E. Bouwman

Effects of donor-, pancreas-, and isolation-related variables on human islet isolation outcome: a systematic review.

Different factors have been reported to influence islet isolation outcome, but their importance varies between studies and are hampered by the small sample sizes in most studies. The purpose of this study was to perform a systematic review to assess the impact of donor-, pancreas-, and isolation-related variables on successful human islet isolation outcome. PubMed, Embase, and Web of Science were searched electronically in April 2009. All studies reporting on donor-, pancreas-, and isolation-related factors relating to prepurification and postpurification islet isolation yield and proportion of successful islet isolations were selected. Seventy-four retrospective studies had sufficient data and were included in the analyses. Higher pre- and postpurification islet yields and a higher proportion of successful islet isolations were obtained when pancreata were preserved with the two-layer method rather than University of Wisconsin solution in donors with shorter cold ischemia times (CITs) [1 h longer CIT resulted in an average decline of prepurification and postpurification yields and proportion of successful isolations of 59 islet equivalents (IEQs)/g, 54 IEQs/g, and 21%, respectively]. Higher prepurification yields and higher percentage of successful islet isolations were found in younger donors with higher body mass index. Lower yields were found in donation after brain death donors compared to donation after cardiac death donors. Higher postpurification yields were found for isolation with Serva collagenase. This review identified donor-, pancreas-, and isolation-related factors that influence islet isolation yield. Standardized reports of these factors in all future studies may improve the power and identify additional factors and thereby contribute to improving islet isolation yield.

Isolation of the Islets of Langerhans From the Human Pancreas With Magnetic Retraction

Low yield and insufficient purity limit the transplantation of human islets of Langerhans. In the rat, a new technique to isolate the islets of Langerhans was developed by intraarterial infusion of iron particles into the islet capillaries. After digestion the iron-loaded islets were purified with magnetic retraction (MR). In the present study, 10 human pancreata not suitable for clinical use were arterially injected with an iron-oxide suspension. After injection a small piece was used for histological analysis, and the tail was intraductally perfused with collagenase and manually digested. The yield was compared with 11 pancreata processed in the standard way. Nine of 10 pancreata were successfully injected with iron-oxide and digested. After MR, enrichment was achieved but the purity was not more than 50%. Similar results between the 2 groups were obtained regarding digestion times (23 +/- 1.1 vs 22.7 +/- 1.5 minutes) and purification yields (1749 +/- 502.1 vs 2111 +/- 501.8 IE/g, P = .6) for the MR vs control groups, respectively. Histological analysis revealed that 60% to 88% of the islets contained iron aggregations with substantially higher concentrations compared with the exocrine tissue. In conclusion, iron-oxide did not influence the isolation outcome before purification. Islet purification with MR gave enrichment but no pure fractions.

Pre‐transplant blood transfusion and cyclosporin A induce long‐term hamster cardiac xenograft survival in immunocompetent rats

Abstract:u2002 Background:u2002 In previous studies we have shown that pre‐transplant hamster blood transfusion (HBT) can induce non‐responsiveness in the T cell independent immunecompartment and result in tolerance towards hamster cardiac xenografts (Xgs) in T cell deficient athymic nude rats. In this study we test the combination of pre‐transplant HBT with cyclosporin A (CSA) in immunocompetent Lewis rats.

Pre‐transplant blood transfusion induces tolerance to hamster cardiac xenografts in athymic nude rats

Abstract: The effects of pre‐transplant blood transfusion vary from induction of antibodies and accelerated graft rejection, to prolonged survival and even tolerance. The beneficial ‘transfusion effect’ in allotransplantation is believed to be merely T‐cell mediated. In xenotransplantation, T‐cell independent mechanisms form a major hurdle. In this study we investigated the effects of pre‐transplant hamster blood transfusion on the survival of hamster cardiac xenografts in T‐cell deficient athymic nude rats. Nude rats rejected xenografts after 3.8u2003±u20030.5u2003d (nu2003=u20038), and immunocompetent Lewis rats after 4.0u2003±u20030.5u2003d (nu2003=u20038), following a similar IgM response (Pu2003=u2003NS). Hamster blood transfusion 3u2003d before transplantation in nude rats led to an IgM response and long‐term xenograft survival in 17/20. Timing was of importance: blood transfusion 7u2003d before transplantation resulted in 45% long‐term xenograft survival (nu2003=u200320). Injection of purified hamster erythrocytes, leukocytes or minced heart also led to survival of xenografts for >u2003100u2003d in nude rats, but not in all cases. Second xenografts transplanted to long‐term survivors did not provoke an IigM response, and were accepted for >u2003100u2003d (nu2003=u20034). Transfer of serum from long‐term survivors to untreated nude rats resulted in survival of xenografts for >u2003100u2003d (nu2003=u20034). In Lewis rats pre‐transplant blood transfusion induced hyperacute rejection of xenografts after 158u2003±u2003128u2003min (nu2003=u20038, Pu2003<u20030.01). We conclude that pre‐transplant hamster blood transfusion can induce long‐term survival of hamster cardiac xenografts in T‐cell deficient athymic nude rats. This blood transfusion effect is mediated by humoral factors and can be transferred by serum. Elucidation of underlying mechanisms might provide some insight into xenotransplantation nonresponsiveness of T‐cell independent immunefactors.

Hamster cardiac xenografts are protected against antibody mediated damage, early after transplantation to Lewis rats

Abstract: Antibodies play a crucial role in the rejection of xenografts. We tested the hypothesis that xenografts are protected against antibody‐mediated attack early after transplantation in a concordant model. We investigated the role of xenoreactive antibodies as a stimulus for protection and the effects of a total blockade of the antibody response by the leflunomide analog malononitrilamide 279. Hamster cardiac xenografts were transplanted to Lewis rat recipients. Second transplants and retransplants of xenografts were performed to untreated rats that had a xenograft in place for 3u2003d. Untreated rats rejected hamster cardiac xenografts after 4.0u2003±u20030.0u2003d. Significant levels of anti‐donor IgM, as measured by flowcytometry, were present on day 3 after transplantation (11.2%u2003±u20032.8 vs. 1.2%u2003±u20030.0 on day 0, Pu2003<u20030.001). ‘Fresh’ second xenografts transplanted to rats that had a first xenograft in place for 3u2003d and had anti‐hamster antibodies, underwent hyperacute rejection. The first xenografts remained functioning. Xenografts that were removed on day 3 from untreated rats and then retransplanted remained functioning. Xenografts that were removed on d 3 from rats that had been treated with malononitrilamide 279, 15u2003mg/kg/d and were retransplanted underwent hyperacute rejection. IgM levels at the time of removal were 1.1%u2003±u20030.5 in these rats and not different from baseline (Pu2003=u20030.96). We conclude that xenografts are protected against antibody‐mediated damage early after transplantation. The presence of anti‐donor antibodies might be an essential stimulus for the induction of protection. There seems to be a delicate balance between the injurious and protective effects of antibodies. Treatment strategies that are designed to block antibody formation completely might prevent the induction of protection.

Porcine islet isolation outcome is not affected by the amount and distribution of collagen in the pancreas.

Hilling DE, Rijkelijkhuizen JKRA, Töns HAM, Terpstra OT, Bouwman E. Porcine islet isolation outcome is not affected by the amount and distribution of collagen in the pancreas.u2028Xenotransplantation 2010; 17: 250–255.

Amount and Distribution of Collagen in the Pancreas Have No Effect on Porcine Islet Isolation Outcome

Xenotransplantation of porcine islets of Langerhans is considered to be a possible alternative for clinical islet transplantation. However, porcine islet isolation procedures have been shown to produce highly variable yields between pigs with similar backgrounds. One of the variables that could account for this is the collagen substrate within the pancreas. We determined the amount and distribution of collagen within porcine pancreata as they determined islet isolation outcomes. This study involved the histological examination of 140 porcine pancreata (64 juvenile and 76 adult) and islet isolation from 58 adult organs. To quantify the amount of collagen, tissue samples were stained with Sirius Red. Collagen distribution was determined by assessing the presence of collagen in the endocrine-exocrine interface (the islet capsule), in tissue samples double-stained with Sirius Red and anti-insulin. Strong variation in total collagen was observed in both adult and juvenile pigs. The mean collagen content in the juvenile group was significantly lower than that in the adult group. Apparently, the pancreas undergoes a process of fibrosis as pigs age. The vast majority of islets from both adult and juvenile pancreata had no or only a limited collagen capsule. However, islet encapsulation was highly variable between pancreata. We observed no significant correlation between total collagen content or the percentage islet encapsulation and islet yield. Although total collagen content and islet encapsulation show great variability between pancreata, neither the amount nor the distribution of collagen affected porcine islet isolation outcome.

Morphological changes of porcine islets of Langerhans after collagenase and HBSS infusion of the pancreas

Hilling DE, Rijkelijkhuizen JK, Marang‐van de Mheen PJ, Töns A, Terpstra OT, Bouwman E. Morphological changes of porcine islets of Langerhans after collagenase and HBSS infusion of the pancreas. Xenotransplantation 2010; 17: 413–417.

Heterogeneity of human pancreata in perspective of the isolation of the islets of langerhans.

The success of human islet isolation is hampered by the varied and unpredictable outcomes of the islet isolation procedure. Pancreata which meet well-defined criteria are no guarantee for success. Interindividual variations may contribute to the differences in isolation outcomes. The present study examined several structural elements in the anatomy of the human pancreas for possible relevance for islet isolation. Sixty pancreata were used for histochemical and immunochemical analyses. We assessed the total percentage of endocrine tissue and the size distribution of the islets. Sirius Red staining quantified total collagen content; the degree of islet encapsulation with collagen was correlated with total collagen. We analyzed the percentage of pancreatic edema and amount of intraparenchymal fat. The percentage of endocrine tissue varied 5-fold with wide variations in islet size distribution. A strong variation was observed for total collagen; its content increased slightly with age. The number of islets totally encapsulated with collagen varied strongly with no relation to age or to total collagen. Pancreatic edema and intraparenchymal fat also showed great differences. These differences justifies continued study to evaluate the correlation of these variables with isolation outcomes.

Hyperemic Islets: A Possible Explanation for Poor Yields in Human and Porcine Islet Isolation

When studying histological characteristics of human and porcine pancreata in relation to islet isolation, we encountered a remarkably high number of hyperemic islets. The abnormalities observed in these islets ranged from a single dilated vessel through multiple widely dilated vessels to hemorrhages extending into the surrounding exocrine tissue. We determined their possible relevance for outcomes of islet isolation. This study involved a histological examination of 143 porcine pancreata (72 juvenile and 71 adult) and islet isolation from 48 adult pancreata. Human pancreata obtained from 71 multiple organ donors yielded islet isolation in 24 cases. To determine their endocrine content, tissue samples were stained with Aldehyde Fuchsin. The presence of hyperemic islets was scored semiquantitatively with pancreata allotted to categories based on the severity. In humans and pigs we observed hyperemic islets in 48% of pancreata, but only 4.0 +/- 2.4% of the islets were hyperemic. In both humans and pigs, significantly higher endocrine content was found in the most severely affected pancreata. When the higher endocrine content was taken into account and isolation results were expressed as ratios of yield and content, we observed significantly lower yields in the most affected pancreata in pigs with a trend toward lower yields in humans. A substantial proportion of human and porcine pancreata contain hyperemic islets. Although the results in humans are preliminary, our data suggest that this phenomenon may contribute to the unpredictable, highly variable islet yields in pigs and humans.