Islet transplantation: overcoming the organ shortage.

BACKGROUND: Type 1 diabetes mellitus (T1D) is a condition resulting from autoimmune destruction of pancreatic ß cells, leading patients to require lifelong insulin therapy, which, most often, does not avoid the most common complications of this disease. Transplantation of isolated pancreatic islets from heart-beating organ donors is a promising alternative treatment for T1D, however, this approach is severely limited by the shortage of pancreata maintained under adequate conditions. METHODS: In order to analyze whether and how this problem could be overcome, we undertook a retrospective study from January 2007 to January 2010, evaluating the profile of brain-dead human pancreas donors offered to our Cell and Molecular Therapy NUCEL Center ( www.usp.br/nucel ) and the basis for organ refusal. RESULTS: During this time period, 558 pancreata were offered by the São Paulo State Transplantation Central, 512 of which were refused and 46 were accepted for islet isolation and transplantation. Due to the elevated number of refused organs, we decided to analyze the main reasons for refusal in order to evaluate the possibility of improving the organ acceptance rate. The data indicate that hyperglycemia, technical issues, age, positive serology and hyperamylasemia are the top five main causes for declination of a pancreas offer. CONCLUSIONS: This study underlines the main reasons to decline a pancreas offer in Sao Paulo-Brazil and provides some guidance to ameliorate the rate of eligible pancreas donors, aiming at improving the islet isolation and transplantation outcome. TRIAL REGISTRATION: Protocol CAPPesq number 0742/02/CONEP 9230.

Diferentes estrategias en la aplicación clínica de los trasplantes de islotes pancreáticos / Different strategies about clinical application of pancreatic islet transplantation

Resumen El trasplante clínico de islotes pancreáticos es un tratamiento para algunos pacientes con diabetes tipo 1 de difícil tratamiento. Se ha logrado restaurar la independencia de la insulina, sin embargo, en la mayoría de los casos es necesario repetir el trasplante, lo que implica el desarrollo de estrategias que permitan superar las limitaciones asociadas a este procedimiento y la integración de métodos que mitiguen la perdida de células con el fin de mejorar el resultado del trasplante. El objetivo es centrarnos en mejorar la eficacia en el trasplante de sistemas de microencapsulación de islotes pancreáticos (TIP) y células madre mesenquimales (CMM) para el tratamiento de la diabetes tipo 1 (DBT1). Se utilizó la base de datos PubMed para identificar los artículos que detallan las aportaciones de investigaciones en el trasplante de islotes pancreáticos y células madre mesenquimales microencapsulados para restablecer la regulación de la glucemia en pacientes diabéticos.

Abstract Clinical islet transplantation is an alternative treatment for selected patients with type 1 Diabetes. With it has accomplished in certain period of time, insulin independence at least for a year although after that the patient will need another transplant. For this reason, researchers have thought strategies to allow improve the islet transplantation. The aim of this review is focus in different ways to improve the efficacy in microencapsulation system with islet transplantation and mesenchymal stem cells for Diabetes Type 1 treatment. We use PubMed database to identify articles that talk about this treatment to adjust the blood glucose in diabetic type 1 patients.

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)

Heat shock protein B1 is required for the prolactin-induced cytoprotective effects on pancreatic islets.

The success of islet transplantation has improved lately. Unfortunately, it is still compromised by cell loss. We have shown that prolactin (PRL) inhibits beta-cell apoptosis and up-regulates the antiapoptotic Heat Shock Protein B1 (HSPB1) in human islets. Since its function in pancreatic islets has not been studied, we explored the role of HSPB1 in PRL-induced beta-cell survival. The significant PRL-induced cytoprotection in control cells was abrogated in HSPB1 silenced cells, overexpression of HSPB1 recovered survival. PRL-mediated inhibition of cytokine-induced caspase activities and cytokine-induced decrease of BCL-2/BAX ratio was significantly reverted in knocked-down cells. Kinetics of HSPB1 and HSF1 expression were studied in primary cultures of murine and human pancreatic islets. These findings are highly relevant for the improvement of clinical islet transplantation success rate since our results demonstrated a key role for HSPB1 pointing it as a promising target for beta-cell cytoprotection through the up-regulation of an endogenous protective pathway.

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

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.

Disruption of Transplant Tolerance by an "Incognito" Form of CD8 T Cell-Dependent Memory.

Several approaches successfully achieve allograft tolerance in preclinical models but are challenging to translate into clinical practice. Many clinically relevant factors can attenuate allograft tolerance induction, including intrinsic genetic resistance, peritransplant infection, inflammation, and preexisting antidonor immunity. The prevailing view for immune memory as a tolerance barrier is that the host harbors memory cells that spontaneously cross-react to donor MHC antigens. Such preexisting "heterologous" memory cells have direct reactivity to donor cells and resist most tolerance regimens. In this study, we developed a model system to determine if an alternative form of immune memory could also block tolerance. We posited that host memory T cells could potentially respond to donor-derived non-MHC antigens, such as latent viral antigens or autoantigens, to which the host is immune. Results show that immunity to a model nonself antigen, ovalbumin (OVA), can dramatically disrupt tolerance despite undetectable initial reactivity to donor MHC antigens. Importantly, this blockade of tolerance was CD8+ T cell-dependent and required linked antigen presentation of alloantigens with the test OVA antigen. As such, this pathway represents an unapparent, or "incognito," form of immunity that is sufficient to prevent tolerance and that can be an unforeseen additional immune barrier to clinical transplant tolerance.

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.

Review of experimental attempts of islet allotransplantation in rodents: parameters involved and viability of the procedure.

The purpose of the present study was to organize the parameters involved in experimental allotransplantation in rodents to elaborate the most suitable model to supply the scarcity of islet donors. We used the PubMed database to systematically search for published articles containing the keywords "rodent islet transplantation" to review. We included studies that involved allotransplantation experiments with rodents' islets, and we reviewed the reference lists from the eligible publications that were retrieved. We excluded articles related to isotransplantation, autotransplantation and xenotransplantation, i.e., transplantation in other species. A total of 25 studies related to allotransplantation were selected for systematic review based on their relevance and updated data. Allotransplantation in rodents is promising and continues to develop. Survival rates of allografts have increased with the discovery of new immunosuppressive drugs and the use of different graft sites. These successes suggest that islet transplantation is a promising method to overcome the scarcity of islet donors and advance the treatment options for type 1 diabetes.

Ileal interposition and viability of pancreatic islets transplanted into intramuscular site of diabetic rats.

BACKGROUND: Assuming that ileal stimulation by food may increase incretin secretion, we aimed to investigate whether ileal interposition obtains adequate pancreatic islet viability and function after intramuscular islet transplantation in diabetic rats. METHODS: We investigated four groups of eight Wistar rats: ileal interposition + islet transplantation, islet transplantation, ileal interposition, and diabetic control. All rats were subjected to streptozotocin-induced diabetes. We used the C-peptide/glucose ratio and islet image to investigate beta cell mass, and plasma glucagon like peptide-1 (GLP-1) measure. RESULTS: Ileal interposition was effective in preserving function and increasing islet mass in animals with islets transplanted into alginate microcapsules. The plasma GLP-1 level in the diabetic control rats was a basal concentration (4.1 ± 1.2 pM). GLP-1 level after ileal interposition + islet transplantation (12.3 ± 3.3 pM) was significantly higher (p < .05) than in the islet transplantation group (8.2 ± 2.4 pM) and ileal interposition group rats (7.6 ± 1.8 pM). CONCLUSIONS: Ileal interposition positively influenced beta cell viability after intramuscular transplantation of pancreatic islets in diabetic rats.

Imunoproteção de ilhotas pancreáticas microencapsuladas em biomateriais inovadores e seu potencial terapêutico no diabetes mellitus tipo 1 / Immunoprotection of pancreatic islets microencapsulated in inovative biomaterials and its therapeutic potential in type 1 Diabetes Mellitus

O transplante de ilhotas microencapsuladas constitui uma alternativa terapêutica interessante para o Diabetes Mellitus tipo 1, permitindo um melhor controle glicêmico e eliminando a necessidade de imunossupressão. Entretanto, a manutenção a longo prazo da viabilidade das células-ß ainda é um desafio. No isolamento, a perda da matriz extracelular e as condições hipóxicas subsequentes afetam decisivamente a sobrevivência e funcionalidade das ilhotas. Objetivo Para diminuir o estresse sobre o enxerto, levando a um sucesso prolongado do transplante, propôs-se a adição de perfluorocarbono (PFC) ou laminina (LN), moléculas associadas respectivamente à oxigenação e interações célula-célula, ao biomaterial baseado em alginato, Biodritina, adequado ao encapsulamento celular. Metodologia Para testar a estabilidade das formulações PFC-Biodritina e LN-Biodritina, microcápsulas foram submetidas a diferentes estresses (rotacional, osmótico, temperatura e cultura) por 7 e 30 dias. A pureza do biomaterial foi avaliada pela coincubação com macrófagos murinos RAW264.7, por 3, 9 e 24h, quando a ativação dos macrófagos foi observada pela expressão gênica de IL- 1ß e TNFα. Microcápsulas implantadas i.p. em camundongos foram recuperadas após 7 ou 30 dias, para análises de biocompatibilidade. A expressão de níveis de mRNA (bax, bad, bcl-2, bcl-XL, xiap, caspase 3, mcp1/ccl2, hsp70, ldh, insulina 1 e 2), proteínas (Bax, Bcl-XL e Xiap) e a atividade de Caspase3 foram avaliadas em ilhotas microencapsuladas com PFC- e LN-Biodritina, após cultura de 48h em condições de normóxia e hipóxia (<2% O2). Camundongos diabéticos foram transplantados com ilhotas encapsuladas nas diferentes formulações e os animais foram monitorados pelas variações de massa corporal, glicêmicas e pela funcionalidade do enxerto (TOTGs). As ilhotas foram recuperadas de animais normo ou hiperglicêmicos e uma análise de biocompatibilidade das cápsulas foi realizada, assim como a avaliação funcional das células-ß. Após o explante, a glicemia dos animais normoglicêmicos foi monitorada para se atestar a eficiência das ilhotas transplantadas. Resultados Microcápsulas de PFC- e LN-Biodritina são tão estáveis e biocompatíveis quanto as de Biodritina. Para ilhotas encapsuladas em ambos os materiais, em normóxia ou hipóxia, observou-se uma modulação gênica que sugere proteção contra apoptose. Adicionalmente, encontrou-se uma diminuição na expressão de genes indicadores de estresse (mcp1, hsp70). Uma diminuição nos níveis de mRNA de ldh foi vista para PFC-Biodritina, mas o oposto foi encontrado para LN-Biodritina. As diferenças encontradas na expressão proteica sugerem o mesmo padrão anti-apoptótico. Caspase3 não foi modulada por nenhum biomaterial. Nos experimentos de transplante, apenas LN-Biodritina levou reversão prolongada do diabetes, com 60% dos animais normoglicêmicos, 198 dias pós-cirurgia, comparado a 9% do grupo Biodritina. O TOTG demonstrou que camundongos transplantados com ilhotas encapsuladas secretaram mais insulina do que controles, 60 (LN-Biodritina) ou 100 (PFC- e LN-Biodritina) dias pós-cirurgia. O explante restabeleceu a hiperglicemia nos camundongos. Microcápsulas recuperadas de animais hiperglicêmicos apresentavam uma extensa adesão celular. Testes de secreção de insulina in vitro demonstraram que somente ilhotas do grupo normoglicêmico responderam às variações da concentração de glicose. Conclusão A adição de moléculas bioativas à Biodritina é capaz de diminuir o estresse em ilhotas isoladas e tem o potencial de melhorar a terapia pelo transplante de ilhotas

Transplantation of microencapsulated islets represents an attractive therapeutical approach to treat type 1 Diabetes Mellitus, accounting for an improved glycemic control and the abolishment of immunosuppressive therapies. However, maintenance of long-term ß-cell viability remains a major problem. During islet isolation, the loss of extracellular matrix interactions and the hypoxic conditions thereafter dramatically affect ß-cell survival and function. Objective To lessen the burden of islet stress and achieve a better outcome in islet transplantation we tested the addition of perfluorocarbon (PFC) or laminin (LN), molecules associated respectively with oxygenation and cell-cell interaction, to Biodritin, an alginate-based material suitable for cell microencapsulation. Methodology To test the stability of PFC-Biodritin and LN-Biodritin composites, microcapsules were subjected to different stresses (rotational, osmotic, temperature and culture) for 7 and 30 days. To assess biomaterial purity microcapsules were co-incubated with RAW264.7 murine macrophage cell line for 3, 9 and 24h and macrophage activation was detected through mRNA levels of IL-1ß and TNFα. Microcapsules were implanted i.p. in mice and retrieved after 7 or 30 days, for biocompatibility analyses. Gene expression at mRNA (bax, bad, bcl-2, bcl-XL, xiap, caspase 3, mcp1/ccl2, hsp70, ldh, insulin 1 and 2) and protein (Bax, Bcl-XL and Xiap) levels, together with Caspase3 activity, were evaluated in islets microencapsulated in PFC- or LN-Biodritin, upon culturing for 48h in normoxic or hypoxic (<2% O2) conditions. Diabetic mice were transplanted with PFC- or LN-Biodritin microencapsulated islets, followed by assessments of body weight, glycemia and graft function by oral glucose tolerance tests (OGTTs). Microencapsulated islets were retrieved from normoglycemic or hyperglycemic mice and biocompatibility analyses of the beads together with a functional assessment of the graft followed. After graft removal, normoglycemic animals had their glycemias monitored to attest the efficacy of the transplanted islets. Results PFC- and LN-Biodritin microcapsules were as stable and biocompatible as Biodritin. For both biomaterials in normoxia and hypoxia a modulation in gene expression was observed in islets associated with a protection against apoptosis. Also, a decreased expression of stress-related genes (mcp1, hsp70) was evidenced. ldh mRNA levels were down-regulated in PFC-Biodritin microencapsulated islets but upregulated in the presence of LN. Increased levels of insulin mRNA were observed. The differences seen in protein expression indicated the same anti-apoptotic pattern. Caspase3 activity was not different between groups. Concerning diabetes reversal experiments, only mice transplanted with LN-Biodritin microencapsulated islets presented a better outcome, with 60% remaining euglycemic at 198 days post-surgery, compared with 9% for the Biodritin group. OGTT showed that mice transplanted with encapsulated islets secreted more insulin than normal mice, 60 (LN-Biodritin) or 100 days (PFC- and LN-Biodritina) posttransplant. Hyperglycemia was achieved after the retrieval of microcapsules showing graft efficacy. Retrieved microcapsules revealed an extensive overgrowth in most beads from hyperglycemic mice. A static glucose stimulated insulin secretion test revealed that only islets from normoglycemic subjects were able to secrete insulin according to glucose concentration. Conclusion- The addition of bioactive molecules to Biodritin may lessen the stress of isolated islets and have the potential to improve islet transplantation therapy.

Transplante de ilhotas pancreáticas humanas: revisão da literatura e implantação de um laboratório de isolamento de ilhotas pancreáticas / Human pancreatic islet transplantation: literature review and establishment of a pancreatic islet isolation laboratory

O diabetes melito tipo 1 (DM1) está associado ao desenvolvimento de complicações crônicas de elevada morbi-mortalidade em indivíduos jovens em idade produtiva. A terapia intensiva com insulina comprovadamente diminui o aparecimento das complicações crônicas da doença. Entretanto, essa terapia ainda está associada ao aumento da incidência de hipoglicemia. Em pacientes com “DM1 lábil”, os quais apresentam hipoglicemias graves sem sintomas de alerta, o transplante de ilhotas pancreáticas humanas é uma das melhores alternativas para restaurar a secreção de insulina e a percepção da hipoglicemia. Cerca de 80% dos pacientes que receberam transplante de ilhotas de mais de um doador, submetidos ao tratamento imunossupressor do protocolo de Edmonton, adquiriram independência de insulina após 1 ano do transplante. Porém, apenas 10% destes pacientes permaneceram livres de insulina após 5 anos. Entretanto, mesmo aqueles pacientes que necessitaram utilizar novamente insulina tiveram a normalização da homeostase glicêmica e da percepção da hipoglicemia, com prevenção da hipoglicemia grave. Sendo assim, o transplante de ilhotas é capaz de diminuir os níveis de glicose plasmática e HbA1c, reduzir a ocorrência de hipoglicemias graves e melhorar a qualidade de vida dos pacientes. O objetivo deste artigo foi fazer uma breve revisão da literatura sobre o isolamento e transplante de ilhotas pancreáticas humanas e relatar a implantação de um laboratório de isolamento de ilhotas humanas no Serviço de Endocrinologia do Hospital de Clínicas de Porto Alegre.

Type 1 diabetes mellitus (DM1) is associated with chronic complications of high morbidity and mortality in young adults in a productive age. Insulin therapy has proved to reduce the chronic complications of diabetes. However, this therapy is still associated to an increased incidence of hypoglycemia. In patients with “brittle DM1”, who have severe hypoglycemia without any symptoms (hypoglycemia unawareness), the pancreatic islet transplantation is one of the best alternatives for restoring insulin secretion and hypoglycemia perception. About 80% of the patients who received islet transplantation from more than one donor, on immunosuppressive treatment with the Edmonton’s protocol, maintained insulin independence 1 year after transplantation. Nevertheless, only 10% of these patients remained free of insulin after 5 years post-transplantation. However, even those patients who returned to insulin treatment had a normalization of the glucose homeostasis and hypoglycemia perception. Therefore, islet transplantation is able to diminish plasmatic glucose and HbA1c levels, to reduce the occurrence of severe hypoglycemia, and to improve the quality of life of the patients. The purpose of this paper is to briefly review islet isolation and transplantation process, and report the establishing of a human islet isolation laboratory in the Endocrine Service at Hospital de Clínicas de Porto Alegre.

O diabetes hiperlábil existe como entidade clínica? / Does brittle diabetes exist as a clinical entity?

A melhor compreensão das causas da instabilidade dos níveis da glicemia em pacientes com diabetes melito tipo 1 (DMT1) e a disponibilidade de novas alternativas para enfrentá-la com sucesso, como a bomba de infusão contínua de insulina e os análogos das insulinas, tornaram relevante o questionamento sobre a existência do diabetes hiperlábil como uma entidade bem como a necessidade de defini-lo. O presente artigo pretendeu descrever o conceito de diabetes hiperlábil à luz dos novos avanços na terapia do DMT1 e propor critérios objetivos para a quantificação da labilidade da glicemia.

The best comprehension about the instability of the glycemia levels in type 1 diabetes mellitus (T1DM) patients and the availability of new alternatives to successfully control it, like insulin pump therapy and the insulin analogues, underlined the questions about the brittle diabetes existence as a clinical entity as well as the necessity of define it. The aim of this article was to describe the concept of brittle diabetes in the light of the latest advances in the treatment of type 1 diabetes and propose objective criteria to evaluate the level of glucose liability.

Transplante de ilhotas na prática clínica: estado atual e perspectivas / Islet transplantation as a clinical tool: present state and future perspectives

O transplante de ilhotas é um procedimento em desenvolvimento, como alternativa para o tratamento do diabetes tipo 1 que está na fronteira entre o experimental e o clínico. É uma terapia celular na qual as células são implantadas em território diferente do fisiológico em que apenas determinado número incerto conseguirá se adaptar. Aperfeiçoar este processo para obter os mesmos resultados que no transplante de pâncreas, representa um desafio para o qual convergem contribuições da biologia celular, da imunologia e das técnicas de laboratório que se entrelaçam de maneira extremamente complexa. Este trabalho revisa a literatura expondo a evolução do procedimento, a sua metodologia atual e os resultados clínicos obtidos. As perspectivas futuras do transplante diante dos recentes avanços também são discutidas.

Islet transplant is an innovative treatment for type 1 diabetic patients, which still lies between experimental and approved transplant therapy. Islet cells are seeded in a non-physiological territory where an uncertain fraction will be able to adapt and survive. Thus, the challenge lies in improving the whole procedure, employing the tools of cell biology, immunology and laboratory techniques, in order to reach the results obtained with whole organ transplant. This review describes the procedure, its progress to the present methodology and clinical results obtained. Future perspectives of islet transplantation in the light of recent biotechnological advances are also focused.

Protocolo clínico para seleção de candidatos ao transplante de ilhotas / Clinical protocol for selection of the candidates for islet transplantation

OBJETIVO: Descrever o perfil clínico dos candidatos ao programa de transplante de ilhotas da Pontifícia Universidade Católica do Paraná (PUC-PR), em Curitiba. MÉTODOS: O processo de avaliação clínica foi estruturado em etapas: triagem, pré-avaliação, avaliação e lista de espera. Os critérios de inclusão utilizados foram: ocorrência de hipoglicemia assintomática, complicações crônicas progressivas da doença, idade entre 18 e 65 anos e pelo menos cinco anos de doença. RESULTADOS: De setembro de 2003 a setembro de 2006 foram avaliados 92 candidatos, dos quais 25 preencheram os critérios de triagem, sendo selecionados para pré-avaliação. O principal motivo de não qualificação foi o uso de insulina em dose > 0,7 UI/kg/d. Dos 25 candidatos incluídos na pré-avaliação, sete não concordaram em assinar o termo de consentimento. Quatro candidatos completaram todas as etapas de seleção, porém apenas dois permanecem em lista de espera. CONCLUSÕES: Os candidatos ao transplante de ilhotas devem ser rigorosamente selecionados. Dois pacientes preencheram todos os critérios e encontram-se em lista de espera.

BACKGROUND: Our intention is to describe the clinical profile of the candidates for islet transplantation in Curitiba, Brazil. METHODS: The clinical evaluation was organized in stages: Screening, Initial Evaluation, Evaluation and Waiting List. Candidates’ inclusion criteria were hypoglycemia unawareness, glycemic imbalance, chronic progressive diabetic complications, 18-65 years of age and at least 5 years of type 1 diabetes evolution. RESULTS: From September 2003 through September 2006, 92 candidates were clinically evaluated, and 25 fulfilled the Screening criteria, being selected at this stage. The main reason for exclusion was insulin requirement of more than 0.7 IU/kg/day. At the Initial Evaluation, seven of the 25 patients were excluded as have not agreed to sign the informed consent. Until now, 4 candidates completed the Evaluation stage and two of them are currently enlisted. CONCLUSIONS: Candidates for islet transplantation must be rigorously evaluated. Two patients fulfilled all the selection criteria and are currently enlisted.

Progression of nephropathy after islet of langerhans transplantation in alloxan-induced diabetic rats

We studied the effects of islet of Langerhans transplantation (IT) on the kidney lesions of rats with alloxan-induced diabetes. Forty-five inbred male Lewis rats were randomly assigned to 3 experimental groups: group Gl included 15 non-diabetic control rats (NC), group GIT included 15 alloxan-induced diabetic rats (DC), and group III included 15 alloxan-induced diabetic rats that received pancreatic islet transplantation prepared by nonenzymatic method from normal donor Lewis rats and injected into the portal vein (IT). Each group was further divided into 3 subgroups of 5 rats which were sacrificed at 1, 3, and 6 months of follow-up, respectively. Clinical and laboratorial parameters were recorded in the mentioned periods in the 3 experimental groups. For histology, the kidneys of all rats of each subgroup were studied and 50 glomeruli and 50 tubules of each kidney were analyzed using light microscopy by two different investigators in a double blind study. The results showed progressive glomerular basement membrane thickening (GBMT), mesangial enlargement (ME), and Bowman's capsule thickening (BCT) in the 3 experimental groups throughout the follow-up. These alterations were significantly more severe in DC rats at 6 months when compared to NC rats (p 0.01). However, the degree of GBMT, ME, and BCT observed in DC rats was not statistically different from IT rats at 1, 3, and 6 months. In addition, Armanni-Ebstein lesions of the tubules (AE) and tubular lumen protein (PRO) observed in DC rats were also observed in IT rats all over the study. These lesions were never present in NC rats. We conclude that IT did not prevent progression of kidney lesions in alloxan-induced diabetic rats within 6 months after transplantation.