti-Pig T, B and NK Cell Responses.

The human leukocyte antigen G1 (HLA-G1), a non-classical class I major histocompatibility complex (MHC-I) protein, is a potent immunomodulatory molecule at the maternal/fetal interface and other environments to regulate the cellular immune response. We created GGTA1-/HLAG1+ pigs to explore their use as organ and cell donors that may extend xenograft survival and function in both preclinical nonhuman primate (NHP) models and future clinical trials. In the present study, HLA-G1 was expressed from the porcine ROSA26 locus by homology directed repair (HDR) mediated knock-in (KI) with simultaneous deletion of α-1-3-galactotransferase gene (GGTA1; GTKO) using the clustered regularly interspersed palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) (CRISPR/Cas9) gene-editing system. GTKO/HLAG1+ pigs showing immune inhibitory functions were generated through somatic cell nuclear transfer (SCNT). The presence of HLA-G1 at the ROSA26 locus and the deletion of GGTA1 were confirmed by next generation sequencing (NGS) and Sanger's sequencing. Fibroblasts from piglets, biopsies from transplantable organs, and islets were positive for HLA-G1 expression by confocal microscopy, flow cytometry, or q-PCR. The expression of cell surface HLA-G1 molecule associated with endogenous ß2-microglobulin (ß2m) was confirmed by staining genetically engineered cells with fluorescently labeled recombinant ILT2 protein. Fibroblasts obtained from GTKO/HLAG1+ pigs were shown to modulate the immune response by lowering IFN-γ production by T cells and proliferation of CD4+ and CD8+ T cells, B cells and natural killer (NK) cells, as well as by augmenting phosphorylation of Src homology region 2 domain-containing phosphatase-2 (SHP-2), which plays a central role in immune suppression. Islets isolated from GTKO/HLA-G1+ genetically engineered pigs and transplanted into streptozotocin-diabetic nude mice restored normoglycemia, suggesting that the expression of HLA-G1 did not interfere with their ability to reverse diabetes. The findings presented here suggest that the HLA-G1+ transgene can be stably expressed from the ROSA26 locus of non-fetal maternal tissue at the cell surface. By providing an immunomodulatory signal, expression of HLA-G1+ may extend survival of porcine pancreatic islet and organ xenografts.

Galactosyl carbohydrate residues on hematopoietic stem/progenitor cells are essential for homing and engraftment to the bone marrow.

The role of carbohydrate chains in leukocyte migration to inflamed sites during inflammation and trafficking to the lymph nodes under physiological conditions has been extensively characterized. Here, we report that carbohydrate chains also mediate the homing and engraftment of hematopoietic stem/progenitor cells (HSPCs) to the bone marrow (BM). In particular, we found that transplanted BM cells deficient in ß-1,4-galactosyltransferase-1 (ß4GalT-1) could not support survival in mice exposed to a lethal dose of irradiation. BM cells obtained from mice deficient in ß4GalT-1 showed normal colony-forming activity and hematopoietic stem cell numbers. However, colony-forming cells were markedly rare in the BM of recipient mice 24 h after transplantation of ß4GalT-1-deficient BM cells, suggesting that ß4GalT-1 deficiency severely impairs homing. Similarly, BM cells with a point mutation in the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase gene, encoding a key enzyme in sialic acid biosynthesis, showed mildly impaired homing and engraftment abilities. These results imply that the galactosyl, but not sialyl residues in glycoproteins, are essential for the homing and engraftment of HSPCs to the BM. These findings suggest the possibility of modifying carbohydrate structures on the surface of HSPCs to improve their homing and engraftment to the BM in clinical application.

Glt25d2 Knockout Directly Increases CD25+CD69- but Decreases CD25-CD69+ Subset Proliferation and is Involved in Concanavalin-Induced Hepatitis.

BACKGROUND/AIMS: The elaborate structure of the extracellular matrix (ECM) and the appropriate surface glycoforms upon it are indispensable to CD4+ T cell regulation. METHODS: To explore the effects of Glcα1,2Galß1 glycosylation mediated by GLT25D2 (Colgalt2) for CD4+ T cell regulation, we prepared C57BL/6J Glt25d2-/- mice. In the induction of hepatitis, after concanavalin A (Con A) challenge for 6, 12, and 24 h, more extensive parenchymal injury was noted in Glt25d2-/- mice than in wild-type (WT) mice at 12 h. Immunohistochemistry and laser scanning confocal microscopy were used to detect GLT25D2 expression, and subsets of CD4+T cells was analyzed by flow cytometry. A total of 26 cytokines in serum samples were determined using Luminex technology. RESULTS: The trend in liver injury score variation was consistent with serum alanine aminotransferase and aspartate aminotransferase levels. The levels of interleukin 4 (IL-4), IL-1ß, IL-9, and several chemokines such as macrophage inflammatory protein-2, eotaxin, and growth-related oncogene α were significantly increased in Glt25d2-/- mice compared with WT mice after Con A challenge. A further phenotype analysis of primary Glt25d2-/- CD4+ T cells showed that Glt25d2 knockout increased the frequency of the CD25+CD69- subset but decreased the frequency of the CD25-CD69+ subset after Con A challenge for 6, 12, and 24 h compared with those of WT CD4+ T cells. Activation-induced apoptosis was also significantly increased in Glt25d2-/- CD4+ T cells after Con A challenge compared with WT CD4+ T cells. Lectin microarray hybridization showed that Glt25d2 knockout increased the binding activity of Narcissus pseudonarcissus lectin to CD4+ T cells but Amaranthus caudatus lectin-binding activity was lost during Con A challenge. CONCLUSION: The present results suggest that collagen glycosylation mediated by GLT25D2 may regulate a subset of CD4+ T cells and be involved in the pathogenesis of Con A-induced hepatitis.

Generation of GTKO Diannan Miniature Pig Expressing Human Complementary Regulator Proteins hCD55 and hCD59 via T2A Peptide-Based Bicistronic Vectors and SCNT.

Pig-to-human organ transplantation has drawn attention in recent years due to the potential use of pigs as an alternative source of human donor organs. While GGTA1 knockout (GTKO) can protect xenografts from hyperacute rejection, complement-dependent cytotoxicity might still contribute to this type of rejection. To prolong the xenograft survival, we utilized a T2A-mediated pCMV-hCD55-T2A-hCD59-Neo vector and transfected the plasmid into GTKO Diannan miniature pig fetal fibroblasts. After G418 selection combined with single-cell cloning culture, four colonies were obtained, and three of these were successfully transfected with the hCD55 and hCD59. One of the three colonies was selected as donor cells for somatic cell nuclear transfer (SCNT). Then, the reconstructed embryos were transferred into eight recipient gilts, resulting in four pregnancies. Three of the pregnant gilts delivered, yielding six piglets. Only one piglet carried hCD55 and hCD59 genetic modification. The expression levels of the GGTA1, hCD55, and hCD59 in the tissues and fibroblasts of the piglet were determined by q-PCR, fluorescence microscopy, immunohistochemical staining, and western blotting analyses. The results showed the absence of GGTA1 and the coexpression of the hCD55 and hCD59. However, the mRNA expression levels of hCD55 and hCD59 in the GTKO/hCD55/hCD59 pig fibroblasts were lower than that in human 293T cells, which may be caused by low copy number and/or CMV promoter methylation. Furthermore, we performed human complement-mediated cytolysis assays using human serum solutions from 0 to 60%. The result showed that the fibroblasts of this triple-gene modified piglet had greater survival rates than that of wild-type and GTKO controls. Taken together, these results indicate that T2A-mediated polycistronic vector system combined with SCNT can effectively generate multiplex genetically modified pigs, additional hCD55 and hCD59 expression on top of a GTKO genetic background markedly enhance the protective effect towards human serum-mediated cytolysis than those of GTKO alone. Thus, we suggest that GTKO/hCD55/hCD59 triple-gene-modified Diannan miniature pig will be a more eligible donor for xenotransplantation.

Functional role of glycosphingolipids in contact inhibition of growth in a human mammary epithelial cell line.

We have demonstrated previously the involvement of certain glycosphingolipids (GSLs) in 'contact inhibition' (dependent on cell-to-cell contact) of cell growth. Here, we examined the roles of specific GSLs in contact inhibition of the human epithelial cell line MCF10A. Contact-inhibited cells show increased expression of the ganglioside GD3 and the globo-series GSL Gb3, and of the mRNAs for the corresponding sialyltransferases ST8SIA1 (GD3 synthase) and galactosyltransferase A4GALT (Gb3 synthase). siRNA knockdown (KD) of ST8SIA1 and/or A4GALT significantly suppresses contact inhibition. Exogenous addition of GD3 or Gb3 inhibits proliferation of low-density cells. Our findings suggest that GSLs play functional roles in contact inhibition of these cells and that Merlin/NF2, a tumor suppressor protein, is involved in the GSL function.

C1GALT1 Seems to Promote In Vitro Disease Progression in Ovarian Cancer.

OBJECTIVE: Aberrant glycosylation affects many cellular properties in cancers. The core 1 ß1,3-galactosyltransferase (C1GALT1), an enzyme that controls the formation of mucin-type O-glycans, has been reported to regulate hepatocellular and mammary carcinogenesis. This study aimed to explore the role of C1GALT1 in ovarian cancer. METHODS: C1GALT1 expression was assessed in a public database based on microarray data from 1287 ovarian cancer patients and ovarian cancerous tissues. Lectin blotting and flow cytometry analysis were conducted to detect changes in O-glycans on ovarian cancer cells. Effects of C1GALT1 on cell growth, migration, and sphere formation were analyzed in C1GALT1 knockdown or overexpressing ovarian cancer cells in vitro. Expression of cancer stemness-related genes was analyzed by quantitative reverse transcription polymerase chain reaction. RESULTS: High C1GALT1 expression shows a trend toward association with poor survival in ovarian cancer patients. C1GALT1 modifies O-glycan expression on surfaces and glycoproteins of ovarian cancer cells. Knockdown of C1GALT1 decreased cell growth, migration, and sphere formation of ES-2 and OVTW59-p4 cells. Conversely, overexpression of C1GALT1 promoted such malignant properties of SKOV3 cells. Furthermore, C1GALT1 regulated the expression of several cancer stemness-related genes, including CD133, CD24, Oct4, Nanog, and SNAI2, in ovarian cancer cells. CONCLUSIONS: C1GALT1 modifies O-glycan expression and enhances malignant behaviors in ovarian cancer cells, suggesting that C1GALT1 plays a role in the pathogenesis of ovarian cancer and targeting C1GALT1 could be a promising approach for ovarian cancer therapy.

Microbial, metabolomic, and immunologic dynamics in a relapsing genetic mouse model of colitis induced by T-synthase deficiency.

Intestinal dysbiosis is thought to confer susceptibility to inflammatory bowel disease (IBD), but it is unknown whether dynamic changes in the microbiome contribute to fluctuations in disease activity. We explored this question using mice with intestine-specific deletion of C1galt1 (also known as T-synthase) (Tsyn mice). These mice develop spontaneous microbiota-dependent colitis with a remitting/relapsing course due to loss of mucin core-1 derived O-glycans. 16S rRNA sequencing and untargeted metabolomics demonstrated age-specific perturbations in the intestinal microbiome and metabolome of Tsyn mice compare with littermate controls at weeks 3 (disease onset), 5 (during remission), and 9 (after relapse). Colitis remission corresponded to increased levels of FoxP3+RORγt+CD4+ T cells in the colonic lamina propria that were positively correlated with operational taxonomic units (OTUs) in the S24-7 family and negatively correlated with OTUs in the Clostridiales order. Relapse was characterized by marked expansion of FoxP3-RORγt+CD4+ T cells expressing IFNγ and IL17A, which were associated with Clostridiales OTUs distinct from those negatively correlated with FoxP3+RORγt+CD4+ T cells. Our findings suggest that colitis remission and relapse in the Tsyn model may reflect alterations in the microbiome due to reduced core-1 O-glycosylation that shift the balance of regulatory and pro-inflammatory T cell subsets. We investigated whether genetic variation in C1galt1 correlated with the microbiome in a cohort of 78 Crohn's disease patients and 101 healthy controls. Polymorphisms near C1galt1 (rs10486157) and its molecular chaperone, Cosmc (rs4825729), were associated with altered composition of the colonic mucosal microbiota, supporting the relevance of core-1 O-glycosylation to host regulation of the microbiome.

The Immune Responses and Calcification of Bioprostheses in the α1,3-Galactosyltransferase Knockout Mouse.

BACKGROUND: The study aim was to evaluate the immune reaction, difference of degenerative calcification, and anti-calcification effect of decellularization with or without α-galactosidase in bovine pericardium and porcine heart valves, using an α1,3-galactosyltransferase (α-Gal) knockout (KO) mouse model. METHODS: In order to elucidate the anti-calcification effect of decellularization with or without α-galactosidase, bovine pericardium and porcine heart valve tissues were assigned to four groups according to the tissue preparation method: (i) glutaraldehyde (GA) fixation only; (ii) decellularization + GA fixation (Decell); (iii) α-galactosidase + GA fixation (α-galactosidase); and (iv) decellularization +α-galactosidase + GA fixation (Decell + α-galactosidase). Each prepared tissue was implanted subcutaneously into α-Gal KO mice. Anti-α-Gal immunoglobulin (Ig) G and IgM antibody titers were monitored prior to implantation and at four, eight and 12 weeks after implantation using an enzyme-linked immunosorbent assay. Calcium contents of explanted tissues were measured at 12 weeks after implantation. RESULTS: There were no significant differences in the anti-α-Gal IgG antibody titers according to the type of bioprosthetic material or tissue preparation method (p >0.05). The calcium content was significantly lower in porcine heart valves than in bovine pericardium when implanted in α-Gal-KO mice (p <0.001). Calcium contents in bovine pericardium and porcine heart valves were significantly lower in the Decell, α-galactosidase and Decell + α-galactosidase groups than in the GA group (all p <0.05). CONCLUSIONS: The porcine heart valve induced lower levels of calcium deposition than did the bovine pericardium, but the anti-α-Gal IgG antibody titers did not differ significantly between the bioprosthetic tissues. Decellularization had significant anticalcification effects in both the bovine pericardium and porcine heart valves, though there was no significant difference in the anti-α-Gal IgG antibody titers among tissue preparation methods.

Loss of intestinal O-glycans promotes spontaneous duodenal tumors.

Mucin-type O-glycans, primarily core 1- and core 3-derived O-glycans, are the major mucus barrier components throughout the gastrointestinal tract. Previous reports identified the biological role of O-glycans in the stomach and colon. However, the biological function of O-glycans in the small intestine remains unknown. Using mice lacking intestinal core 1- and core 3-derived O-glycans [intestinal epithelial cell C1galt1(-/-);C3GnT(-/-) or double knockout (DKO)], we found that loss of O-glycans predisposes DKO mice to spontaneous duodenal tumorigenesis by ∼1 yr of age. Tumor incidence did not increase with age; however, tumors advanced in aggressiveness by 20 mo. O-glycan deficiency was associated with reduced luminal mucus in DKO mice before tumor development. Altered intestinal epithelial homeostasis with enhanced baseline crypt proliferation characterizes these phenotypes as assayed by Ki67 staining. In addition, fluorescence in situ hybridization analysis reveals a significantly lower bacterial burden in the duodenum compared with the large intestine. This phenotype is not reduced with antibiotic treatment, implying O-glycosylation defects, rather than bacterial-induced inflammation, which causes spontaneous duodenal tumorigenesis. Moreover, inflammatory responses in DKO duodenal mucosa are mild as assayed with histology, quantitative PCR for inflammation-associated cytokines, and immunostaining for immune cells. Importantly, inducible deletion of intestinal O-glycans in adult mice leads to analogous spontaneous duodenal tumors, although with higher incidence and heightened severity compared with mice with O-glycans constitutive deletion. In conclusion, these studies reveal O-glycans within the small intestine are critical determinants of duodenal cancer risk. Future studies will provide insights into the pathogenesis in the general population and those at risk for this rare but deadly cancer.

Chimeric 2C10R4 anti-CD40 antibody therapy is critical for long-term survival of GTKO.hCD46.hTBM pig-to-primate cardiac xenograft.

Preventing xenograft rejection is one of the greatest challenges of transplantation medicine. Here, we describe a reproducible, long-term survival of cardiac xenografts from alpha 1-3 galactosyltransferase gene knockout pigs, which express human complement regulatory protein CD46 and human thrombomodulin (GTKO.hCD46.hTBM), that were transplanted into baboons. Our immunomodulatory drug regimen includes induction with anti-thymocyte globulin and αCD20 antibody, followed by maintenance with mycophenolate mofetil and an intensively dosed αCD40 (2C10R4) antibody. Median (298 days) and longest (945 days) graft survival in five consecutive recipients using this regimen is significantly prolonged over our recently established survival benchmarks (180 and 500 days, respectively). Remarkably, the reduction of αCD40 antibody dose on day 100 or after 1 year resulted in recrudescence of anti-pig antibody and graft failure. In conclusion, genetic modifications (GTKO.hCD46.hTBM) combined with the treatment regimen tested here consistently prevent humoral rejection and systemic coagulation pathway dysregulation, sustaining long-term cardiac xenograft survival beyond 900 days.

Immunization of A4galt-deficient mice with glycosphingolipids from renal cell cancers resulted in the generation of anti-sulfoglycolipid monoclonal antibodies.

In this study, we immunized Gb3/CD77 synthase gene (A4galt) knockout (KO) mice with glycosphingolipids (GSLs) extracted from 3 renal cell cancer (RCC) cell lines to raise monoclonal antibodies (mAbs) reactive with globo-series GSLs specifically expressed in RCCs. Although a number of mAbs reactive with globo-series GSLs were generated, they reacted with both RCC cell lines and normal kidney cells. When we analyzed recognized antigens by mAbs that were specifically reactive with RCC, but not with normal kidney cells at least on the cell surface, many of them turned out to be reactive with sulfoglycolipids. Eight out of 11 RCC-specific mAbs were reactive with SM2 alone, and the other 3 mAbs were more broadly reactive with sulfated glycolipids, i.e. SM3 and SM4 as well as SM2. In the immunohistochemistry, these anti-sulfoglycolipids mAbs showed RCC-specific reaction, with no or minimal reaction with adjacent normal tissues. Thus, immunization of A4galt KO mice with RCC-derived GSLs resulted in the generation of anti sulfated GSL mAbs, and these mAbs may be applicable for the therapeutics for RCC patients.

The Role of Liver Sinusoidal Endothelial Cells in Induction of Carbohydrate Reactive B Cells Tolerance Through the Programmed Death 1/Programmed Death Ligand 1 Pathway.

BACKGROUND: A spontaneous tolerance of B cells responding to blood group antigens frequently develops in ABO-incompatible pediatric liver transplantation (LT). Liver sinusoidal endothelial cells (LSECs), which exclusively express blood group antigens in the liver, possess a capacity to induce alloantigen-specific tolerance. In this study, we elucidated the role of LSECs in the tolerance induction of blood group antigen-reactive B cells after ABO-incompatible LT using mice that lack galactose-α(1,3)galactose (Gal) epitopes resembling blood group carbohydrate antigens. METHODS: Using adoptive transfer of LSECs from wild type (WT) C57BL/6J mice to congenic α1,3-galactosyltransferase gene knockout (GalT) mice, we established orthotropic GalT → GalT LSEC chimera mice. Anti-Gal Ab (antibody) production was evaluated after immunization of GalT → GalT LSEC chimera mice with Gal rabbit RBC. RESULTS: Adoptive transfer of LSECs isolated from WT GalT mice via the portal vein resulted in persistent engraftment of Gal LSECs in congenic GalT mouse livers. Only when GalT mice were splenectomized before LSEC inoculation, the GalT → GalT LSEC chimera lost the ability to produce anti-Gal Abs. The administration of blocking monoclonal Abs (mAbs) against programmed death ligand 1 to the splenectomized GalT → GalT LSEC chimera resulted in the recovery of anti-Gal Ab production. CONCLUSIONS: These findings suggest that LSECs take a part in tolerization of immature but not mature B cells specifically for Gal. Furthermore, the programmed death 1/programmed death ligand 1 pathway likely plays a crucial role in the mechanisms underlying spontaneous tolerization of B cells responding to ABO-blood group antigens in LT.

Pig kidney graft survival in a baboon for 136 days: longest life-supporting organ graft survival to date.

The longest survival of a non-human primate with a life-supporting kidney graft to date has been 90 days, although graft survival > 30 days has been unusual. A baboon received a kidney graft from an α-1,3-galactosyltransferase gene-knockout pig transgenic for two human complement-regulatory proteins and three human coagulation-regulatory proteins (although only one was expressed in the kidney). Immunosuppressive therapy was with ATG+anti-CD20mAb (induction) and anti-CD40mAb+rapamycin+corticosteroids (maintenance). Anti-TNF-α and anti-IL-6R were administered. The baboon survived 136 days with a generally stable serum creatinine (0.6 to 1.6 mg/dl) until termination. No features of a consumptive coagulopathy (e.g., thrombocytopenia, decreased fibrinogen) or of a protein-losing nephropathy were observed. There was no evidence of an elicited anti-pig antibody response. Death was from septic shock (Myroides spp). Histology of a biopsy on day 103 was normal, but by day 136, the kidney showed features of glomerular enlargement, thrombi, and mesangial expansion. The combination of (i) a graft from a specific genetically engineered pig, (ii) an effective immunosuppressive regimen, and (iii) anti-inflammatory agents prevented immune injury and a protein-losing nephropathy, and delayed coagulation dysfunction. This outcome encourages us that clinical renal xenotransplantation may become a reality.

In vitro exposure of pig neonatal isletlike cell clusters to human blood.

BACKGROUND: Pig islet grafts have been successful in treating diabetes in animal models. One remaining question is whether neonatal pig isletlike cell clusters (NICC) are resistant to the early loss of islets from the instant blood-mediated inflammatory reaction (IBMIR). METHODS: Neonatal isletlike cell clusters were harvested from three groups of piglets-(i) wild-type (genetically unmodified), (ii) α1,3-galactosyltransferase gene-knockout (GTKO)/CD46, and (iii) GTKO/CD46/CD39. NICC samples were mixed with human blood in vitro, and the following measurements were made-antibody binding; complement activation; speed of islet-induced coagulation; C-peptide; glutamic acid decarboxylase (GAD65) release; viability. RESULTS: Time to coagulation and viability were both reduced in all groups compared to freshly drawn non-anticoagulated human blood and autologous combinations, respectively. Antibody binding to the NICC occurred in all groups. CONCLUSIONS: Neonatal isletlike cell clusters were subject to humoral injury with no difference associated to their genetic characteristics.

In vivo efficacy for novel combined anticalcification treatment of glutaraldehyde-fixed cardiac xenograft using humanized mice.

The animal immune response against Galα1,3-Galß1-4GlcNAc-R(α-Gal) epitopes gives an important cause for the failure of glutaraldehyde(GA)-fixed cardiac xenografts. This study aimed to assess the in vivo effect of our novel combined anticalcification treatment, which includes immunologic modification, using α1,3-galactosyltransferase knock-out mice to mimic human immunologic environment. Bovine pericardia were cross-linked with GA and treated with decellularization, immunologic modification with α-galactosidase, space-filler with polyethylene glycol, organic solvent, and detoxification. The bovine pericardia were subcutaneously implanted into humanized and wild type mice, and titers of anti α-Gal IgM and IgG were evaluated at various time intervals. In vivo calcification and immunohistochemistry staining was assessed for the explanted xenografts several months after implantation. In humanized mice, titers for anti α-Gal IgM and IgG increased as the period of implantation increased, and reduced with our anticalcification treatments. The humanized mice had more in vivo calcification in GA-fixed xenografts treated with our anticalcification protocol compared with wild type mice. In humanized mice, in vivo calcification reduced with our combined anticalcification treatment, and the immunohistochemistry of the harvested xenografts proved the compatible findings with the results of in vivo immunogenicity and calcification. Humanized mice are effective model for the assessment of in vivo calcification, and our combined anticalcification treatments reduced in vivo calcification as well as in vivo immunogenicity in humanized mice group, suggesting that the animal immune reaction is the cause for calcification. Our novel combined anticalcification strategies of decellularization, immunologic modification, space-filler, organic solvent, and detoxification have possible promise to prolong the lifespan of cardiac xenograft.

Glucose metabolism in pigs expressing human genes under an insulin promoter.

BACKGROUND: Xenotransplantation of porcine islets can reverse diabetes in non-human primates. The remaining hurdles for clinical application include safe and effective T-cell-directed immunosuppression, but protection against the innate immune system and coagulation dysfunction may be more difficult to achieve. Islet-targeted genetic manipulation of islet-source pigs represents a powerful tool to protect against graft loss. However, whether these genetic alterations would impair islet function is unknown. METHODS: On a background of α1,3-galactosyltransferase gene-knockout (GTKO)/human (h)CD46, additional genes (hCD39, human tissue factor pathway inhibitor, porcine CTLA4-Ig) were inserted in different combinations under an insulin promoter to promote expression in islets (confirmed by immunofluorescence). Seven pigs were tested for baseline and glucose/arginine-challenged levels of glucose, insulin, C-peptide, and glucagon. RESULTS: This preliminary study did not show definite evidence of ß-cell deficiencies, even when three transgenes were expressed under the insulin promoter. Of seven animals, all were normoglycemic at fasting, and five of seven had normal glucose disposal rates after challenge. All animals exhibited insulin, C-peptide, and glucagon responses to both glucose and arginine challenge; however, significant interindividual variation was observed. CONCLUSIONS: Multiple islet-targeted transgenic expression was not associated with an overtly detrimental effect on islet function, suggesting that complex genetic constructs designed for islet protection warrants further testing in islet xenotransplantation models.

Expanding the clinical spectrum of B4GALT7 deficiency: homozygous p.R270C mutation with founder effect causes Larsen of Reunion Island syndrome.

First described as a variant of Larsen syndrome in Reunion Island (LRS) in the southern Indian Ocean, 'Larsen of Reunion Island syndrome' is characterized by dwarfism, hyperlaxity, multiple dislocations and distinctive facial features. It overlaps with Desbuquois dysplasia, Larsen syndrome and spondyloepiphyseal dysplasia with dislocations ascribed to CANT1, FLNB and CHST3 mutations, respectively. We collected the samples of 22 LRS cases. After exclusion of CANT1, FLNB and CHST3 genes, an exome sequencing was performed in two affected second cousins and one unaffected sister. We identified a homozygous missense mutation in B4GALT7, NM_007255.2: c.808C>T p.(Arg270Cys) named p.R270C, in the two affected cases, not present in the unaffected sister. The same homozygous mutation was subsequently identified in the remaining 20 LRS cases. Our findings demonstrate that B4GALT7 is the causative gene for LRS. The identification of a unique homozygous mutation argues in favor of a founder effect. B4GALT7 encodes a galactosyltransferase, required for the initiation of glycoaminoglycan side chain synthesis of proteoglycans. This study expands the phenotypic spectrum of B4GALT7 mutations, initially described as responsible for the progeroid variant of Ehlers-Danlos syndrome. It further supports a common physiopathological basis involving proteoglycan synthesis in skeletal disorders with dislocations.

Genetically engineered pigs and target-specific immunomodulation provide significant graft survival and hope for clinical cardiac xenotransplantation.

OBJECTIVES: Cardiac transplantation and available mechanical alternatives are the only possible solutions for end-stage cardiac disease. Unfortunately, because of the limited supply of human organs, xenotransplantation may be the ideal method to overcome this shortage. We have recently seen significant prolongation of heterotopic cardiac xenograft survival from 3 to 12 months and beyond. METHODS: Hearts from genetically engineered piglets that were alpha 1-3 galactosidase transferase knockout and expressed the human complement regulatory gene, CD46 (groups A-C), and the human thrombomodulin gene (group D) were heterotropically transplanted in baboons treated with antithymocyte globulin, cobra venom factor, anti-CD20 antibody, and costimulation blockade (anti-CD154 antibody [clone 5C8]) in group A, anti-CD40 antibody (clone 3A8; 20 mg/kg) in group B, clone 2C10R4 (25 mg/kg) in group C, or clone 2C10R4 (50 mg/kg) in group D, along with conventional nonspecific immunosuppressive agents. RESULTS: Group A grafts (n = 8) survived for an average of 70 days, with the longest survival of 236 days. Some animals in this group (n = 3) developed microvascular thrombosis due to platelet activation and consumption, which resulted in spontaneous hemorrhage. The median survival time was 21 days in group B (n = 3), 80 days in group C (n = 6), and more than 200 days in group D (n = 5). Three grafts in group D are still contracting well, with the longest ongoing graft survival surpassing the 1-year mark. CONCLUSIONS: Genetically engineered pig hearts (GTKOhTg.hCD46.hTBM) with modified targeted immunosuppression (anti-CD40 monoclonal antibody) achieved long-term cardiac xenograft survival. This potentially paves the way for clinical xenotransplantation if similar survival can be reproduced in an orthotopic transplantation model.

Anti-non-Gal-specific combination treatment with an anti-idiotypic Ab and an inhibitory small molecule mitigates the xenoantibody response.

BACKGROUND: B-cell depletion significantly extends survival of α-1,3-galactosyltranferase knockout (GTKO) porcine organs in pig-to-primate models. Our previous work demonstrated that the anti-non-Gal xenoantibody response is structurally restricted. Selective inhibition of xenoantigen/xenoantibody interactions could prolong xenograft survival while preserving B-cell-mediated immune surveillance. METHODS: The anti-idiotypic antibody, B4N190, was selected from a synthetic human phage display library after enrichment against a recombinant anti-non-Gal xenoantibody followed by functional testing in vitro. The inhibitory small molecule, JMS022, was selected from the NCI diversity set III using virtual screening based on predicted xenoantibody structure. Three rhesus monkeys were pre-treated with anti-non-Gal-specific single-chain anti-idiotypic antibody, B4N190. A total of five monkeys, including two untreated controls, were then immunized with GTKO porcine endothelial cells to initiate an anti-non-α-1,3-Gal (non-Gal) xenoantibody response. The efficacy of the inhibitory small molecule specific for anti-non-Gal xenoantibody, JMS022, was tested in vitro. RESULTS: After the combination of in vivo anti-id and in vitro small molecule treatments, IgM xenoantibody binding to GTKO cells was reduced to pre-immunization levels in two-thirds of animals; however, some xenoantibodies remained in the third animal. Furthermore, when treated with anti-id alone, all three experimental animals displayed a lower anti-non-Gal IgG xenoantibody response compared with controls. Treatment with anti-idiotypic antibody alone reduced IgM xenoantibody response intensity in only one of three monkeys injected with GTKO pig endothelial cells. In the one experimental animal, which displayed reduced IgM and IgG responses, select B-cell subsets were also reduced by anti-id therapy alone. Furthermore, natural antibody responses, including anti-laminin, anti-ssDNA, and anti-thyroglobulin antibodies were intact despite targeted depletion of anti-non-Gal xenoantibodies in vivo indicating that selective reduction of xenoantibodies can be accomplished without total B-cell depletion. CONCLUSIONS: This preliminary study demonstrates the strength of approaches designed to selectively inhibit anti-non-Gal xenoantibody. Both anti-non-Gal-specific anti-idiotypic antibody and small molecules can be used to selectively limit xenoantibody responses.