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Islet Transplantation

Optimizing enzyme blends for maximizing islet yield

Clinical islet transplantations are offering the hope of a greater quality of life to patients suffering from chronic pancreatitis and diabetes with the potential of insulin independence. The main factor determining the likelihood of insulin independence after islet transplantation is the islet mass that is transplanted. Determining suitable enzyme blends, specifically, the combination, concentration, and optimal ratio of collagenases and proteases are critical for obtaining proper islet release from the surrounding acinar tissue. We have successfully optimized and utilized SERVA enzymes for our clinical autologous islet isolation and transplantation [American Journal of Transplantation. 2009; 9(10): 2383]. In a later study, based on the biochemical properties of class 1 and class 2 collagenases, we successfully introduced the VITACYTE enzymes for clinical allo-transplants [Transplantation. 2010;27; 89(8):954]. We then further refined these enzymes for obtaining high islet yield for both auto- and allo-graft preparations. Based on biochemical properties and enzyme behavior during islet isolations, we conceived a new enzyme mixture by combining enzyme components from the two different suppliers: Clostridium histolyticum neutral protease (SERVA), and purified class I (C1) and class II (C2) collagenases (VITACYTE). This new enzyme mixture consistently provided high islet yields of better, morphologically intact islets for successful autologous and allograft clinical transplantation [Transplantation. 2012;15; 93(7):693]. Many islets laboratories around the world are using this new enzyme mixture for clinical islet isolation. Recently, we have tested recombinant class 1 (rC1) and class 2 (rC2) collagenases performance on the recovery of functional human islets. Results from this study indicate the effectiveness of this animal-free enzyme mixture to recover islets at doses of collagenase that are approximately 50% of those used commonly for human islet isolation.

Other previous studies include: identifying the harmful effects of isolation enzyme [American Journal of Transplantation. 2005; 5(11): 2671], successful isolation of high islet numbers from sub-optimal human pancreases [American Journal of Transplantation. 2003; 3(9): 1135] and successful isolation from pediatric donors [Diabetologia. 2006; 49(8): 1845]. New methods have also been established for a simple method for monkey islet isolation [Diabetes Research and Clinical Practice 2004; 66(1): 13] as well as improved pig islet isolation techniques [Xenotransplantation. 2007; 14(1): 74].

Islet Transplantation

Alpha-1 Antitrypsin for improving islet function

Research is also focused on overcoming some of the obstacles faced during the islet cell transplantation process, such as islet cell culture after isolation and poor islet graft function after transplant. These issues arise from the utilization of proteases during isolation and prolonged exposure to these enzymes afterwards, evidenced by the problems becoming exacerbated in cases of impure islet preparations compared to pure islet preparations. To overcome, we have successfully utilized a clinical grade protease inhibitor, alpha-1 antitrypsin. Alpha-1 antitrypsin treatment to islet graft recipients could block the negative effect of proteases and improve the islet graft function [Transplantation. 2011; 92(11): 1222].

Islet Transplantation

Improved islet purification technique

We developed an analytical test gradient system (ATGS) to determine the density distribution of exocrine and islet tissue dissociated from human pancreases before the actual COBE purification process. Our ATGS-guided method maximizes the islet purification recovery for successful transplantations by reducing acinar contamination in allograft preparations and by reducing sedimentation and loss of islets in the COBE bag in autograft preparations [Transplantation. 2011; 91(5): 508-14].

Islet Transplantation

Anti-oxidative treatment of islets with SOD-mimic

The process of human islet isolation triggers a cascade of stressful events in the islets involving activation of apoptosis, necrosis, and the production of pro-inflammatory molecules that negatively impact islet yield and function after islet transplantation. We reduced the stress to islets and improved the survival of human islets in culture and in vivo by using SOD-mimic anti-oxidative compound. [Diabetes. 2002; 51(8): 2561][Diabetes. 2004; 53(10): 2559]. We have also used curcumin for human islet culture and observed that curcumin or its analogues could be used to induce cellular defense against oxidative stress and improve islet transplantation outcomes [Pancreas. 2009; 38(4): 454-60].

Islet Transplantation

Ductal cell of human chronic-pancreatitis pancreas for beta cell neogenesis

In our laboratory during islet isolation from chronic pancreatitis (CP) pancreases, we have observed many interesting and unique cell release patterns. These unusual results prompted immunofluorescence investigation of three CP young donor pancreases, which showed strong evidence for a ductal origin of islet neogenesis [Acta Diabetologia. 2011 Jul 20].

Islet Transplantation

Publications

Anazawa T, Balamurugan AN, Bellin M, Zhang HJ, Matsumoto S, Yonekawa Y, Tanaka T, Loganathan G, Papas KK, Beilman GJ, Hering BJ, Sutherland DE. Human islet isolation for autologous transplantation: comparison of yield and function using SERVA/Nordmark versus Roche enzymes. American Journal of Transplantation. 2009; 9:2383-91.

Balamurugan AN, Breite AG, Anazawa T, Loganathan G, Wilhelm JJ, Papas KK, Dwulet FE, McCarthy RC, Hering BJ. Successful human islet isolation and transplantation indicating the importance of class 1 collagenase and collagen degradation activity assay. Transplantation. 2010; 89:954-61.

Balamurugan AN, Loganathan G, Bellin MD, Wilhelm JJ, Harmon J, Anazawa T, Soltani SM, Radosevich DM, Yuasa T, Tiwari M, Papas KK, McCarthy R, Sutherland DE, Hering BJ. A new enzyme mixture to increase the yield and transplant rate of autologous and allogeneic human islet products. Transplantation. 2012; 93:693-702.

Balamurugan AN, Green ML, Breite AG, Loganathan G, Wilhelm JJ, Tweed B, Vargova L, Lockridge A, Hughes M, Williams SK, Hering BJ, Dwulet FE, McCarthy RC. Identifying effective enzyme activity targets for recombinant class I and class II collagenase for successful human islet isolation. Transplantation Direct. (Published online 23 December 2015).

Balamurugan AN, Chang Y, Fung JJ, Trucco M, Bottino R. Flexible management of enzymatic digestion improves human islet isolation outcome from sub-optimal donor pancreata. American Journal of Transplantation. 2003; 3:1135-42.

Balamurugan AN, Ramakrishna B, Gunasekaran S. Insulin secretory characteristics of monkey pancreatic islets: a simple method of islet isolation and the effect of various density gradients on separation. Diabetes research and clinical practice. 2004; 66:13-21.

Balamurugan AN, He J, Guo F, Stolz DB, Bertera S, Geng X, Ge X, Trucco M, Bottino R. Harmful delayed effects of exogenous isolation enzymes on isolated human islets: relevance to clinical transplantation. American Journal of Transplantation. 2005; 5:2671-81.

Balamurugan AN, Chang Y, Bertera S, Sands A, Shankar V, Trucco M, Bottino R. Suitability of human juvenile pancreatic islets for clinical use. Diabetologia. 2006; 49:1845-54.

Bottino R, Balamurugan AN, Smetanka C, Bertera S, He J, Rood PP, Cooper DK, Trucco M. Isolation outcome and functional characteristics of young and adult pig pancreatic islets for transplantation studies. Xenotransplantation. 2007; 14:74-82.

Loganathan G, Dawra RK, Pugazhenthi S, Guo Z, Soltani SM, Wiseman A, Sanders MA, Papas KK, Velayutham K, Saluja AK, Sutherland DE, Hering BJ, Balamurugan AN: Insulin degradation by acinar cell proteases creates a dysfunctional environment for human islets before/after transplantation: benefits of alpha-1 antitrypsin treatment. Transplantation. 2011; 92:1222-30.

Anazawa T, Matsumoto S, Yonekawa Y, Loganathan G, Wilhelm JJ, Soltani SM, Papas KK, Sutherland DE, Hering BJ, Balamurugan AN: Prediction of pancreatic tissue densities by an analytical test gradient system before purification maximizes human islet recovery for islet autotransplantation/allotransplantation. Transplantation. 2011; 91:508-14.

Bottino R, Balamurugan AN, Bertera S, Pietropaolo M, Trucco M, Piganelli JD. Preservation of human islet cell functional mass by anti-oxidative action of a novel SOD mimic compound. Diabetes. 2002; 51:2561-7.

Bottino R, Balamurugan AN, Tse H, Thirunavukkarasu C, Ge X, Profozich J, Milton M, Ziegenfuss A, Trucco M, Piganelli JD. Response of human islets to isolation stress and the effect of antioxidant treatment. Diabetes. 2004; 53:2559-68.

Balamurugan AN, Akhov L, Selvaraj G, Pugazhenthi S. Induction of antioxidant enzymes by curcumin and its analogues in human islets: implications in transplantation. Pancreas 2009; 38:454-60.

Soltani SM, O'Brien TD, Loganathan G, Bellin MD, Anazawa T, Tiwari M, Papas KK, Vickers SM, Kumaravel V, Hering BJ, Sutherland DE, Balamurugan AN. Severely fibrotic pancreases from young patients with chronic pancreatitis: evidence for a ductal origin of islet neogenesis. Acta diabetologica. 2013; 50:807-14.

Balamurugan AN, Gu Y, Tabata Y, Miyamoto M, Cui W, Hori H, Satake A, Nagata N, Wang W, Inoue K. Bioartificial pancreas transplantation at prevascularized intermuscular space: effect of angiogenesis induction on islet survival. Pancreas. 2003; 26(3): 279-85.

Other Contributions

Balamurugan AN, Naziruddin B, Lockridge A, Tiwari M, Loganathan G, Takita M, Matsumoto S, Papas K, Trieger M, Rainis H, Kin T, Kay TW, Wease S, Messinger S, Ricordi C, Alejandro R, Markmann J, Kerr-Conti J, Rickels MR, Liu C, Zhang X, Witkowski P, Posselt A, Maffi P, Secchi A, Berney T, O’Connell PJ, Hering BJ and Barton FB. Islet Product Characteristics and Factors Related to Successful Human Islet Transplantation From the Collaborative Islet Transplant Registry (CITR) 1999–2010. American Journal of Transplantation. 2014; 11: 2595-606.

Balamurugan AN, Nelson EJ, Ramakrishna B, Gunasekaran S. Effect of various immunosuppressive monotherapies on survival and histopathology of monkey islet xenografts in rats. Xenotransplantation. 2007; 14:316-22.

Balamurugan AN, Ramakrishna B, Gunasekaran S. Insulin secretory characteristics of monkey pancreatic islets: a simple method of islet isolation and the effect of various density gradients on separation. Diabetes Research and Clinical Practice. 2004; 66:13-21.