Recent advances in cell replacement therapies for the treatment of type 1 diabetes.

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Recent advances in cell replacement therapies for the treatment of type 1 diabetes Kathryn Cogger1, and Maria Cristina Nostro1,2 1. Toronto General Research Institute and McEwen Centre for Regenerative Medicine, University Health Network, Toronto, ON, M5G 1L7; Canada; 2. Department of Physiology, University of Toronto, Toronto, ON, M5S 1A8; Canada

Exogenous insulin administration is currently the only treatment available to all type 1 diabetes patients, but it is not a cure. By helping to regulate circulating blood glucose levels, it has significantly improved life expectancy, but there are still long-term complications associated with the disease, that may be preventable with a treatment strategy that can provide better glycemic control. Isolated islet (or whole pancreas) transplantation, xenotransplantation, and the transplant of human pluripotent stem cell-derived ␤-cells provide the potential to restore endogenous insulin production and re-establish normoglycemia. Here we discuss the latest advances in these fields, including the use of encapsulation technology, as well as some of the hurdles that still need to be overcome for these alternative therapies to become mainstream and/or progress to clinical development.

ype 1 diabetes mellitus (T1DM) is an endocrine disorder that affects millions of people worldwide. It is caused by an autoimmune response that targets and destroys the insulin producing ␤-cells that reside in the islets of Langerhans of the pancreas, resulting in insulin deficiency. Patients showing clinical manifestations of T1DM, such as fasting hyperglycemia, have reduced ␤-cell mass (1) and there is a growing body of evidence to support a role for ␤-cell dysfunction in the disease pathogenesis (2). There have been several theories as to the exact trigger for the immune-associated destruction (3), but it is currently accepted that there is no single cause, but rather many multifactorial mechanisms that could trigger T1DM onset such as virus infection, introduction of toxic agents through diet and/or beta cell stress (4). To date, the only treatment available to most T1DM patients consists of lifelong exogenous administration of human recombinant insulin. Although insulin treatment has substantially improved life expectancy, there is still substantial morbidity and mortality associated with the disease (5). While helping to control blood glucose levels, administration of exogenous insulin does not provide as precise regulation as functional islets, resulting in fluctu-

ations between hypo- and hyper-glycemic states. Severe hypoglycemic episodes can result in coma, or even death, while frequent recurrent hyperglycemia can lead to serious long-term complications, such as heart disease, kidney failure and blindness (6, 7). Current efforts in regenerative medicine are focused on establishing alternative therapies, which will provide better glycemic control, and help prevent long-term complications. Here we summarize the latest progress in the use of islet transplantation, xenotransplantation and the generation of human embryonic stem cell (ESC) (hESC)- or human induced pluripotent stem cell (hiPSC)-derived ␤-cells to re-establish endogenous regulation of insulin production.

ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright © 2014 by the Endocrine Society Received August 14, 2014. Accepted November 7, 2014.

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Whole pancreas and islet transplantation Whole pancreas transplant is a successful approach to treat T1DM as it can provide a reliable and durable restoration of normoglycemia (8). Currently, graft survival for pancreas transplants alone is 82% at one year, and 58% at five years. These figures are increased to 89% and 71% respectively for dual pancreas and kidney transplants (9). However, this is a complicated procedure, and

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Cell therapies for T1DM treatment

exposes patients to surgical risks, as well as the need for lifelong immunosuppression, and is therefore only performed in cases of severe brittle diabetes, or in patients already undergoing a kidney transplant. To overcome the need for major surgery, transplantation of islet cells isolated from human cadaver pancreata has been developed as an alternative therapeutic approach. As this technique also requires lifelong immunosuppression, and due to the limited availability of islets, the cohort of patients generally used for these studies only include those with metabolic instability and refractory hypoglycemia, but it has been used to successfully re-establish normoglycemia in a number of diabetic patients (10 –12). The isolated islets are most commonly transplanted into the hepatic portal vein. They then travel the short distance to the liver, where they become highly vascularized and start to produce insulin to regulate blood glucose. However, the transplanted cells must withstand both an instant blood-mediated inflammatory reaction (IBMIR), and humoral and cell-mediated immune responses led by T and B lymphocytes (7). IBMIR is thought to be triggered by the cell surface expression of inflammatory mediators, such as tissue factor, on the transplanted islets, resulting in activation of platelets, coagulation and complement systems, and infiltration of leukocytes (7, 13, 14). Even with immunosuppression there could be a 50%–70% loss of cells almost immediately after islet infusion, due to IBMIR (13, 14). The first successful transplantation of isolated human pancreatic islets into a human diabetic patient, with resulting insulin independence (albeit transient), was reported in 1990 by Scharp et al (15). Between then and 2000, when Shapiro et al established the Edmonton transplantation protocol, islet transplantation had very low success rates, probably due to inefficient isolation techniques, inadequate numbers of islets per transplant, and the use of harsh immunosuppressive therapies. The Edmonton protocol revolutionized the field of islet transplantation by implementing a method to reduce alloimmune reactivity, and improve islet survival and functionality (16 –18). With this novel protocol, the group was able to demonstrate that insulin independence could be achieved in most patients after transplantation of an adequate number of islets, and insulin production could be maintained long-term. They also showed this technique could provide glycemic stability and normalize glycosylated hemoglobin (HbA1c) levels, which is not often achieved with exogenous insulin therapy (19). Although insulin independence was, for the most part, unsustainable after the first year post-transplantation, patients with residual islet function had improved symptoms than prior

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to the treatment, and were protected from severe hypoglycemic episodes. Together with immunomodulatory techniques, it is now possible to achieve 5-year islet graft survival and insulin independence in certain specialized centers (10, 20, 21). In a transplant cohort study, Bellin et al ascertained that patients receiving potent induction therapy (T cell depleting antibodies in conjunction with TNF-␣ inhibition, or an anti-CD3 antibody alone) as part of their immunosuppressive regimen, had far better graft survival rates than those receiving IL-2 receptor antibodies alone, with 50% of patients now achieving 5 year insulin independence, and 2 patients still independent at 10 years (10). Despite the significant benefits of whole pancreas and islet transplantation in preventing long-term diabetic complications, the risks associated with lifelong immunosuppression, such as increased susceptibility to infection and cancer (22, 23), are deemed to be too great for these treatments to be made available to all T1DM patients. Aside from this, the likelihood that many patients would require islets from more than one pancreata, combined with the severe lack of donors, means that as it stands, islet transplantation is not a practical alternative therapy. To address this first issue, and reduce graft rejection without the need for immunosuppressive drugs, much work has been done on developing encapsulation devices. Encapsulation aims to create an artificial immune-privileged site, protecting the grafts from the host immune system. A successful device should contain membranes with selective permeability, to exclude large host immune cells and immunoglobulins, while permitting small molecules such as oxygen, carbon dioxide and essential nutrients to reach the cells, and insulin to be secreted in response to high blood glucose levels (5, 24, 25). Graft site is also an important consideration when designing an encapsulation device, as engrafted islets/cells must be able to respond rapidly to elevated circulating glucose levels. Research into encapsulation devices started in the 1980’s, and there are currently eleven companies working on developing the technology (14), as well as the recently created JDRF Encapsulation Consortium which aims to bring encapsulation therapies to clinical trials in diabetes (26, 27). One of the more successful devices, which consists of a clinical grade alginate-polyornithine microcapsule system (28), has made its way to clinical trials. In the pilot trial, encapsulated human islets were transplanted into the peritoneum of four T1DM patients. Although insulin independence was never fully achieved, blood glucose levels were stabilized, resulting in a reduction in the requirement for exogenous insulin, and normalization of HbA1c plasma levels (25, 29). Throughout the 5 year follow-up period, there was no evidence of an immune re-


doi: 10.1210/en.2014-1691

sponse generated by the grafts as measured by anti-MHC (major histocompatibility complex (MHC), class I and II), anti-GAD65 and islet cell antibodies. The transient nature of the metabolic benefits indicates there are still areas to develop and refine this technology; however, this study does show that it is possible to create an implantable encapsulation device that is biocompatible and capable of immunoisolating the graft. While encapsulation may prevent an immune response being generated by, and directed towards the transplanted graft, it can also impair cell survival and function due to poor oxygen supply (11, 30, 31). The “␤-air” bioartificial pancreas implantation device from ␤-O2 Technologies aims to overcome the issue of oxygen deficit by providing an internal oxygen supply (32, 33). In this system, two islet-containing modules surround a central gas chamber lined with a rubber silicon oxygen-permeable membrane. The islets are encapsulated in an alginate-hydrogel, and are separated from the recipient’s tissue by a hydrophilized polytetrafluoroethylene (PTFE) membrane. Oxygen, which is infused daily via 2 ports implanted subcutaneously along with the device, diffuses from the chamber across the membrane to reach the islets. This macrochamber has been used in rats to successfully reverse streptozotocin (STZ)-induced diabetes for up to 6 months, which returns upon removal of the device or the oxygen supply (32–34). Remarkably, both isogeneic and allogeneic islet transplantations have been carried out, with no visible signs of an inflammatory or fibrotic reaction after explantation (32, 33, 35). The company is now focusing on translating this technology to larger diabetic animal models (14, 35), and thus far has been able to restore normoglycemia for 30 days, in diabetic mini pigs (34). Most recently, this device was used in an individual treatment approach for one T1DM patient (36). With the aim of assessing islet allograft survival, the patient was transplanted with 2100 islet equivalents (IEQs) per kilogram of bodyweight within the chamber, without immunosuppressive therapy, and was followed for a period of 10 months. Given the low dose of islets received, only mild improvements in HbA1C levels and exogenous insulin requirements were observed, however, the graft was well preserved and retained functionality. The device did not trigger an immune response, and on explantation there were no signs of inflammation. For insulin independence to be achieved in humans, it is anticipated that 300 – 500 000 IEQs would be required per transplant. The device used in this study has a maximum capacity if 150 000 IEQs, and thus ␤-O2 Technologies is now tackling ways to increase islet capacity and density, as well as improving islet preparation techniques to optimize islet viability and


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functionality. It is anticipated that human clinical trials could begin in the next couple of years. Xenotransplantation In the search for alternative sources of donor islets, much interest has been focused on the use of porcine islets. Not only was pig insulin previously used to treat diabetic patients prior to the production of recombinant human (rh) insulin (37), but porcine glucose physiology is not too dissimilar from that of humans, indicating their functional compatibility (6, 38). Pigs can also be bred for this specific purpose, creating a large, readily available source of islets. To prevent rejection of xenografts, encapsulation devices have been used to protect against the immune response generated by porcine cell surface antigens. Encapsulation can also provide protection from xenosis, which has been a concern in the use of animal cells in humans (5, 7, 24, 34). Unencapsulated pig islets with systemic immune suppression were first used in Sweden in 1994 to treat diabetic patients who underwent kidney transplant (39). However, current trials, run by Living Cell Technologies, are testing the safety and efficacy of encapsulated pig islets transplanted into human T1DM patients without immunosuppression (5, 24, 40, 41). The islets are sourced from a pathogen-free, isolated herd of Auckland Islands pigs and encapsulated in alginate microcapsules (42, 43). The initial pilots showed no signs of inflammation or fibrosis, and no porcine endogenous retroviral infection, with a reduction in daily insulin dose and HbA1c levels in the transplanted patients. Furthermore, microcapsules that were retrieved 9.5 years after transplantation contained viable cells that produced insulin in response to glucose stimulation (5, 24, 40). In 2009 the company initiated a Phase I/IIa dose-finding study (NCT00940173) with 14 patients, for which they reported improved hypoglycemic awareness and elimination of hypoglycemic convulsions in one patient in 2011 (5). This study was completed in October 2013, and we expect their findings to be published imminently. The same group is currently overseeing two additional clinical trials (NCT01739829, NCT01736228), aimed at determining the safety and efficacy of this encapsulation system. These studies indicate that islet function can be maintained within the microcapsules for many years, with no transmission of porcine endogenous retrovirus or microorganisms to any of the patients studied (44 – 47). While ultimately, maintenance of normoglycemia and insulin independence long-term would be a fantastic achievement, it is clear that even in their absence, there are significant improvements in hypoglycemic awareness yielded from this xenotransplantation approach. Aside from encapsulation, attempts have been made to


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genetically modify donor islets to prevent them from eliciting an immune response. Knock-out of the Gal xenoantigen gene GalT in pigs (GTKO) better protects neonatal porcine islets from IBMIR, allowing increased rates of engraftment and insulin-independent normoglycemia when transplanted into STZ-induced diabetic nonhuman primates (48). Transgenic pigs expressing the human complement-regulatory proteins (hCRPs) CD46, CD55 and CD59, have also been created (7, 49). Adult hCD46 expressing porcine islets again exhibit prolonged graft survival and insulin independence when transplanted into STZ-induced diabetic cynomolgus monkeys compared to nontransgenic islets (50). Multitransgenic pigs are now available combining GTKO with expression of hCRPs, human anti-inflammatory and antithrombotic proteins (CD39, tissue factor pathway inhibitor, thrombomodulin), and immunosuppressive factors (CTLA4-Ig) (7, 51). In an ongoing trial, STZ-induced diabetic monkeys receiving intraportal islet infusions from adult GTKO/hCD46/ hTFPIIns/pCTLA4-IgIns or GTKO/hCD46/hCD39Ins/hTFPIIns/pCTLA4-IgIns pigs have reduced IBMIR and early islet loss, as well as prolonged graft survival and function, maintaining euglycemia for up to 1 year (7, 52). However, most trials using genetically engineered islets include systemic immunosuppression, which will limit the clinical applicability of this approach. In a recent study by Brady et al porcine islets have been generated that produce an anti-CD2 antibody, and cause local T-cell depletion at the site of engraftment in a humanized mouse model (53). Potentially, by combining a multitude of different genetic manipulations, effectively masking the porcine islets from detection by the recipient’s immune system, while also producing local immunosuppressors, it might be feasible to reduce systemic immunosuppressive protocols, while maintaining graft survival and function (50, 53). hESC derived ␤-like cells Another promising source of islet-like cells comes from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), as they hold the potential to generate an unlimited supply of ␤-cells that could be used for the treatment of T1DM. The current challenge in the field of ES cell biology is to identify the signaling pathways and culture conditions controlling pancreatic differentiation as well as ␤-cell development and maturation. To date, the most successful approach to generate pancreatic cells in vitro aims at recapitulating the embryonic events leading to pancreatic morphogenesis; starting with the induction of definitive endoderm, followed by generation of a PDX1⫹ pancreatic progenitor pool, specification of endocrine pancreatic progenitors, and ultimately the induction of ␤-cells. Using such an approach, the first

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iteration of insulin-producing cells was generated in vitro from hESCs (54). However, these initial endocrine cells tended to be polyhormonal, coexpressing glucagon and/or somatostatin and were unresponsive to glucose challenges, indicating they lacked functional maturity (55– 58). Through the use of a hESC reporter line where GFP is expressed under the control of the insulin promoter (INSgfp/w) (59), it was possible to purify hESC-derived insulin:GFP⫹ cells by fluorescence-activated cell sorting (FACS) and demonstrate that, when transplanted into immunocompromised NSG mice, FACS-purified hESC-derived insulin:GFP⫹ cells did not give rise to insulin-positive cells in vivo, they instead committed to glucagon-positive cells (60). These results are in agreement with previous data reporting the generation of ␣-like cells following transplantation of a heterogeneous population of hESCderived pancreatic cells (56, 61), and confirm that human poly-hormonal cells generated in vitro do not give rise to functional ␤-cells in vivo, similarly to the poly-hormonal cells detected in the mouse embryo during the first wave of endocrine development (62). Of note poly-hormonal cells have also been detected in sections of human fetal pancreata (63, 64). As an alternative to generating mature, functional insulin-positive cells suitable for transplantation, scientists are leaning towards the transplantation of hPSC-derived pancreatic progenitors for the treatment of T1DM, as the use of these cells would lead to the generation of not only ␤-cells, but entire islets after in vivo development and maturation. Highly enriched populations of pancreatic endoderm have been generated in vitro from hESCs (61, 65– 67). On transplantation of these pancreatic cultures into immunocompromised mice and rats, these cells mature into mono-hormonal cells, with the formation of islet-like clusters within the transplanted grafts (61, 65– 67). Immunohistochemical analysis of the grafts indicates the presence of all the mature pancreatic lineages, including ductal, enzyme-producing exocrine, and hormone-producing endocrine cells. Human C-peptide and insulin have been detected in the serum by 3 months post-transplant, and are sufficient to ameliorate hyperglycemia in STZinduced diabetic mice (65, 67, 68). Furthermore, Bruin and colleagues demonstrated that development and functionality of the hESC-derived pancreatic progenitors is not affected by encapsulation into a macro-device (TheraCyteTM), supporting the potential use of such a system for the treatment of T1DM (69). Indeed, based on these exciting data, Viacyte, a regenerative medicine company based in California, has been granted FDA approval to launch the first clinical evaluation of a stem cell-derived islet replacement therapy for the treatment of patients with T1DM (70).


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In keeping with the goal to generate mature ␤-cells in vitro, a recent study showed that mono-hormonal insulinproducing cells with the capacity to respond to glucose challenge can be generated in culture from hESC-derived endodermal progenitor cell lines (EPCs) (71). EPCs have been established by culturing hESC-derived definitive endoderm in the presence of a serum-free medium containing VEGF, EGF, FGF2 and BMP4 on matrigel-coated plates, with mouse embryonic fibroblasts. Remarkably, in such conditions, EPCs can be expanded, without loss of hepatic, intestinal and pancreatic potential. Similarly to hESC-derived definitive endoderm, upon pancreatic differentiation, EPCs from the earlier passages generated poly-hormonal cells in vitro. However, differentiation of late passage EPCs led to the in vitro production of up to 30% glucose-responsive, mono-hormonal, insulin-positive cells, suggesting that the molecular changes acquired during EPCs expansion could play a role in endocrine maturation. While it remains to be tested whether the EPCderived insulin-positive cells can survive in vivo and restore normoglycemia in a diabetic animal model, the identification of the intrinsic molecular differences between hESC-derived definitive endoderm and late passage-EPCs may shed light on the process of cell maturation. More recently, a very interesting approach was used to generate functional ␤-cells in vitro from partially reprogrammed somatic cells. Murine fibroblasts were partially reprogrammed with inducible oct4, klf4, sox2 and c-myc, and then cultured with a histone methyltransferase inhibitor (Bix-01 294), recombinant activin A and LiCl, to generate definitive endoderm-like cells. Following treatment with retinoic acid, TGF-␤ and Shh inhibitors, ascorbic acid, and in the final stage a p38 MAP kinase inhibitor (SB203580), the authors were able to generate up to 2% insulin-positive, glucose-responsive cells. A similar protocol applied to murine iPSC-derived definitive endoderm did not generate glucose responsive, insulin-positive cells, suggesting that the definitive endoderm-generated from partially reprogrammed mouse fibroblasts was poised to generate ␤-cells. Remarkably, transplantation of these pancreatic-like cells into STZ-treated immunocompromised mice led to amelioration of hyperglycemia (72), suggesting that, if this approach can be translated to human fibroblasts, it could lead to a faster way to generate patient-specific human ␤-like cells for the treatment of T1DM. Using directed differentiation strategies; two groups recently reported a novel and efficient approach to generate ␤-like cells, in vitro, from hESCs, as well as hiPSCs (albeit with lower efficiencies) (73, 74). This remarkable breakthrough delivers a straightforward differentiation proto-


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col to generate cells that closely resemble bona fide human ␤-cells. In both studies, thyroid hormone and inhibition of TGF␤ signaling using the inhibitor ALK5iII were used to generate approximately 20%–50% insulin (c-peptide)positive cells, within a 30 to 40 days differentiation protocol. Through a careful characterization, Rezania and colleagues demonstrated that these ␤-like cells share many of the molecular and functional characteristics of mature ␤-cells, including MAFA expression and the capacity to secrete insulin in response to glucose challenge in static conditions, but fail to do so in a perifusion assay (73). Upon transplantation into immunocompromised mice, the graft (composed of endocrine and ductal cells) restored glycemia within a 2-weeks (74) and a 6-weeks (73) period following transplantation; a tremendous improvement compared to the 23-weeks period required following transplantation of hESC-derived pancreatic progenitors (73). The similarities and differences between ␤-like cells generated by the two groups remain to be elucidated by a direct comparison at the molecular level. Nevertheless, the shortened time required to restore normoglycemia following in vivo transplantation, suggests that, compared to current strategies, both these populations may represent a superior cell product for the use in clinical settings. In preparation for these trials, scientists will have to demonstrate that, similarly to the hESC-derived pancreatic progenitors, the cell population containing ␤-like cells can functionally develop in vivo within a micro- or macroencapsulation device. Alongside investigating ways to improve and refine techniques for differentiating hESCs towards a ␤-like cell phenotype, considerations should also be made as to the scalability of the approach. As it is anticipated that each transplant would require at least 300 000 IEQs for insulin independence to be achieved in humans, this may be equivalent to 90 million ␤-cells. If, as described using EPCs and the recent protocols developed by the Kieffer and Melton laboratories, 20%–50% of the cells produced after differentiation in vitro are glucose-responsive, mono-hormonal, insulin-positive cells, an average of 300 million cells would be needed per patient. The ability to differentiate hESCs in large scale may be facilitated by culturing cells in a 3D-suspension system (68, 74), although the ability to passage EPCs while retaining their differentiation potential certainly provides a window for their expansion. Furthermore, a clinically relevant cell product will need to be generated in good manufacturing procedure (GMP) conditions. While Pagliuca and colleagues, demonstrated that it is possible to generate large number of cells using bioreactors, the final stages of differentiation were carried out in media containing fetal bovine serum. The use of a serum-free media, similar to the one devel-


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oped by Rezania, will be crucial for the development of a cell population that could be endorsed for clinical use.

Conclusions Current treatment options for T1DM patients are limited, and do not eliminate long-term complications associated with the disease. Therefore, alternative therapeutic strategies are being investigated. Significant progress has been made with the use of isolated-islets for transplantation, largely due to improved immunosuppression strategies and islet isolation techniques. However, the need to improve graft reliability, durability, and functionality advocates the search for alternative transplantation sites/modalities and the replacement of systemic immunosuppressive regimens. Encapsulation technology is striving to resolve the latter, with frequent reports of new developments in the field. Nevertheless there are still some key points to address, such as; maintenance of longterm cell survival within the devices, including appropriate oxygen delivery, and the creation of a device that can contain the adequate number of islets/cells to maintain normoglycemia, while not being so large as to impair patient movement or cause discomfort when implanted. Consideration must also be given to the site of engraftment with regard to accessibility; either to remove engrafted cells, or to replenish them as needed, and to access to vasculature to ensure a rapid glucose response (11). HESC and hiPSCs hold tremendous potential to give rise to all cell types of the body, and there is no doubt that in vitro-generated pancreatic progenitors and/or ␤-cells will change the way we treat T1DM. Transplantation of these cells into humans would also likely involve encapsulation technology, not only to prevent destruction of the grafted cells by the host immune system, but also to contain the graft itself. The TheraCyteTM macroencapsulation device has already been tested in mice with positive outcomes (67, 69, 75), but further testing to ensure complete immunoisolation and containment of engrafted cells, as well as its long-term durability would be necessary for the translation of this technology into humans using hESC-derived cells. Clinical trials of the ViaCyte VC01TM combination product, in which PEC-01TM hESCderived proprietary pancreatic endoderm cells are transplanted within the Encaptra® drug delivery system, are planned to commence this year. This trial will evaluate the safety of the system, and aims to identify the optimum dose of PEC-01TM cells required to establish normoglycemia (76). As an alternative to encapsulation, it may be possible to use immune tolerance strategies to prevent auto-immune destruction of the ␤-cells, making it feasible to use

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iPSCs to generate ␤-cells from the patient themselves, avoiding allogenic rejection (77). In either case, for the medical development of these technologies we need to generate large quantities of clinically relevant cells, in GMP regulated facilities, with secure quality assurance (QA) measures. It is encouraging to witness that the tremendous efforts in the fields of islet and stem cell biology, pancreatic development, transplantation, immunology and bioengineering are currently being combined and translated into new potential therapeutic approaches that will impact the life of many diabetic patients. Furthermore, the recent progress is taking us a step closer to develop a system to generate bona fide ␤-cells in vitro. Achieving this goal will empower disease modeling and provide a platform for drug screening, which might one day allow us to develop new drugs to prevent and/or treat both type 1 and type 2 diabetes.

Acknowledgments Address all correspondence and requests for reprints to: Maria Cristina Nostro PhD, E-mail: [email protected], Toronto General Research Institute/UHN - Experimental Therapeutics, 101 College St, TMDT, 3–918, Toronto, ON, CANADA M5G 1L7, Phone: ⫹14165817595. Disclosure Summary: K.C. and M.C.N. have nothing to declare This work was supported by .

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