Haneulnari Lee

The role of the alternative complement pathway in early graft loss after intraportal porcine islet xenotransplantation.

Background Intraportal islet transplantation (ITx) causes instant blood-mediated inflammatory reaction (IBMIR), resulting in an early loss of transplanted islets. Porcine islets, transplanted intraportally into nonhuman primates (NHPs), induce complement activation, contributing to the development of IBMIR; however, the exact mechanism is not clear. Methods Complement activation were compared after incubation of purified adult porcine islets in 20% human serum with or without complement inhibitors, C1 esterase inhibitor (C1E-inh), anti-factor B, and purified human factor H. Intraportal porcine ITx was performed in diabetic NHPs to which cobra venom factor (CVF), factor H, or none of complement inhibitor was administered during the peritransplant period. The extent of complement activation and function of islet grafts were monitored after ITx. Results The incubation of porcine islets with human serum resulted in generation of C3a, C4d, and factor Bb in the fluid phase. However, the generation of C3a after incubation was suppressed by anti-factor B or factor H, but not by C1E-inh. Moreover, in NHPs with porcine ITx, the administration of CVF or factor H ameliorated the increase in plasma C3a and factor Bb levels, as well as early release of porcine C-peptide after ITx. Furthermore, the functional survival of islet grafts was prolonged in the recipients of the CVF group compared to those in the control group. Conclusions The alternative complement pathway contributes to the development of IBMIR and the early loss of grafts in NHPs with porcine ITx. Complement inhibition during the peritransplant period may be beneficial for the survival of islet grafts.

The role of phagocytosis in IL-8 production by human monocytes in response to lipoproteins on Staphylococcus aureus.

Cytokine responses to microbes are triggered by pattern recognition receptors, such as Toll-like receptors (TLRs), which sense pathogen-associated molecular patterns. Cell wall-associated triacylated lipoproteins in Staphylococcus aureus are known to be native TLR2 ligands that mediate host inflammatory responses against S. aureus. However, the mechanism by which these lipidated lipoproteins, which are buried under the thick S. aureus cell wall, work to stimulate TLR2 remains unclear. Heat-killed wild type S. aureus cells activated human monocytic THP-1 cells to produce proinflammatory cytokines, including interleukin (IL)-8, whereas the lipoprotein lipidation-deficient lgt mutant induced less than an eighth of the amount of IL-8 induced by the wild type. IL-8 induction in response to heat-killed S. aureus cells in THP-1 cells was not inhibited by a blocking antibody against cell surface TLR2, suggesting that intracellular TLR2 might be involved in the induction of IL-8 by S. aureus lipoprotein. The relationship between phagocytosis and IL-8 production in THP-1 cells was analyzed on a single-cell level by flow cytometry using fluorescein-labeled S. aureus cells and phycoerythrin-labeled anti-IL-8 antibody. Production of intracellular IL-8 was correlated with phagocytosis of S. aureus cells in THP-1 cells and in human peripheral blood mononuclear cells. Opsonization of S. aureus cells enhanced both the phagocytosis of S. aureus cells and the production of intracellular IL-8 in THP-1 cells. These results suggest that lipidated lipoproteins on S. aureus cells stimulate human monocytes after phagocytosis.

Dissociation between anti-porcine albumin and anti-Gal antibody responses in non-human primate recipients of intraportal porcine islet transplantation.

To understand humoral responses elicited after xenotransplantation, we compared the induction of anti‐non‐Gal antibodies vs. anti‐Gal antibodies in non‐human primates (NHPs) after intraportal porcine islet transplantation (PITX).

Increased human tumor necrosis factor-α levels induce procoagulant change in porcine endothelial cells in vitro.

Lee KG, Lee H, Ha JM, Lee YK, Kang HJ, Park C‐G, Kim SJ. Increased human tumor necrosis factor‐α levels induce procoagulant change in porcine endothelial cells in vitro. Xenotransplantation 2012; 19: 186–195.

Increase in Anti-Gal IgM Level is Associated With Early Graft Failure in Intraportal Porcine Islet Xenotransplantation

Background Anti-Gal is a major antibody induced in non-human primates (NHPs) after xenotransplantation. To understand the mechanism of graft rejection, we investigated the association between anti-Gal responses and graft failure in NHP recipients of porcine islet transplantation (PITx). Methods Intraportal PITx was performed in 35 diabetic NHPs, and graft function was monitored. Early graft failure (EGF) was defined as loss of graft function within a month after PITx. Seven, 19, nine NHPs received immunosuppression (IS) without CD40 pathway blockade (Group I), with anti-CD154 (Group II), and with anti-CD40 (Group III), respectively. The anti-Gal levels on day 0 and day 7 of PITx were measured by ELISA. Results The frequency of EGF was significantly lower in Group II (26.3%) than in Group I (100%, P=0.0012) and Group III (77.8%, P=0.0166). While levels of anti-Gal IgG in Group I and anti-Gal IgM in Group III increased on day 7 compared with day 0 (P=0.0156 and 0.0273), there was no increase in either on day 7 in Group II. The ratio of anti-Gal IgM or IgG level on day 7 to that on day 0 (Ratio7/0) was significantly higher in recipients with EGF than without EGF (P=0.0009 and 0.0027). ROC curve analysis of anti-Gal IgM Ratio7/0 revealed an area under the curve of 0.789 (P=0.0003). Conclusions IS with anti-CD154 suppressed anti-Gal responses and prevented EGF in PITx. Anti-Gal IgM Ratio7/0, being associated with EGF, is a predictive marker for EGF.

The Value of Glycated Albumin for Monitoring Graft Function in the Non-Human Primate Porcine Islet Transplantation Model

Background The development of a precise and simple-to-use monitoring tool for 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 re-transplanted) 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 (Ins-Ind). Blood was 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 < 0.0001). 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) correlated with Ins-Ind duration (rho [&rgr;] = -0.605, P = 0.0028 and &rgr; = 0.662, P = 0.0008, respectively]. The GA level strongly correlated with KG, the glucose disappearance rate during intravenous glucose tolerance testing (&rgr; = -0.76, P < 0.0001). The GA levels at post-transplant week (PTW) 3 and at PTW 4 in recipients with long-term Ins-Ind (>90 days) were significantly lower than those with short-term Ins-Ind, revealing excellent performance for prediction of long-term Ins-Ind, similar to that of porcine C-peptide (historic data). Conclusions As a surrogate indicator for graft function, serial measurement of GA may provide supplemental information to conventional monitoring techniques for graft function in assessing porcine islet grafts in NHP models.

D‐dimer level, in association with humoral responses, negatively correlates with survival of porcine islet grafts in non‐human primates with immunosuppression

Several immunosuppression (IS) regimens achieve long‐term graft survival in non‐human primates (NHPs) after porcine islet transplantation (PITx), but their success rates vary. To understand the mechanism of graft loss, we investigated the relationships between graft survival and humoral or inflammatory responses for maintenance IS in NHPs after PITx.