In Vitro Assessment of Human Islet Vulnerability to Instant Blood-Mediated Inflammatory Reaction (IBMIR) and Its Use to Demonstrate a Beneficial Effect of Tissue Culture.

Culture of human pancreatic islets is now routinely carried out prior to clinical islet allotransplantation, using conditions that have been developed empirically. One of the major causes of early islet destruction after transplantation is the process termed instant blood-mediated inflammatory reaction (IBMIR). The aim of this study was to develop in vitro methods to investigate IBMIR and apply them to the culture conditions used routinely in our human islet isolation laboratory. Freshly isolated or precultured (24 h, 48 h) human islets were incubated in either ABO-compatible allogeneic human blood or Hank's buffered salt solution (HBSS) for 1 h at 37°C. Tissue factor (TF) expression and leukocyte migration were assessed by light microscopy. TF was also quantified by ELISA. To assess ß-cell function, glucose-stimulated insulin secretion (GSIS) assay was carried out. The extent of islet ß-cell damage was quantified using a proinsulin assay. Islets cultured for 24 h had higher GSIS when compared to freshly isolated or 48-h precultured islets. Freshly isolated islets had significantly higher TF content than 24-h and 48-h precultured islets. Incubation of freshly isolated human islets in allogeneic human blood released 6.5-fold higher level of proinsulin in comparison to freshly isolated human islets in HBSS. The high level of proinsulin released was significantly attenuated when precultured islets (24 h or 48 h) were exposed to fresh blood. Histological examination of fresh islets in blood clot showed that some islets were fragmented, showing signs of extraislet insulin leakage and extensive neutrophil infiltration and necrosis. These features were markedly reduced when the islets were cultured for 24 h. These results suggest that our standard 24-h islet culture is markedly beneficial in attenuating IBMIR, as evidenced by increased GSIS, lower content of TF, decrease islet fragmentation, and proinsulin release.

Collagenase does not persist in human islets following isolation.

Optimal human islet isolation requires the delivery of bacterial collagenase to the pancreatic islet-exocrine interface. However, we have previously demonstrated the presence of collagenase within human islets immediately following intraductal collagenase administration. This potentially has significant implications for patient safety. The present study aimed to determine if collagenase becomes internalized into islets during the isolation procedure and if it remains within the islet postisolation. Islet samples were taken at various stages throughout 14 clinical human islet isolations: during digest collection, following University of Wisconsin solution incubation, immediately postisolation, and after 24 h of culture. Samples were embedded in agar, cryosectioned, and then assessed by immunolabeling for collagenase and insulin. Immunoreactivity for collagenase was not observed in isolated islets in any preparation. Collagenase labeling was detected in one sample taken at the digest collection phase in one islet preparation only. No collagenase-specific labeling was seen in islets sampled at any of the other time points in any of the 14 islet preparations. Collagenase that enters islets during intraductal administration is washed out of the islets during the collection phase of the isolation process and thus does not remain in islets after isolation. This observation alleviates some of the important safety concerns that collagenase remains within islet grafts.

Collagenase penetrates human pancreatic islets following standard intraductal administration.

BACKGROUND: : To optimize human islet isolation, it is important to improve our understanding of the collagenase digestion phase. Previous studies of collagenase action were mostly concerned with optimizing its composition, but the delivery and distribution of collagenase at the islet-exocrine interface is likely to be important for liberation of intact islets. The aim of this study was to characterize collagenase distribution in relation to islets in infused human pancreases. METHODS: : Human pancreases were retrieved from multiorgan donors with appropriate consent. Tissue samples were taken from the neck, body, and tail regions before and after collagenase infusion by manual syringe-loading (n=10) or recirculating perfusion (n=8), and snap frozen in liquid nitrogen. Frozen sections were immunolabeled for collagenase, insulin, CK19, collagen VI and CD31, then assessed by confocal microscopy. RESULTS: : Collagenase labeling was widespread throughout the pancreas, associated with collagen VI, and adjacent to CK19-labeled ducts. Collagenase was found within 67%+/-2% of islets ("intraislet"), associated with capillaries (CD31-positive). Intraislet collagenase was observed in 70%+/-3% of islets in the pancreatic tail, compared with 58%+/-2% and 53%+/-2% of islets in the body and neck, respectively (P<0.05 tail vs. neck), and was more prevalent in islets with diameters more than 150 microm (98%+/-1% of islets >150 microm vs. 52%+/-2% of islets <150 microm, P<0.05). There was no difference in intraislet collagenase labeling between perfused and syringe-loaded pancreases. CONCLUSIONS: : Using current infusion techniques, collagenase penetrates the islet interior. This could cause islet fragmentation, and consequently, low islet yields. This study underlies the need to optimize collagenase delivery to preserve intact islets.

Characterisation of collagen VI within the islet-exocrine interface of the human pancreas: implications for clinical islet isolation?

BACKGROUND: To optimize the methods used for human islet isolation for transplantation, it is important to improve our understanding of the structure of the islet-exocrine interface. In this study, the composition of collagen subtypes in the interface have been characterized and quantified in human pancreas. METHODS: Human adult pancreases were retrieved from older (mean age 55.7+/-3.0 yrs) and young donors (mean age 21.8+/-3.2 yrs). Tissue from the body of each pancreas was examined by quantitative immunohistochemistry. Collagen within the islet-exocrine interface was identified by immunolabeling for collagen I, IV, V or VI and islets identified either morphologically or by immunolabeling for insulin. Collagen subtypes were quantified and data expressed as collagen area at the interface relative to the islet area. Statistical analysis was by ANOVA or Mann Whitney U test. RESULTS: In older pancreases, collagen IV, V and VI were present throughout the islet-exocrine interface, whereas collagen I was more variable. The mean peri-islet collagen VI proportion was significantly greater than that of collagen I or IV. Mean islet area and the proportional collagen VI content in specimens from younger subjects were not significantly different to those in older subjects. CONCLUSIONS: Collagen VI is a major component of the islet-exocrine interface of the adult pancreas, the content being more than double that of collagen I or IV. However, the proportional collagen VI content was not dependent on the age of the donor. These data may facilitate the design of new collagenases, targeting major substrates such as collagen VI in order to improve clinical islet isolation.

Adverse outcome of human islet-allogeneic blood interaction.

BACKGROUND: A report of inflammatory damage when islets come into contact with allogeneic blood prompted us to confirm the finding. METHODS: Fresh handpicked human islets were incubated in blood group matched, nonsensitized allogeneic blood. Destruction was quantified by assaying the supernatants for proinsulin release and by blood clot histology. The effect on global hemostasis was assessed by thromboelastography (TEG), and heparin-bonded tubing was used to assess the effect on blood cellular counts. In separate experiments, islets were incubated in allogeneic blood with heparin or Reopro (monoclonal anti-GpIIbIIIa). Islets were also incubated in serum, and cryosections were stained for C1q, C4, C3, C5b-9, immunoglobulin (Ig)M, and IgG binding using immunohistochemistry. RESULTS: Histologic assessment showed severe destruction in 37% of islets in contact with allogeneic blood versus none in controls and a sevenfold increase in proinsulin release from controls (n = 6)(P < 0.005). TEG (n = 11) showed accelerated coagulation in the presence of islets (P < 0.001). Analysis of blood cellular counts (n = 3) showed consumption of platelets, neutrophils, and monocytes in the presence of islets (P < 0.001). Inhibition of coagulation with heparin (n = 3) or inhibition of platelet aggregation with Reopro (n = 3), separately or together (n = 3), did not make a substantial improvement in the destruction in terms of histology or proinsulin release. Immunohistochemical staining (n = 4) revealed C1q, C4, C3, and C5b-9 deposition along with IgG binding. IgM binding was weak if anything. CONCLUSION: This study confirms and extends the finding that human islet-allogeneic blood interaction results in significant destruction of islet tissue with activation of the coagulation cascade and platelet, neutrophil, and monocyte consumption. There was evidence for activation of complement by the classical pathway along with IgG binding.

The interaction between primate blood and mouse islets induces accelerated clotting with islet destruction.

BACKGROUND: Mouse islets transplanted under the renal subcapsular space of cynomolgus monkeys are subject to a form of hyperacute rejection, the mechanism of which is unclear. As islets are in contact with whole blood at the time of transplantation, the effect of platelets and the coagulation cascade on islet destruction was assessed. METHODS: Coagulation was assessed using thromboelastography on citrated/recalcified human blood samples with freshly isolated C57/Bl6 mouse islets. A dynamic islet perifusion system was used to assess the effect of islets on blood cells and coagulation factors. Cytotoxicity was evaluated using (51)Cr labelled islets incubated with human blood and islet destruction was also evaluated using a histological grading system. Continuous PO(2) measurements were made in a static incubation system to assess the role of hypoxia in islet destruction. RESULTS: Mouse islets incubated in human blood induced accelerated coagulation and rapid consumption of platelets within 15 min. Within 1 h of incubation, 52% of mouse islets exposed to xenogeneic human blood showed features of severe damage with necrosis when compared with islets incubated in syngeneic blood. Specific lysis of the xenogeneic islets was demonstrable (Mean percentage lysis: 48%, P < 0.05 vs. control) after 4 h incubation in human blood. Oxygen levels remained constant at a level adequate to maintain islet viability in separate experiments. CONCLUSION: Mouse islets induce rapid activation of the clotting cascade and platelet consumption in vitro when exposed to human blood, which correlated with histological evidence of significant destruction demonstrable within 1 h of exposure to human or non-human primate blood. This in vitro model has features which appear to correlate with the islet destruction seen in vivo and could be a useful model for the study of the mechanisms underlying the rapid destruction of xenogeneic islets in primate recipients.

Lecithinized superoxide dismutase reduces cold ischemia-induced chronic allograft dysfunction.

BACKGROUND: Chronic renal allograft failure (CAF) is influenced by both allo-dependent and independent factors and is a major cause of graft loss in clinical renal transplantation. We evaluated a novel membrane-bound free radical scavenger, lecithinized superoxide dismutase (lec-SOD), to determine its potential in limiting the harmful effects of ischemia/reperfusion injury on CAF. METHODS: Fisher rat kidneys were stored for either 1 hour or 18 hours in cold Marshall's preservation solution either with or without lec-SOD and transplanted into Lewis recipients. RESULTS: Within 3 days of transplantation, an early inflammatory response involving granulocytes and macrophages was detected in renal allografts exposed to 18 hours cold ischemia that was significantly reduced by preservation with lec-SOD. By 24 weeks post-transplantation, elevated proteinuria and detection of apoptotic cells was observed in kidneys exposed to 18 hours of cold ischemia, that was attenuated by preservation with lec-SOD (P < 0.05). However, up-regulated expression of intracellular adhesion molecule-1 (ICAM-1) and major histocompatibility complex (MHC) Class II together with a T lymphocyte infiltration were observed at 24 weeks that was not prevented by preservation with lec-SOD. CONCLUSIONS: These results demonstrate that ischemia/reperfusion injury, apoptotic cell death and allo-immune responses may be exacerbated by cold ischemia and accelerate the development of CAF. Preservation with lec-SOD may protect against the early damage induced by cold ischemia and reperfusion injury.