Effects of different routes of heparin on instant blood-mediated inflammatory reaction after portal vein islet transplantation / 中南大学学报(医学版)

OBJECTIVES@#Heparin is mainly used as an anticoagulant in clinic, and it also has a certain anti-inflammatory effect. At present, after portal vein islet transplantation in diabetic patients, heparin is mainly infused through the peripheral veins of the limbs to achieve the purpose of anticoagulation and protection of the graft, rather than through the portal vein. In this study, animal experiments were conducted to investigate the effect of heparin infusion via the portal vein and marginal ear vein on the instant blood-mediated inflammatory reaction (IBMIR) after portal vein islet transplantation, which is the choice of anticoagulation methods for clinical islet transplantation to provide a basis for decision-making.@*METHODS@#A total of 50 neonatal pigs (Xeno-1 type, 3-5 days) were selected. Islets were isolated and purified from the pancreas of neonatal pigs. Ten non-diabetic Landrace pigs (1.5-2.0 months) served as recipients, and 12 000 IEQ/kg neonatal porcine islets were transplanted into the liver through the portal vein. All recipients received bolus injection of 50 U/kg of heparin 10 minutes before transplantation. After the bolus injection of heparin, the experimental group received heparin via the portal vein [10 U/(kg·h), 5 recipients], and the control group received heparin via the marginal ear vein [10 U/(kg·h), 5 recipients]. The superior vena cava blood was collected from the 2 groups pre-operation at 1, 3, 24 h post-operation of the transplantation. The portal vein blood was collected from the experimental group at 1 and 3 h after the transplantation as well. The levels of complement C3a, C5a, thrombin-antithrombin complex (TAT), β-thromboglobulin (β-TG), and D-dimer as well as activated partial thromboplastin time (APTT) in superior vena cava blood from 1 and 3 h post-transplantation were detected in the 2 groups, and the levels of anti-Xa and anti-IIa in the portal vein and superior vena cava blood from 1 and 3 h post-transplantation in the experimental group were detected. Twenty four hours after the transplantation, the liver tissues in the 2 groups were collected for pathological examination to observe the inflammatory cell infiltration and peripheral thrombosis around the islets graft in liver.@*RESULTS@#Before transplantation, there was no statistically significant difference in C3a, C5a, TAT, β-TG, D-dimer levels and APTT between the 2 groups (all P>0.05). At 1 and 3 h after transplantation, the C3a, TAT, and D-dimer levels in the experimental group were significant decreased than those in the control groups (all P<0.05), and at 3 h after transplantation the C5a was significant decreased than that in the control group (P<0.05). At 1 and 3 h after transplantation, the anti-Xa and anti-IIa levels in the portal vein blood were significantly increased than those in the superior vena cava blood in the experimental group (all P<0.05). Pathological results showed the presence of islet cell clusters in the liver blood vessels. The thrombus formation and neutrophil infiltration around islet graft was not obvious in the experimental group, while massive thrombus formation and neutrophil infiltration in the control group.@*CONCLUSIONS@#Compared with marginal ear vein infusion of heparin, the direct infusion of heparin in the portal vein has a certain inhibitory effect on complement system, coagulation system activation and inflammatory cell infiltration in portal vein islet transplantation, which may attenuate the occurrence of IBMIR.

Comparison of two techniques for assessing pancreatic islet viability: flow cytometry and fluorescein diacetate/propidium iodine staining

Introduction: The success of islet transplantation for patients with unstable type 1 diabetes mellitus depends, in part, on the number of isolated islets and their quality, which is assessed by functional and viability tests. The test currently employed to evaluate islet viability, used by the Collaborative Islet Transplant Registry to release products for transplantation, is fluorescein diacetate/propidium iodide (FDA/PI) staining. However, the efficacy of this method relies on researcher experience; in this context, a quantitative method may be useful. The aim of this study was to compare islet viability as assessed by flow cytometry and the FDA/PI assay. Methods: Viability was analyzed in islets isolated from 10 male Wistar rats. Upon FDA/PI staining, 50 islets from each animal were analyzed under fluorescence microscopy by two well-trained researchers. For flow cytometry, islets were dispersed and 100 000 single cells were incubated with the 7-amino-actinomycin D (7AAD) fluorophore (dyes necrotic and late apoptotic cells) and the Annexin V-APC antibody (marks early apoptotic cells). Results: A moderate correlation was found between techniques (r = 0.6; p = 0.047). The mean islet viability measured by flow cytometry was higher than that estimated using FDA/PI staining (95.5 ± 1.4% vs 89.5 ± 5.0%; p = 0.002). Conclusions: Although flow cytometry is more expensive and time-consuming than FDA/PI staining, it is a quantitative technique with greater reproducibility that is less subject to inter-observer variability than FDA/PI. Therefore, flow cytometry appears to be the technique of choice when aiming for a more precise determination of islet viability. (AU)

Terapias alternativas para o diabetes mellitus tipo 1: caracterização funcional do gene Txnip na diferenciação ß-pancreática e desenvolvimento de biomaterial inovador para microencapsulamento celular / Alternative therapies for type 1 diabetes mellitus: functional characterization of Txnip gene during -pancreatic differentiation and generation of an innovative biomaterial for cell microencapsulation

O diabetes mellitus do tipo 1 (DM1) é uma doença causada pela destruição autoimune das células-ß produtoras de insulina do pâncreas. O transplante de ilhotas pancreáticas é um procedimento tecnicamente simples sendo uma alternativa terapêutica interessante para o DM1. Entretanto, a oferta limitada de pâncreas de doadores falecidos e a necessidade de imunossupressão crônica são fatores que limitam a aplicabilidade dessa modalidade de transplante. Neste trabalho foram estudadas duas estratégias que visam oferecer soluções aos fatores limitantes do transplante de ilhotas pancreáticas. Na primeira parte do trabalho, o mecanismo molecular que dirige o processo de diferenciação de células-tronco embrionárias murinas (murine embryonic stem cells, mESCs) em células produtoras de insulina (insulin producing cells, IPCs) foi analisado visando otimizar o processo de diferenciação. Nós selecionamos o gene Thioredoxin interacting protein (Txnip), diferencialmente expresso ao longo da diferenciação ß-pancreática, para realizar um estudo funcional através da modificação genética de mESCs. Os resultados obtidos permitiram verificar que a inibição de Txnip na diferenciação ß-pancreática pode induzir a diferenciação de IPCs com maior expressão de marcadores de células- e mais responsivas ao estímulo de glicose. Além disso, o modelo de zebrafish permitiu elucidar in vivo o papel de Txnip durante a organogênese pancreática, revelando que a inibição desse gene é capaz de aumentar a massa de células-ß através do estimulo de células presentes no ducto extra-pancreático. Dessa forma, a inibição de Txnip pode aprimorar os protocolos para obtenção de IPCs a partir de células-tronco pluripotentes. A exposição crônica a agentes imunossupressores diabetogênicos e a perda de componentes de matriz extracelular durante o isolamento de ilhotas pancreáticas são causas para a perda de funcionalidade do enxerto. Dessa forma, na segunda parte do trabalho, um biomaterial inovador foi desenvolvido, contendo um polímero de laminina (polilaminina, PLn) para o encapsulamento e a imunoproteção de ilhotas pancreáticas. As cápsulas produzidas com o biomaterial desenvolvido, Bioprotect-Pln, são térmica- e mecanicamente estáveis, além de serem biocompatíveis e capazes de imunoproteger ilhotas pancreáticas humanas in vitro. O encapsulamento com Bioprotect-Pln preserva a funcionalidade de ilhotas pancreáticas. Além disso, quando cápsulas vazias de Bioprotect-Pln foram implantadas em camundongos imunocompetentes, houve atenuação da resposta inflamatória ao implante, uma das principais causas para perda de funcionalidade de enxertos encapsulados. Os resultados obtidos indicam que a presença de polilaminina na malha capsular induz uma resposta anti-inflamatória que pode beneficiar a preservação do enxerto de ilhotas pancreáticas encapsuladas. Atualmente, o transplante de ilhotas pancreáticas é visto como a terapia celular mais promissora para atingir a independência de insulina em pacientes de DM1, porém, a aplicabilidade desse transplante ainda é limitada. Este trabalho contribuiu para a elucidação dos mecanismos moleculares que podem aprimorar o processo de diferenciação de célulastronco pluripotentes em IPCs, estabelecendo uma fonte alternativa de células para a terapiade reposição, e, também, estabeleceu um biomaterial inovador, capaz de diminuir a resposta inflamatória ao implante de microcápsulas e de imunoproteger células microencapsuladas. Desta forma, este trabalho contribui para o estabelecimento da terapia de reposição celular para pacientes de DM1

Type 1 diabetes mellitus (DM1) is a disease caused by the autoimmune destruction of insulin-producing pancreatic ß-cells. Pancreatic islet transplantation is a technically simple procedure and an interesting alternative therapy for DM1, however, the limited supply of cadaveric donated pancreas and the need of life-long immunosuppression are factors which limit its applicability. In the present work, two strategies were employed aiming at establishing viable solutions for the factors limiting pancreatic islet transplantation. In the first part of this study, the molecular mechanism which drives differentiation of murine embryonic stem cells (mESCs) into insulin producing cells (IPCs) was analyzed in order to optimize the differentiation process. The Thioredoxin interacting protein (Txnip) gene, which is differentially expressed along -pancreatic differentiation, was selected to undergo a functional analysis by genetically modifying mESCs. The results allowed us to verify that Txnip inhibition during the ß-pancreatic differentiation process can induce differentiation of IPCs displaying higher expression of ß-cell markers and being more responsive to glucose stimuli. In addition, the zebrafish model allowed us to elucidate in vivo the role of Txnip during pancreatic organogenesis, revealing that its inhibition is able to increase the mass of ß-cells through stimulation of extra-pancreatic ductal cells. Therefore, Txnip inhibition may turbinate IPCs differentiation from pluripotent stem cells. The chronic exposure to diabetogenic immunosuppressive agents and the loss of extracellular matrix components during isolation of pancreatic islets are probable causes for the loss of pancreatic islet graft functionality. Therefore, in the second part of this study, an innovative biomaterial was developed by incorporating a laminin polymer (polylaminin, PLn) for the encapsulation and immunoprotection of pancreatic islets. The capsules produced with the novel biomaterial, Bioprotect-Pln, are biocompatible, thermally and mechanically stable and are able to immunoprotect human pancreatic islets in vitro. Encapsulation with Bioprotect-Pln preserves the functionality of pancreatic islets. In addition, when empty Bioprotect-Pln capsules were implanted into immunocompetent mice, an attenuation of the inflammatory response to the implant occurred, this being one of the main causes of encapsulated graft loss. The results indicate that polylaminin addition to the capsular mesh induces an anti-inflammatory response which may favor preservation of the engrafted encapsulated pancreatic islets. Pancreatic islet transplantation is currently seen as the most promising cell therapy to achieve insulin independence in DM1 patients, however, the applicability of this transplant is still limited. This work contributed to the elucidation of the molecular mechanisms which can turbinate the differentiation of pluripotent stem cells into IPCs, establishing an alternative source of cells for the replacement therapy, and, also, established an innovative biomaterial which is able to decrease the inflammatory response to the graft, thereby immunoprotecting the microencapsulated cells. Therefore, this work contributes to the establishment of the cell replacement therapy for DM1 patients

Mecanismos moleculares mediadores da citoproteção de células beta pancreáticas induzidos por prolactina / Role of HSPB1 in PRL-induced cytoprotective effects on beta cells

A manutenção da célula de ilhotas in vitro aparece como uma estratégia atraente para aumentar o resultado do transplante de ilhotas pancreáticas. Entretanto, o destino das ilhotas em cultura é determinado pelo equilíbrio entre mediadores pró e antiapoptóticos. Nós mostramos anteriormente que os níveis de HSPB1 são aumentados pela prolactina (PRL) tanto nas células beta pancreáticas humanas quanto nas células de insulinoma murino MIN6. Além disso, mostramos que os efeitos pró- sobrevivência induzidos pela prolactina nas células beta pancreáticas são mediados pela HSPB1. Uma vez que o papel da HSPB1 nas células beta não foi estudado diretamente, procuramos explorar os mecanismos moleculares pelos quais a HSPB1 medeia a citoproteção da célula beta induzida pela PRL. Para isso, células MIN6 derivadas de um insulinoma de camundongo e cultura primária de ilhotas pancreáticas murinas (I), silenciadas ou superexpressando HSPB1 foram submetidas à privação de soro e então pré- tratadas na presença ou na ausência de PRL (300 ng / mL) e expostos a ou citocinas (IL-1ß (0,8 ng / mL), IFN-γ (4 ng / mL) e TNF-α (8 ng / mL) por 16 ou 24 h. Após esses períodos de tempo foi avaliada a viabilidade celular. De fato, as células silenciadas para HSPB1 tiveram maiores porcentagens de morte celular em comparação aos controles. No entanto, a superexpressão de HSPB1 sozinha imita os efeitos citoprotectores da Prolactina em ambas as células MIN6 e nas culturas primárias das ilhotas. Estes resultados mostram o papel fundamental da HSPB1 no efeito citoprotetor inibindo a apoptose inducida pelo tratamento com citocinas pró-inflamatórias. Além disso, os lisados de células Min6 tratadas com citocinas na presença ou na ausência de PRL durante 6 h foram sujeitos a imunoprecipitação de HSPB1. Proteínas coimmunoprecipitadas separadaspor SDS-PAGE e posteriormente identificadas por nano-HPLC acoplado à espectrometria de massas. Células pré-tratadas com PRL apresentaram um enriquecimento de proteínas que coprescipitaram com HSPB1 relacionadas em processos de resistência ao estresse oxidativo, degradação proteica e metabolismo de carboidratos. Células MIN6, silenciadas ou superexpressando HSPB1 foram expostas á menadiona e peróxido de hidrogênio e parâmetros oxidativos foram analisados. O silenciamento de HSPB1 promoveu células mais sensíveis ao estresse oxidativo e levou a uma redução da capacidade antioxidante, enquanto que prolactina induziu citoproteção mediada por HSPB1 contra o estresse oxidativo. A superexpressão de HSPB1, no entanto, levou a efeitos opostos. O tratamento com PRL, o silenciamento ou superexpressão de HSPB1 não mudou a expressão de enzimas antioxidantes, mas os níveis proteicos de HSPB1 estão relacionados com a modulação da razão GSH/GSSG e a atividade de G6PD. Dado de estudos recentes reportam que o perfil respiratório das ilhotas prévias ao transplante pode predizer seu desempenho e que não se sabe nada sobre se a PRL poderia modular a função mitocondrial nas células beta; no presente projeto foi investigado se o tratamento hormonal poderia aumentar a eficiência mitocondrial das células beta. Observamos que o tratamento com citocinas pró-inflamatórias produziu uma diminuição na eficiência do consumo de oxigênio mitocondrial estar relacionado à síntese de ATP. Esses resultados foram significativamente revertidos a valores similares ao obtidos nas células submetidas Às condições de máxima viabilidade após o tratamento com PRL. Além disso, os resultados mostraram que os níveis elevados de HSPB1 medeiam este efeito, uma vez que a falta desta proteína anulou significativamente a recuperação da função mitocondrial induzida pelo tratamento hormonal. Visto que as taxas de síntese de ATP mitocondrial são as responsáveis pela elevação na sua concentração intracelular e que esse evento está diretamente relacionado com a secreção de insulina nas células beta, analisamos se diferentes níveis proteicos de HSPB1 poderia modificar a função secretora de células beta. Para isso foram calculados os índices de estímulo da secreção de insulina em resposta ao aumento da concentraçãode glicose no meio de cultura tanto em células parentais MIN6 como em culturas primárias de ilhotas pancreáticas murinas que foram submetidas ou não ao silenciamento ou superexpressão de HSPB1. Nossos resultados mostraram que nem a presença de citocinas, Prolactina, ou a ausência ou superexpressão de HSPB1 nas culturas celulares analisadas apresentaram diferença significativa em relação aos índices de estímulo da secreção e conteúdo de insulina. Esses resultados sugerem que nem a falta, nem a superexpressão de HSPB1 poderia alterar a função de célula beta. Nós mostramos a relevância da HSPB1 em ambos os efeitos pró- sobrevivência da PRL contra a morte da célula beta induzida tanto por citocinas quanto por indução de estresse oxidativo. Este último efeito poderia também estar relacionado com a participação da HSPB1 na recuperação da função mitocondrial observada após o tratamento hormonal corroborando assim parte dos resultados obtidos nos experimentos de immunoprecipitação. Finalmente, nossos resultados destacam a importância de mais estudos visando um entendimento mais profundo das funções da HSPB1 nas células beta, uma vez que elas poderiam levar à mitigação da morte da célula beta através da regulação positiva de uma via de proteção endógena, que não é dependente da modulação do sistema imunológico

The success of islet transplantation has improved lately. Unfortunately, it is still compromised by cell loss. Maintaining islet cell in vitro appears as an attractive strategy to increase the outcome of pancreatic islet transplantation. However, islet fate in culture is determined by the balance between pro- and anti- apoptotic mediators. We have previously shown that Heat Shock Protein B1 (HSPB1) levels are increased by prolactin (PRL) on both human pancreatic beta cells and MIN6 murine insulinoma cells. Furthermore, we have demonstrated the prolactin-induced pro-survival effects on pancreatic beta-cells are mediated by HSPB1. Since HSPB1 role in beta cells has not been directly studied, we set out to explore the molecular mechanisms by which HSPB1 mediates PRL-induced beta cell cytoprotection. For this purpose, MIN6 insulinoma mouse cells and primary culture of murine pancreatic islets (I) wild type, HSPB1 silenced or overexpressing the chaperone were subjected to serum starvation and then pre-treated in the presence or in the absence of PRL (300 ng/mL) and exposed to or cytokines (IL-1ß (0,8 ng/mL), IFN-γ (4 ng/mL) and TNF-α (8 ng/mL)) for 16 or 24h. Then, we analyse cell viability. HSPB1silenced cells presented higher percentages of cell death compared to controls. However, the overexpression of HSPB1, independently of hormonal treatment, was able mimic the cytoprotective effects of Prolactin. These results point at the key role of HSPB1 in the cytoprotective effect against proinflammatory cytokines-induced beta cell death. In addition, lysates from Min6 cells incubated for 6 hours in the presence of a cocktail of cytokines and/or PRL were subjected to HSPB1 immunoprecipitation. Co-precipitated proteins were identified by SDS-PAGE coupled to mass spectrometry. We found an enrichment of proteins relatedto signaling pathways involved in a response against oxidative and endoplasmic reticulum stress induction. Moreover, we also identified antiapoptotic effects and carbohydrate metabolism related proteins. Indeed, HSPB1 knockdown rendered cells more sensitive to oxidative stress and led to a reduced antioxidant capacity, while prolactin induced an HSPB1- mediated cytoprotection against ROS induced beta-cell apoptosis. One again, HSPB1 overexpression mimic PRL- induced cytoprotection. While hormonal treatment, HSPB1 silencing or overexpression did not change the expression of antioxidant enzymes; this conditions influenced reduced glutathione cell content and G6DP activity. Since recent studies have pointed that islets respiratory profile prior to transplantation may predict their performance; we also investigated whether PRL treatment could increase beta-cell mitochondrial efficiency. We observed a cytokine-induced increase of mitochondrial oxygen consumption rate not related to ATP synthesis, which was significantly decreased upon PRL treatment. HSPB1 was a key mediator of this effect since the lack of this protein significantly abrogated PRL-induced mitochondrial function recovery. The secretory function was then analysed in wild type MIN6 cells as well as in primary cultures of pancreatic islets either HSPB1 silenced or overexpressing the chaperone. Cells were subjected to serum starvation and then pre-treated in the presence or in the absence of PRL and exposed to cytokines for 16 or 24h. We didn´t found significant differences in both glucose induced-insulin secretion and insulin content between the hormonal treatment, HSPB1 silencing or overexpression. These results suggest that neither lack, nor overexpression of HSPB1 could alter beta cell function. Altogether our results have shown the importance of HSPB1 on PRL prosurvival effects as well as on maintenance of mitochondrial efficiency against both cytokine treatment and oxidative-stress-induced beta cell damage. These results are in accordance with the PRL-induced enrichment of HSPB1 interacting proteins displaying functions related to protein degradation, oxidative stress protection or mitochondrial carbohydrate metabolism.Finally, our results outline the importance of further studies aiming at a deeper understanding of HSPB1 functions on beta cells, since they could lead to the mitigation of beta cell death through the up-regulation of an endogenous protective pathway, which is not dependent on the modulation of the immune system

Alginate-Catechol Cross-Linking Interferes with Insulin Secretion Capacity in Isolated Murine Islet Cells

Over the past three decades, human pancreatic islet isolation and transplantation techniques have developed as a routine clinical procedure for selected patients with type 1 diabetes mellitus. However, due to the donor shortage and required chronic systemic immunosuppression, the widespread application of islet transplantation is limited. To overcome these limitations, providing a physical barrier to transplanted islet cells with encapsulating biomaterial has emerged as a promising approach to enhance engraftment and promote islet survival post-transplantation. Alginate has been considered to be a reliable biomaterial, as it enhances islet survival and does not hamper hormone secretion. Alginate-catechol (Al-CA) hydrogel was reported to provide high mechanical strength and chemical stability without deformation over a wide range of pH values. In this study, we, demonstrated, for the first time in the literature, that encapsulation of murine pancreatic islet cells with Al-CA hydrogel does not induce cytotoxicity ex vivo for an extended period; however, it does markedly abate glucose-stimulated insulin secretion. Catechol should not be considered as a constituent for alginate gelation for encapsulating islet cells in the application of islet transplantation.

Human islet xenotransplantation in rodents: A literature review of experimental model trends

Among the innovations for the treatment of type 1 diabetes, islet transplantation is a less invasive method of treatment, although it is still in development. One of the greatest barriers to this technique is the low number of pancreas donors and the low number of pancreases that are available for transplantation. Rodent models have been chosen in most studies of islet rejection and type 1 diabetes prevention to evaluate the quality and function of isolated human islets and to identify alternative solutions to the problem of islet scarcity. The purpose of this study is to conduct a review of islet xenotransplantation experiments from humans to rodents, to organize and analyze the parameters of these experiments, to describe trends in experimental modeling and to assess the viability of this procedure. In this study, we reviewed recently published research regarding islet xenotransplantation from humans to rodents, and we summarized the findings and organized the relevant data. The included studies were recent reports that involved xenotransplantation using human islets in a rodent model. We excluded the studies that related to isotransplantation, autotransplantation and allotransplantation. A total of 34 studies that related to xenotransplantation were selected for review based on their relevance and current data. Advances in the use of different graft sites may overcome autoimmunity and rejection after transplantation, which may solve the problem of the scarcity of islet donors in patients with type 1 diabetes.

Pancreatitis, genes y autotrasplante de islotes: actualizaciones y nuevos horizontes / Pancreatitis, genes and islet cells auto transplant: updates and new horizons

La pancreatitis es una inflamación del páncreas que puede progresar de una presentación aguda, a una presentación aguda recurrente y eventualmente a pancreatitis crónica, caracterizada por cambios morfológicos y formación de cicatriz los cuales son irreversibles. La entidad conocida como pancreatitis hereditaria ha sido reconocida en la literatura por años y ciertamente el hallazgo del gen PRSS1 en 1996 marcó el inicio de una era de descubrimientos genéticos asociados a dicha enfermedad. Desde entonces, múltiples genes han sido descritos como causa de pancreatitis hereditaria o modificadores de la enfermedad, entre los que se destacan el PRSS1, SPINK1, CFTR, CASR, CTRC, CLDN2 y CPA1 entre otros. La selección de pacientes a la que se le va a practicar los estudios genéticos correspondientes debe ser guiada por las recomendaciones de los expertos y debe cumplir con los criterios correspondientes. El tratamiento, una vez hecho el diagnóstico, debe ser adaptado en base a las necesidades particulares de cada paciente. Ciertamente, el advenimiento de la pancreatectomía total con auto trasplante, cuyo principal objetivo es aliviar el dolor ocasionado por la pancreatitis crónica a la vez que se reduce la severidad de la diabetes inducida por la pancreatectomía, es una alternativa atractiva en el tratamiento y manejo de pacientes con diagnóstico de pancreatitis hereditaria. Sin embargo, estrictos criterios y un manejo interdisciplinario son esenciales ya que éste es un procedimiento irreversible y tiene consecuencias médicas por el resto de la vida que todo paciente tiene que conocer y que a su vez, el equipo médico debe saber identificar y tratar a tiempo. Sin embargo, existen muchas áreas potenciales en este campo para investigaciones que sean mayores, mejores y a la vanguardia para lograr un mejor entendimiento y desarrollar potenciales curas para esta enfermedad

Pancreatitis is an inflammation of the pancreas that can progress from an acute presentation to an acute recurring presentation and eventually to chronic pancreatitis, which is characterized by irreversible morphological changes and scarring of the pancreas. The entity known as hereditary pancreatitis has been recognized in the literature for years and certainly the discovery of the PRSS1 gene in 1996 marked the beginning of a new era of genetic discoveries associated with the disease. Since then, multiple genes have been described as the causing agents of pancreatitis or disease modifiers, some of the most important ones being the PRSS1, SPINK1, CFTR, CASR, CTRC, CLDN2, and CPA1. The patient selection process for genetic testing should be guided by the current experts’ recommendations and should meet specific corresponding criteria. Once the diagnosis has been made, treatment should be tailored to each patient’s particular needs. Certainly, the advent of the total pancreatectomy with auto islet cell transplantation, which has the main goal to improve the pain caused by the chronic pancreatitis and simultaneously reduce the severity of the pancreatectomy induced diabetes, is an attractive alternative in the treatment and management of patient with a diagnosis of hereditary pancreatitis. However, strict criteria and an interdisciplinary management are essential since this surgery is irreversible and carries lifetime health consequences that all patients must be aware of and the medical team must be able to early recognize these and treat accordingly. However, there are many potential areas in this field for more, better and forefront research to be developed aiming for a better understanding of the disease process and for the development of a cure

Is islet transplantation a realistic approach to curing diabetes?

Since the report of type 1 diabetes reversal in seven consecutive patients by the Edmonton protocol in 2000, pancreatic islet transplantation has been reappraised based on accumulated clinical evidence. Although initially expected to therapeutically target long-term insulin independence, islet transplantation is now indicated for more specific clinical benefits. With the long-awaited report of the first phase 3 clinical trial in 2016, allogeneic islet transplantation is now transitioning from an experimental to a proven therapy for type 1 diabetes with problematic hypoglycemia. Islet autotransplantation has already been therapeutically proven in chronic pancreatitis with severe abdominal pain refractory to conventional treatments, and it holds promise for preventing diabetes after partial pancreatectomy due to benign pancreatic tumors. Based on current evidence, this review focuses on islet transplantation as a realistic approach to treating diabetes.

Stepwise Approach to Problematic Hypoglycemia in Korea: Educational, Technological, and Transplant Interventions

Impaired awareness of hypoglycemia has been found to be prevalent in 20% to 40% of people with type 1 diabetes. If a similar prevalence exists in Koreans with type 1 diabetes, at a minimum, thousands of people with type 1 diabetes suffer at least one unpredicted episode of severe hypoglycemia per year in Korea. For patients with problematic hypoglycemia, an evidence-based stepwise approach was suggested in 2015. The first step is structured education regarding multiple daily injections of an insulin analog, and the second step is adding a technological intervention, such as continuous subcutaneous insulin infusion or real-time continuous glucose monitoring. The next step is a sensor-augmented pump, preferably with a low glucose suspension feature or very frequent contact, and the final step is islet or pancreas transplantation. In Korea, however, none of these treatments are reimbursed by the National Health Insurance, and thus have not been widely implemented. The low prevalence of type 1 diabetes means that Korean physicians are relatively unfamiliar with the new technologies in this field. Therefore, the roles of new technologies and pancreas or islet transplantation in the treatment of problematic hypoglycemia need to be defined in the current clinical setting of Korea.

Polyglycolic Acid Fibrous Scaffold Improving Endothelial Cell Coating and Vascularization of Islet / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Improving islet graft revascularization has become a crucial task for prolonging islet graft survival. Endothelial cells (ECs) are the basis of new microvessels in an isolated islet, and EC coating has been demonstrated to improve the vascularization and survival of an islet. However, the traditional method of EC coating of islets has low efficiency in vitro. This study was conducted to evaluate the effect of a polyglycolic acid (PGA) scaffold on the efficiency of islet coating by ECs and the angiogenesis in the coated islet graft.</p><p><b>METHODS</b>A PGA fibrous scaffold was used for EC coating of islet culture and was evaluated for its efficiency of EC coating on islets and islet graft angiogenesis.</p><p><b>RESULTS</b>In in vitro experiments, we found that apoptosis index of ECs-coating islet in PGA group (27% ± 8%) was significantly lower than that in control group (83% ± 20%, P < 0.05) after 7 days culture. Stimulation index was significantly greater in the PGA group than in the control group at day 7 after ECs-coating (2.07 ± 0.31 vs. 1.80 ± 0.23, P < 0.05). vascular endothelial growth factor (VEGF) level in the PGA group was significantly higher than the coating in the control group after 7 days culture (52.10 ± 13.50 ng/ml vs. 16.30 ± 8.10 ng/ml, P < 0.05). Because of a tight, circumvallated, adhesive and three-dimensional growth microenvironment, islet cultured in a PGA scaffold had higher coating efficiency showing stronger staining intensity of enzyme than those in the control group after 14 days of culture following ECs-coating. For in vivo study, PGA scaffold significantly prolonged the average survival time of EC-coated islet graft after transplantation compared with control group (15.30 ± 5.60 days vs. 8.30 ± 2.45 days, P < 0.05). The angiogenesis and area of survived grafts were more in the PGA group compared with the control group by measuring the mean microvessel density (8.60 ± 1.21/mm2 vs. 5.20 ± 0.87/mm2, P < 0.05). In addition, expression of VEGF and tyrosin-protein kinase receptor (Tie-2) gene increased in PGA scaffold group than that in control group by real-time reverse transcription-polymerase chain reaction analysis.</p><p><b>CONCLUSIONS</b>These results demonstrate that the efficiency of EC coating of islets was successfully increased by culturing ECs on a PGA scaffold. This method enhances the function, survival, and vascularization of isolated islets in vitro and in vivo.</p>

Management of Hypoglycemic Unawareness and the Current Status of Clinical Allogeneic Islet Transplantation / 임상당뇨병

Problematic hypoglycemia is defined as a condition in which episodes of severe hypoglycemia are unpredictable and/or cannot be easily explained or prevented, typically associated with impaired awareness of hypoglycemia. The treatment algorithm for patients with type 1 diabetes and problematic hypoglycemia emphasizes the stepwise approach including structured education regarding multiple daily injections of insulin, use of technology such as sensor-augmented pump with low glucose suspension, and islet or pancreas transplantation. Although the prevalence of insulin independence at 5 years is 25~50% in most recent clinical trials of islet transplantation, both islet and pancreas transplantation are equally efficient to cure severe hypoglycemia for more than 5 years in about 70% of the recipients. To date, international cohorts of clinical islet transplantation such as the French-Swiss GRAGIL Network have successfully reproduced the long-term C-peptide positivity initially achieved with the Edmonton protocol, with long-term insulin independence demonstrated in selected cases. Several cases with partial islet graft function have been reported in Korea, with the first case of long-term insulin independence being reported in late 2015. Therefore, islet transplantation can offer freedom from life-threatening severe hypoglycemia for type 1 diabetes patients with problematic hypoglycemia, even in non-responders to the latest technology-based treatment.

Implications of Calcineurin/NFAT Inhibitors' Regulation of Dendritic Cells and Innate Immune Cells in Islet Xenotransplantation

Calcineurin inhibitors, such as cyclosporine and tacrolimus (FK506) are broadly used in organ transplantations as immune suppressants. As the calcineurin/NFAT signaling pathway has been identified as critical pathway in the interleukin-2 (IL-2) production of T cells, inhibition of T-cell derived IL-2 has been considered the major mechanism of calcineurin inhibitors. However, there is increasing evidence that NFAT transcription factor is involved in multiple functions of dendritic cells and innate immune cells as well. NFAT expression is not restricted to T cells, and IL-2 can be produced in dendritic cells and macrophages through the calcineurin/NFAT pathway. Furthermore, it has been discovered that NFAT regulates expressions of several inflammatory mediators, including TNF-α and cyclooxygenase-2 in innate immune cells. Therefore, calcineurin inhibitors may have much broader effects in the transplant recipients than previously being considered. In this review, we reviewed recently discovered roles of NFAT pathway in dendritic cells and innate immune cells, and discussed positive and negative implications of calcineurin inhibitors' broader effects with a focus on islet xenotransplantation.

Gastric submucosa is inferior to the liver as transplant site for autologous islet transplantation in pancreatectomized diabetic Beagles / 华中科技大学学报（医学）（英德文版）

Intraportal transplantation of islets is no longer considered to be an ideal procedure and finding the extrahepatic alternative site is becoming a subject of high priority. Herein, in this study, we would introduce our initial outcomes of using gastric submucosa (GS) and liver as sites of islet autotransplantation in pancreatectomized diabetic Beagles. Total pancreatectomy was performed in Beagles and then their own islets extracted from the excised pancreas were transplanted into GS (GS group, n=8) or intrahepatic via portal vein (PV group, n=5). Forty-eight hours post transplantation, graft containing tissue harvested from the recipients revealed the presence of insulin-positive cells. All recipients in GS group achieved euglycemia within 1 day, but returned to a diabetic state at 6 to 8 days post-transplantation (mean survival time, 7.16±0.69 days). However, all of the animals kept normoglycemic until 85 to 155 days post-transplantation in PV group (mean survival time, 120±28.58 days; P<0.01 vs. GS group). The results of intravenous glucose tolerance test (IVGTT) confirmed that the marked improvement in glycometabolism was obtained in intrahepatic islet autotransplantation. Thus, our findings indicate that the liver is still superior to the GS as the site of islet transplantation, at least in our islet autotransplant model in pancreatectomized diabetic Beagles.

Insight into Microenvironment Remodeling in Pancreatic Endocrine Tissue Engineering: Biological and Biomaterial Approaches

The treatment of diabetes mellitus, as a chronic and complicated disease, is a valuable purpose. Islet transplantation can provide metabolic stability and insulin independence in type 1 diabetes patients. Diet and insulin therapy are only diabetes controllers and cannot remove all of the diabetes complications. Moreover, islet transplantation is more promising treatment than whole pancreas transplantation because of lesser invasive surgical procedure and morbidity and mortality. According to the importance of extracellular matrix for islet viability and function, microenvironment remodeling of pancreatic endocrine tissue can lead to more success in diabetes treatment by pancreatic islets. Production of bioengineered pancreas and remodeling of pancreas extracellular matrix provide essential microenvironment for re-vascularization, re-innervation and signaling cascades triggering. Therefore, islets show better viability and function in these conditions. Researchers conduct various scaffolds with different biomaterials for the improvement of islet viability, function and transplantation outcome. The attention to normal pancreas anatomy, embryology and histology is critical to understand the pancreatic Langerhans islets niche and finally to achieve efficient engineered structure. Therefore, in the present study, the status and components of the islets niche is mentioned and fundamental issues related to the tissue engineering of this structure is considered. The purpose of this review article is summarization of recent progress in the endocrine pancreas tissue engineering and biomaterials and biological aspects of it.

Development of the endocrine pancreas and novel strategies for beta-cell mass restoration and diabetes therapy

Diabetes mellitus represents a serious public health problem owing to its global prevalence in the last decade. The causes of this metabolic disease include dysfunction and/or insufficient number of β cells. Existing diabetes mellitus treatments do not reverse or control the disease. Therefore, β-cell mass restoration might be a promising treatment. Several restoration approaches have been developed: inducing the proliferation of remaining insulin-producing cells, de novo islet formation from pancreatic progenitor cells (neogenesis), and converting non-β cells within the pancreas to β cells (transdifferentiation) are the most direct, simple, and least invasive ways to increase β-cell mass. However, their clinical significance is yet to be determined. Hypothetically, β cells or islet transplantation methods might be curative strategies for diabetes mellitus; however, the scarcity of donors limits the clinical application of these approaches. Thus, alternative cell sources for β-cell replacement could include embryonic stem cells, induced pluripotent stem cells, and mesenchymal stem cells. However, most differentiated cells obtained using these techniques are functionally immature and show poor glucose-stimulated insulin secretion compared with native β cells. Currently, their clinical use is still hampered by ethical issues and the risk of tumor development post transplantation. In this review, we briefly summarize the current knowledge of mouse pancreas organogenesis, morphogenesis, and maturation, including the molecular mechanisms involved. We then discuss two possible approaches of β-cell mass restoration for diabetes mellitus therapy: β-cell regeneration and β-cell replacement. We critically analyze each strategy with respect to the accessibility of the cells, potential risk to patients, and possible clinical outcomes.

Human pancreatic islet transplantation: an update and description of the establishment of a pancreatic islet isolation laboratory

Type 1 diabetes mellitus (T1DM) is associated with chronic complications that lead to high morbidity and mortality rates in young adults of productive age. Intensive insulin therapy has been able to reduce the likelihood of the development of chronic diabetes complications. However, this treatment is still associated with an increased incidence of hypoglycemia. In patients with “brittle T1DM”, who have severe hypoglycemia without adrenergic symptoms (hypoglycemia unawareness), islet transplantation may be a therapeutic option to restore both insulin secretion and hypoglycemic perception. The Edmonton group demonstrated that most patients who received islet infusions from more than one donor and were treated with steroid-free immunosuppressive drugs displayed a considerable decline in the initial insulin independence rates at eight years following the transplantation, but showed permanent C-peptide secretion, which facilitated glycemic control and protected patients against hypoglycemic episodes. Recently, data published by the Collaborative Islet Transplant Registry (CITR) has revealed that approximately 50% of the patients who undergo islet transplantation are insulin independent after a 3-year follow-up. Therefore, islet transplantation is able to successfully decrease plasma glucose and HbA1c levels, the occurrence of severe hypoglycemia, and improve patient quality of life. The goal of this paper was to review the human islet isolation and transplantation processes, and to describe the establishment of a human islet isolation laboratory at the Endocrine Division of the Hospital de Clínicas de Porto Alegre – Rio Grande do Sul, Brazil.

Assessment of nursing care using indicators generated by software / Avaliação da assistência de enfermagem utilizando indicadores gerados por um software / Evaluación de la asistencia de enfermería utilizando indicadores generados por un software

OBJECTIVE: to analyze the efficacy of the Nursing Process in an Intensive Care Unit using indicators generated by software. METHOD: cross-sectional study using data collected for four months. RNs and students daily registered patients, took history (at admission), performed physical assessments, and established nursing diagnoses, nursing plans/prescriptions, and assessed care delivered to 17 patients using software. Indicators concerning the incidence and prevalence of nursing diagnoses, rate of effectiveness, risk diagnoses, and rate of effective prevention of complications were computed. RESULTS: the Risk for imbalanced body temperature was the most frequent diagnosis (23.53%), while the least frequent was Risk for constipation (0%). The Risk for Impaired skin integrity was prevalent in 100% of the patients, while Risk for acute confusion was the least prevalent (11.76%). Risk for constipation and Risk for impaired skin integrity obtained a rate of risk diagnostic effectiveness of 100%. The rate of effective prevention of acute confusion and falls was 100%. CONCLUSION: the efficacy of the Nursing Process using indicators was analyzed because these indicators reveal how nurses have identified patients' risks and conditions, and planned care in a systematized manner. .

OBJETIVO: analisar a eficácia do Processo de Enfermagem em uma Unidade de Terapia Intensiva, utilizando indicadores gerados por um software. MÉTODO: estudo transversal, cujos dados foram coletados durante quatro meses. Enfermeiros e acadêmicos realizaram, diariamente, cadastro e anamnese (na admissão), exame físico, diagnósticos de enfermagem, planejamento/prescrição de enfermagem e avaliação da assistência de 17 pacientes, utilizando um software. Calculou-se os indicadores incidência e prevalência de diagnósticos de enfermagem, taxa de efetividade diagnóstica de risco e taxa de efetividade na prevenção de complicações. RESULTADOS: o Risco de desequilíbrio na temperatura corporal foi o diagnóstico mais incidente (23,53%) e o menos incidente foi o Risco de constipação (0%). O Risco de integridade da pele prejudicada foi prevalente em 100% dos pacientes, enquanto o Risco de confusão aguda foi o menos prevalente (11,76%). Risco de constipação e Risco de integridade da pele prejudicada obtiveram taxa de efetividade diagnóstica de risco de 100%. A taxa de efetividade na prevenção de confusão aguda e de queda foi de 100%. CONCLUSÃO: analisou-se a eficácia do Processo de Enfermagem utilizando indicadores, pois retratam como o enfermeiro tem identificado os problemas e riscos do paciente, e planejado a assistência de forma sistematizada. .

OBJETIVO: analizar la eficacia del Proceso de Enfermería en una Unidad de Terapia Intensiva, utilizando indicadores generados por un software. MÉTODO: estudio transversal, cuyos datos fueron recolectados durante cuatro meses. Enfermeros y académicos realizaron, diariamente, registro y anamnesis (en la admisión), examen físico, diagnósticos de enfermería, planificación/prescripción de enfermería y evaluación de la asistencia en 17 pacientes, utilizando un software. Se calculó los indicadores incidencia y prevalencia de diagnósticos de enfermería, la tasa de efectividad diagnóstica de riesgo y la tasa de efectividad en la prevención de complicaciones. RESULTADOS: el Riesgo de desequilibrio en la temperatura corporal fue el diagnóstico más prevalente (23,53%) y el menos prevalente fue el Riesgo de constipación (0%). El Riesgo de integridad de la piel perjudicada fue prevalente en 100% de los pacientes, en cuanto el Riesgo de confusión aguda fue el menos prevalente (11,76%). El Riesgo de constipación y el Riesgo de integridad de la piel perjudicada obtuvieron una tasa de efectividad diagnóstica de riesgo de 100%. La tasa de efectividad en la prevención de confusión aguda y de caída fue de 100%. CONCLUSIÓN: se analizó la eficacia del Proceso de Enfermería utilizando indicadores, ya que retratan cómo el enfermero ha identificado los problemas y riesgos del paciente, y planificado la asistencia de forma sistematizada. .

Ultrasound molecular detection of immediately blood-mediated inflammatory reaction induced by islets transplantation in vitro / 中南大学学报(医学版)

OBJECTIVE@#To study the feasibility of ultrasonic molecular imaging of immediately blood-mediated inflammatory reaction (IBMIR) in vitro.
@*METHODS@#IBMIR models in vitro were divided into 3 groups: Group A, no microbubbles were added; Group B, non-targeted micro-bubbles were added; Group C, Lys-Gly-Asp-Ser (KGDS)-targeted microbubbles (MBK) were added. The ultrasonic enhancement of IBMIR in loops by ultrasonic contrast imaging was evaluated.
@*RESULTS@#The contrast-enhanced US imaging did not show thrombus formation in the group A, whereas the thrombus was found in the Group B and C with a change in filling defects or ring enhancement, respectively. The time for detecting thrombosis was (7.3 ± 0.5) min and (13.2 ± 0.6) min in Group B and Group C, respectively (P<0.05). The average-gray scales of thrombus in Group B and Group C were 31.22 ± 3.56 and 75.85 ± 5.21, respectively (P<0.05). The fluorescence microscope also showed that MBK was attached to thrombus surrounding islets.
@*CONCLUSION@#IBMIR model in vitro showed that KGDS-targeted ultrasound contrast agent could adhere to thrombus shell surrounding islets and molecular target ultrasonography could image these thrombi noninvasively and effectively.

Current Prospects of RNA Interference-based Therapy in Organ Transplantation / 대한이식학회지

RNA interference (RNAi) is a normal cellular process in which small RNAs control gene expression. siRNAs introduced into cells suppress gene expression through their recognition and cleavage of cognate mRNAs in a sequence specific manner. Due to its highly specific mode of action, RNAi has recently been tested for treatment or prevention of various diseases including organ transplantation as well as basic biomedical research. However, to achieve clinical success, there are some important issues that should be fully validated. First, siRNAs should be properly designed to avoid off-target effects. Second, siRNAs must be modified so as not to induce innate immune responses. Third, selective delivery of siRNA into desired organs or tissues is required. Despite such prerequisites, siRNAs are thought to be superior to traditional small molecule drug in terms of new drug development. In addition, in case of heart and islet transplantation which probably requires preservation of organs or cultivation of tissues for a while, siRNAs can be added to preserving solution or medium to control target gene expression during this period. In many research studies, mediators of innate immune response, inflammation, and cell death have been tested for alleviation of tissue injury and immune rejection after transplantation as potent targets of RNAi. We suggest that elucidation of exact mechanisms for tissue injury and immune rejection and subsequent selection and validation of target of RNAi in future studies might be helpful in enabling RNAi-based therapy in clinical organ transplantation to become a reality.

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.