Clinical Trial Protocol for Porcine Islet Xenotransplantation in South Korea.

Background: Islet transplantation holds promise for treating selected type 1 diabetes mellitus patients, yet the scarcity of human donor organs impedes widespread adoption. Porcine islets, deemed a viable alternative, recently demonstrated successful longterm survival without zoonotic risks in a clinically relevant pig-to-non-human primate islet transplantation model. This success prompted the development of a clinical trial protocol for porcine islet xenotransplantation in humans. Methods: A single-center, open-label clinical trial initiated by the sponsor will assess the safety and efficacy of porcine islet transplantation for diabetes patients at Gachon Hospital. The protocol received approval from the Gachon Hospital Institutional Review Board (IRB) and the Korean Ministry of Food and Drug Safety (MFDS) under the Investigational New Drug (IND) process. Two diabetic patients, experiencing inadequate glycemic control despite intensive insulin treatment and frequent hypoglycemic unawareness, will be enrolled. Participants and their family members will engage in deliberation before xenotransplantation during the screening period. Each patient will receive islets isolated from designated pathogen-free pigs. Immunosuppressants and systemic infection prophylaxis will follow the program schedule. The primary endpoint is to confirm the safety of porcine islets in patients, and the secondary endpoint is to assess whether porcine islets can reduce insulin dose and the frequency of hypoglycemic unawareness. Conclusion: A clinical trial protocol adhering to global consensus guidelines for porcine islet xenotransplantation is presented, facilitating streamlined implementation of comparable human trials worldwide.

Characterization of Escherichia coli Strains for Novel Production of Plasmodium ovale Lactate Dehydrogenase.

Malaria is one of the most prevalent diseases worldwide with high incidence and mortality. Among the five species that can infect humans, Plasmodium ovale morphologically resembles Plasmodium vivax, resulting in misidentification and confusion in diagnosis, and is responsible for malarial disease relapse due to the formation of hypnozoites. P. ovale receives relatively less attention compared to other major parasites, such as P. falciparum and P. vivax, primarily due to its lower pathogenicity, mortality rates, and prevalence rates. To efficiently produce lactate dehydrogenase (LDH), a major target for diagnosing malaria, this study used three Escherichia coli strains, BL21(DE3), BL21(DE3)pLysS, and Rosetta(DE3), commonly used for recombinant protein production. These strains were characterized to select the optimal strain for P. ovale LDH (PoLDH) production. Gene cloning for recombinant PoLDH production and transformation of the three strains for protein expression were performed. The optimal PoLDH overexpression and washing buffer conditions in nickel-based affinity chromatography were established to ensure high-purity PoLDH. The yields of PoLDH expressed by the three strains were as follows: BL21(DE3), 7.6 mg/L; BL21(DE3)pLysS, 7.4 mg/L; and Rosetta(DE3), 9.5 mg/L. These findings are expected to be highly useful for PoLDH-specific diagnosis and development of antimalarial therapeutics.

Long-term control of diabetes by tofacitinib-based immunosuppressive regimen after allo islet transplantation in diabetic rhesus monkeys that rejected previously transplanted porcine islets.

Porcine islet xenotransplantation has been highlighted as an alternative to allo islet transplantation. Despite the remarkable progress that has been made in porcine-islet pre-clinical studies in nonhuman primates, immunological tolerance to porcine islets has not been achieved to date. Therefore, allo islet transplantation could be required after the failure of porcine islet xenotransplantation. Here, we report the long-term control of diabetes by allogeneic pancreatic islet transplantation in diabetic rhesus monkeys that rejected previously transplanted porcine islets. Four diabetic male rhesus monkeys received the porcine islets and then allo islets (5700-19 000 IEQ/kg) were re-transplanted for a short or long period after the first xeno islet rejection. The recipient monkeys were treated with an immunosuppressive regimen consisting of ATG, humira, and anakinra for induction, and sirolimus and tofacitinib for maintenance therapy. The graft survival days of allo islets in these monkeys were >440, 395, >273, and 127, respectively, similar to that in allo islet transplanted cynomolgus monkeys that received the same immunosuppressive regimen without xeno sensitization. Taken together, it is likely that prior islet xenotransplantation does not affect the survival of subsequent allo islets under clinically applicable immunosuppressants.

Expression, Purification, and Characterization of Plasmodium vivax Lactate Dehydrogenase from Bacteria without Codon Optimization.

Plasmodium vivax is the most widespread cause of malaria, especially in subtropical and temperate regions such as Asia-Pacific and America. P. vivax lactate dehydrogenase (PvLDH), an essential enzyme in the glycolytic pathway, is required for the development and reproduction of the parasite. Thus, LDH from these parasites has garnered attention as a diagnostic biomarker for malaria and as a potential molecular target for developing antimalarial drugs. In this study, we prepared a transformed Escherichia coli strain for the overexpression of PvLDH without codon optimization. We introduced this recombinant plasmid DNA prepared by insertion of the PvLDH gene in the pET-21a(+) expression vector, into the Rosetta(DE3), an E. coli strain suitable for eukaryotic protein expression. The time, temperature, and inducer concentration for PvLDH expression from this E. coli Rosetta(DE3), containing the original PvLDH gene, were optimized. We obtained PvLDH with a 31.0 mg/L yield and high purity (>95%) from this Rosetta(DE3) strain. The purified protein was characterized structurally and functionally. The PvLDH expressed and purified from transformed bacteria without codon optimization was successfully demonstrated to exhibit its potential tetramer structure and enzyme activity. These findings are expected to provide valuable insights for research on infectious diseases, metabolism, diagnostics, and therapeutics for malaria caused by P. vivax.

Long-term control of diabetes in a nonhuman primate by two separate transplantations of porcine adult islets under immunosuppression.

Porcine islet transplantation is an alternative to allo-islet transplantation. Retransplantation of islets is a routine clinical practice in islet allotransplantation in immunosuppressed recipients and will most likely be required in islet xenotransplantation in immunosuppressed recipients. We examined whether a second infusion of porcine islets could restore normoglycemia and further evaluated the efficacy of a clinically available immunosuppression regimen including anti-thymocyte globulin for induction; belimumab, sirolimus, and tofacitinib for maintenance and adalimumab, anakinra, IVIg, and tocilizumab for inflammation control in a pig to nonhuman primate transplantation setting. Of note, all nonhuman primates were normoglycemic after the retransplantation of porcine islets without induction therapy. Graft survival was >100 days for all 3 recipients, and 1 of the 3 monkeys showed insulin independence for >237 days. Serious lymphodepletion was not observed, and rhesus cytomegalovirus reactivation was controlled without any serious adverse effects throughout the observation period in all recipients. These results support the clinical applicability of additional infusions of porcine islets. The maintenance immunosuppression regimen we used could protect the reinfused islets from acute rejection.

Enhancement of anti-inflammatory and immunomodulatory effects of adipose-derived human mesenchymal stem cells by making uniform spheroid on the new nano-patterned plates.

Human mesenchymal stem cells (MSCs) are known to have anti-inflammatory and immunomodulatory functions; thus, several MSC products have been applied as cell therapy in clinical trials worldwide. Recent studies have demonstrated that MSC spheroids have superior anti-inflammatory and immunomodulatory functions to a single cell suspension. Current methods to prepare MSC spheroids include hanging drop, concave microwell aggregation, spinner flask, and gravity circulation. However, all these methods have limitations such as low scalability, easy cell clumping, low viability, and irregular size distribution. Here, we present a nano-patterned culture plasticware named PAMcell™ 3D plate to overcome these limitations. Nano-sized silica particles (700 nm) coated with RGD peptide were arrayed into fusiform onto the PLGA film. This uniform array enabled the seeded MSCs to grow only on the silica particles, forming uniform-sized semi-spheroids within 48 h. These MSC spheroids have been shown to have enhanced stemness, anti-inflammatory, and immunomodulatory functions, as revealed by the increased expression of stem cell markers (Oct4, Sox2, and Nanog), anti-inflammatory (IL-10, TSG6, and IDO), and immunomodulatory molecules (HGF, VEGF, CXCR4) both at mRNA and protein expression levels. Furthermore, these MSC spheroids demonstrated an increased palliative effect on glycemic control in a multiple low-dose streptozotocin-induced diabetes model compared with the same number of MSC single cell suspensions. Taken together, this study presents a new method to produce uniform-sized MSC spheroids with enhanced anti-inflammatory and immunomodulatory functions in vitro and in vivo.

Long-term porcine islet graft survival in diabetic non-human primates treated with clinically available immunosuppressants.

BACKGROUND: Although pancreatic islet transplantation is becoming an effective therapeutic option for patients with type 1 diabetes (T1D) who suffer from a substantially impaired awareness of hypoglycemia, its application is limited due to the lack of donors. Thus, pig-to-human islet xenotransplantation has been regarded as a promising alternative due to the unlimited number of "donor organs." Long-term xenogeneic islet graft survival in pig-to-non-human primate (NHP) models has mainly been achieved by administering the anti-CD154 mAb-based immunosuppressant regimen. Since the anti-CD154 mAb treatment has been associated with unexpected fatal thromboembolic complications in clinical trials, the establishment of a new immunosuppressant regimen that is able to be directly applied in clinical trials is an urgent need. METHODS: We assessed an immunosuppressant regimen composed of clinically available agents at porcine islet transplantation in consecutive diabetic NHPs. RESULTS: Porcine islet graft survival in consecutive diabetic NHPs (n = 7; >222, >200, 181, 89, 62, 55, and 34 days) without severe adverse events. CONCLUSION: We believe that our study could contribute greatly to the initiation of islet xenotransplantation clinical trials.

CD4+ /CD8+ T-cell ratio correlates with the graft fate in pig-to-non-human primate islet xenotransplantation.

BACKGROUND: Xenogeneic islet transplantation using porcine pancreata has been a promising option for substituting human islet transplantation. Moreover, recent advances in pre-clinical results have put islet xenotransplantation closer to the possibility of clinical application. While preparing for the era of clinical xenotransplantation, developing non-invasive immune monitoring method which could predict the graft fate could benefit the patient. However, there are few reports showing predictive immune parameters associated with the fate of the graft in islet xenotransplantation. METHODS: The absolute number and ratio of T-cell subsets have been measured via flow cytometry from the peripheral blood of 16 rhesus monkeys before and after porcine islet xenotransplantation. The correlation between the graft survival and the absolute number or ratio of T cells was retrospectively analyzed. RESULTS: The ratio of CD4+ versus CD8+ T cells was significantly reduced due to CD8+ effector memory cells' increase. Correlation analyses revealed that CD4+ /CD8+ , CD4+ /CD8+ naïve, CD4+ naïve/CD8+ naïve, and CD4+ central memory/CD8+ naïve cell ratios negatively correlated with the duration of graft survival. Conversely, further analyses discovered strong, positive correlation of CD4+ /CD8+ cell ratios within the early graft-rejected monkeys (≤60 days). CONCLUSIONS: This retrospective study demonstrated that CD4+ /CD8+ ratios correlated with graft survival, especially in recipients which rejected the graft in early post-transplantation periods. CD4+ /CD8+ ratios could be used as a surrogate marker to predict the graft fate in pig-to-NHP islet xenotransplantation.

Bioinformatic analysis of peripheral blood RNA-sequencing sensitively detects the cause of late graft loss following overt hyperglycemia in pig-to-nonhuman primate islet xenotransplantation.

Clinical islet transplantation has recently been a promising treatment option for intractable type 1 diabetes patients. Although early graft loss has been well studied and controlled, the mechanisms of late graft loss largely remains obscure. Since long-term islet graft survival had not been achieved in islet xenotransplantation, it has been impossible to explore the mechanism of late islet graft loss. Fortunately, recent advances where consistent long-term survival (≥6 months) of adult porcine islet grafts was achieved in five independent, diabetic nonhuman primates (NHPs) enabled us to investigate on the late graft loss. Regardless of the conventional immune monitoring methods applied in the post-transplant period, the initiation of late graft loss could rarely be detected before the overt graft loss observed via uncontrolled blood glucose level. Thus, we retrospectively analyzed the gene expression profiles in 2 rhesus monkey recipients using peripheral blood RNA-sequencing (RNA-seq) data to find out the potential cause(s) of late graft loss. Bioinformatic analyses showed that highly relevant immunological pathways were activated in the animal which experienced late graft failure. Further connectivity analyses revealed that the activation of T cell signaling pathways was the most prominent, suggesting that T cell-mediated graft rejection could be the cause of the late-phase islet loss. Indeed, the porcine islets in the biopsied monkey liver samples were heavily infiltrated with CD3+ T cells. Furthermore, hypothesis test using a computational experiment reinforced our conclusion. Taken together, we suggest that bioinformatics analyses with peripheral blood RNA-seq could unveil the cause of insidious late islet graft loss.

A combination regimen of low-dose bortezomib and rapamycin prolonged the graft survival in a murine allogeneic islet transplantation model.

As the first FDA-approved proteasome inhibitor drug, bortezomib has been used for the treatment of multiple myeloma and lymphoma. However, its effects alone or in combination with other immunosuppressants on allogeneic islet transplantation have not been reported so far. In this study, we showed that the short-term combination treatment of low-dose bortezomib and rapamycin significantly prolonged the survival of islet allografts. Short-term treatment of low-dose (0.05 mg/kg or 0.1 mg/kg) bortezomib reduced the MHC class II expression in dendritic cells (DCs) of alloantigen-sensitized mice, and prolonged the islet allograft survival for up to 50 days in diabetic mice. Notably, when bortezomib was combined with rapamycin, it induced islet-specific immunological tolerance which allowed the acceptance of a second graft without additional immunosuppression. This regimen dramatically reduced the alloantigen-specific IFN-γ-producing T cells in the spleen, and increased regulatory T cells both at the graft site and in the spleen. Therefore, we propose that short-term treatment of low-dose bortezomib and rapamycin could be a new tolerance-promoting immunosuppressive regimen for allogeneic islet transplantation.

JAK3 inhibitor-based immunosuppression in allogeneic islet transplantation in cynomolgus monkeys.

Islet transplantation is efficacious to prevent severe hypoglycemia and glycemic liability of selected patients of type 1 diabetes. However, since calcineurin inhibitor (CNI) causes ß-cell and nephrotoxicity, alternative drug(s) with similar potency and safety profile to CNI will be highly desirable. Here we tested whether JAK3 inhibitor, tofacitinib could be used instead of tacrolimus in CIT07 immunosuppression regimen in cynomolgus nonhuman primate (NHP) model. Five independent streptozotocin (STZ)-induced diabetic monkeys were transplanted with MHC-mismatched allogeneic islets and three animals were further re-transplanted upon insufficient glycemic control or early islet graft rejection. After islet transplantation, blood glucose levels were quickly stabilized and maximal islet graft survival as measured by serum C-peptide concentration was >330, 98, >134, 31, or 22 days, respectively, after transplantation (median survival day; 98 days). Cellular and humoral immune responses were efficiently suppressed by JAK3 inhibitor-based immunosuppression during the follow-up periods. Although intermittent increases of the genome copy number of cynomolgus cytomegalovirus (CMV) were detected by quantitative real-time PCR analyses, serious infections or posttransplant lymphoproliferative disease (PTLD) was not found in all animals. Taken together, we have shown that JAK3 inhibitor could be used in replacement of tacrolimus in a highly translatable NHP islet transplantation model and these results suggest that JAK3 inhibitor will be potentially incorporated in human allogeneic islet transplantation.

Galectin-4 Interaction with CD14 Triggers the Differentiation of Monocytes into Macrophage-like Cells via the MAPK Signaling Pathway.

Galectin-4 (Gal-4) is a ß-galactoside-binding protein mostly expressed in the gastrointestinal tract of animals. Although intensive functional studies have been done for other galectin isoforms, the immunoregulatory function of Gal-4 still remains ambiguous. Here, we demonstrated that Gal-4 could bind to CD14 on monocytes and induce their differentiation into macrophage-like cells through the MAPK signaling pathway. Gal-4 induced the phenotypic changes on monocytes by altering the expression of various surface molecules, and induced functional changes such as increased cytokine production and matrix metalloproteinase expression and reduced phagocytic capacity. Concomitant with these changes, Gal-4-treated monocytes became adherent and showed elongated morphology with higher expression of macrophage markers. Notably, we found that Gal-4 interacted with CD14 and activated the MAPK signaling cascade. Therefore, these findings suggest that Gal-4 may exert the immunoregulatory functions through the activation and differentiation of monocytes.

Peri-graft porcine-specific CD4+ FoxP3+ regulatory T cells by CD40-CD154 blockade prevented the rejection of porcine islet graft in diabetic mice.

BACKGROUND: Anti-CD154 monoclonal antibody (mAb) treatment has been known to have potential to induce immune tolerance in organ transplantation. Several studies have suggested the involvement of CD4+ regulatory T cells (Treg s) in xeno-immune tolerance. However, the characteristics of Treg s and the mechanisms of their regulatory functions in islet xenotransplantation have not been clearly defined. METHOD: Adult porcine islet cells were isolated and purified, and were transplanted under the kidney capsule of diabetic C57BL/6J mice with the administration of 0.5 mg/mouse of anti-CD154 mAb on 0, 1, 3, 5, and 7 days post-transplantation (DPT). The graft survival was monitored by blood glucose level. The islet graft and recipients' cells were analyzed by immunohistochemistry (IHC), flow cytometry, enzyme-linked immunosorbent spot (ELISPOT) assay, and mixed-lymphocyte reaction. RESULTS: Short-term anti-CD154 mAb monotherapy enabled the porcine islet graft to survive indefinitely in diabetic mice (n = 18). Immunohistochemical staining showed significantly higher ratio of CD4+ Foxp3+ Treg s in the peri-graft site, but not in the spleen and kidney-draining lymph node of the recipient mice. Depletion of Treg s evoked graft rejection, and adoptive transfer of Treg s from anti-CD154 mAb-treated recipients provided protection to the graft from rejection. These Treg s showed more potent suppressive capacity than those from the untreated control and were found to be porcine antigen-specific. CONCLUSIONS: In this study, we showed that anti-CD154 mAb monotherapy resulted in indefinite porcine islet graft survival in mice. The porcine-specific CD4+ Foxp3+ Treg s in the peri-graft site played the critical role in the protection of islet graft from rejection, which suggests a prospective immunosuppressive strategy for islet xenotransplantation.

Ascites formation accompanied by portal vein thrombosis after porcine islet xenotransplantation via the portal vein in Rhesus macaque (Macaca mulatta).

Pig-to-nonhuman primate (NHP) islet transplantation has been widely conducted as a preclinical xenotransplantation model prior to human clinical trial. Portal vein thrombosis is one of the complications associated with islet infusion through the portal vein into the liver. Here, we briefly report severe case of ascites formation accompanied by portal vein thrombi after pig-to-NHP islet xenotransplantation in a rhesus monkey. Meticulous prophylactic treatment such as continuous heparin infusion should be implemented to prevent portal vein thrombi in pig-to-NHP islet transplantation models.

Vascularization of PLGA-based bio-artificial beds by hypoxia-preconditioned mesenchymal stem cells for subcutaneous xenogeneic islet transplantation.

BACKGROUND: Subcutaneous tissue is an attractive extra-hepatic heterotopic site for islet transplantation; however, poor oxygen tension and blood supply during early engraftment of implanted islets have limited the use of this site in clinical applications. METHODS: This study investigated the vascularization potential of hypoxia-preconditioned mesenchymal stem cells (3% O2 ; hypo-MSCs) in PLGA-based bio-artificial beds for subsequent subcutaneous islet transplantation. Sheet-typed polymeric PLGA scaffolds coated with hypo-MSCs or normo-MSCs (MSCs cultured under normoxia conditions, 21% O2 ) were implanted subcutaneously in mice. RESULTS: Compared to normo-MSCs, hypo-MSCs significantly enhanced vasculogenesis, both on the interior and exterior surfaces of the implanted PLGA devices, which peaked 4 weeks after implantation. Further, infusion of porcine islets inside the prevascularized PLGA bed restored normal glycemic control in 6 of 6 STZ-induced diabetic mice. The mass of the marginal islet was approximately 2000 IEQs, which is comparable to that required for the renal subcapsular space, a highly vascularized site. CONCLUSIONS: Therefore, PLGA-based bio-artificial devices prevascularized with hypo-MSCs could be a useful modality for successful subcutaneous islet transplantation, which is of high clinical relevance.

Absence of spontaneous regeneration of endogenous pancreatic ß-cells after chemical-induced diabetes and no effect of GABA on α-to-ß cell transdifferentiation in rhesus monkeys.

ß-cell deficiency is common feature of type 1 and late-stage of type 2 diabetes mellitus. Thus, ß-cell replacement therapy has been the focus of regenerative medicine past several decades. Particularly, evidences suggest that ß-cell regeneration via transdifferentiation from sources including α-cells is promising. However, data using higher mammals besides rodents are scarce. Here, we examined whether endogenous pancreatic ß-cells could regenerate spontaneously or under normoglycemia following porcine islet transplantation for varied periods up to 1197 days after streptozotocin-induced diabetes, and remaining α-cells transdifferentiate into ß-cells by GABA treatment in vivo and in vitro. The results showed that endogenous ß-cells rarely regenerate in both conditions as evidenced by stagnant serum C-peptide levels and ß-cell number in the pancreas, and the remaining α-cells did not transdifferentiate into ß-cells by GABA treatment. Collectively, we concluded that monkey ß-cells had relatively low regenerative potential compared with rodent counterpart and GABA treatment could not induce α-to-ß-cell transdifferentitation.

Tacrolimus-induced asymptomatic thrombotic microangiopathy diagnosed by laboratory tests in pig-to-rhesus corneal xenotransplantation: A case report.

Tacrolimus-associated thrombotic microangiopathy (TA-TMA) is a rare complication. TA-TMA is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and organ damage due to thrombus. We report asymptomatic TA-TMA diagnosed by laboratory tests in pig-to-rhesus corneal xenotransplantation. Corneal transplantation had been conducted from a wild-type SNU miniature pig to a rhesus macaque. The veterinary records were retrospectively reviewed in this case. The immunosuppressive regimen consisted of rituximab, basiliximab, and IVIg as inductive therapies, and steroids with tacrolimus (0.1 mg/kg/day) as maintenance therapies. Although there were no clinical symptoms, increased levels of lactate dehydrogenase, total bilirubin, blood urea nitrogen, and creatinine and decreased levels of hemoglobin and platelet were observed in laboratory tests on Day (D) 61. Systemic TA-TMA was tentatively diagnosed. Tacrolimus was discontinued starting on D71. Dalteparin, clopidogrel bisulfate (D77-D99), and IVIg (D72) were administered as a conservative treatment. Abnormal laboratory results were reversed on D99. When low-dose tacrolimus (0.07 mg/kg/day) was re-administered on D131 to prevent rejection of the graft, TMA was detected again by laboratory tests on D161, confirming the initial diagnosis. Discontinuation of tacrolimus on D162 and re-administration of Dalteparin (D161-D196) corrected the laboratory values on D161. This report shows that in pig-to-rhesus corneal xenotransplantation, clinically asymptomatic TMA can be induced by tacrolimus, and the discontinuation of tacrolimus and administration of anticoagulant seems sufficient to correct the laboratory TMA.

Construction of EMSC-islet co-localizing composites for xenogeneic porcine islet transplantation.

Pancreatic islet transplantation is an ultimate solution for treating patients with type 1 diabetes (T1D). The pig is an ideal donor of islets for replacing scarce human islets. Besides immunological hurdles, non-immunological hurdles including fragmentation and delayed engraftment of porcine islets need solutions to succeed in porcine islet xenotransplantation. In this study, we suggest a simple but effective modality, a cell/islet co-localizing composite, to overcome these challenges. Endothelial-like mesenchymal stem cells (EMSCs), differentiated from bone-marrow derived mouse mesenchymal stem cells (MSCs), and MSCs evenly coated the surface of porcine islets (>85%) through optimized culture conditions. Both MSCs and EMSCs significantly reduced the fragmentation of porcine islets and increased the islet masses, designated as islet equivalents (IEQs). In fibrin in vitro and in vivo angiogenesis analysis, constructed EMSC-islet composites showed higher angiogenic potentials than naked islets, MSC-islet composites, or human endothelial cell-islet composites. This novel delivery method of porcine islets may have beneficial effects on the engraftment of transplanted islets by prevention of fragmentation and enhancement of revascularization.

The value of glycated albumin for the prediction of graft outcome in the non-human primate porcine islet transplantation model.

BACKGROUND: The development of a precise and easy-to-use tool for monitoring islet graft function is important in clarifying the causes of graft loss, identifying appropriate therapy, and ensuring graft survival in the nonhuman primate (NHP) model of porcine islet transplantation (PITx). Glycated albumin (GA) is an indicator of intermediate-term changes in blood glucose control and is useful in clinical diabetes management. The validity of GA for monitoring graft function in NHP recipients of PITx was evaluated using a retrospective analysis of cohort samples. METHODS: Data from a total of 23 PITxs performed in 20 recipients (3 were retransplanted) were included in this study. Islet clusters purified from adult wild-type pigs were transplanted via the intraportal route into streptozotocin-induced diabetic rhesus monkeys with immune suppression. Blood samples were obtained once per week from the recipients until they lost insulin-independence. Blood samples were also obtained from 69 non-diabetic monkeys that served as a control group. The levels of GA and albumin in stored plasma aliquots were measured using each enzymatic method, and the GA result was expressed as the percentage of GA level to the total albumin level. RESULTS: The median level of GA in the recipients on the day of PITx (median 18.6%, 95% confidence interval [CI] 16.7%-20.4%) was significantly higher than that of healthy controls (median 9.14%, 95% CI 9.0%-9.3%, P < .0001). However, the level decreased after PITx and remained low or increased depending on the extent of residual graft function. The GA level at a nadir (median 11.6%, 95% CI 10.8%-13.0%) and the time to reach a nadir (median 43 days, 95% CI 21.7-69.3 days) both correlated with the duration of insulin-independence (rho [ρ] = -.605, P = .0028 and ρ = .662, P = .0008, respectively). The GA level strongly correlated with KG , the glucose disappearance rate during intravenous glucose tolerance testing (ρ = -.76, P < .0001). At post-transplant week (PTW) 3 and at PTW 4, the GA levels in recipients with long-term insulin-independence (>90 days) were significantly lower than those with short-term insulin-independence, which revealed the excellent performance for the prediction of long-term insulin-independence that is comparable to that of porcine C-peptide (historic data). CONCLUSIONS: As a surrogate indicator for graft function, serial measurement of GA may provide Supporting Information to that obtained from conventional monitoring techniques of graft function for assessing porcine islet grafts in NHP models.