Survival of encapsulated islets:More than a membrane story

At present, proven clinical treatments but no cures are available for diabetes, a global epidemic with a huge economic burden. Transplantation of islets of Langerhans by their infusion into vascularized organs is an experimental clinical protocol, the first approach to attain cure. However, it is associated with lifelong use of immunosuppressants. To overcome the need for immunosuppression, islets are encapsulated and separated from the host immune system by a permselective membrane. The lead material for this application is alginate which was tested in many animal models and a few clinical trials. This review discusses all aspects related to the function of transplanted encapsulated islets such as the basic requirements from a permselective membrane (e.g., allowable hydrodynamic radii, implications of the thickness of the membrane and relative electrical charge). Another aspect involves adequate oxygen supply, which is essential for survival/performance of transplanted islets, especially when using large retrievable macro-capsules implanted in poorly oxygenated sites like the subcutis. Notably, islets can survive under low oxygen tension and are physiologically active at > 40 Torr. Surprisingly, when densely crowded, islets are fully functional under hyperoxic pressure of up to 500 Torr (> 300% of atmospheric oxygen tension). The review also addresses an additional category of requirements for optimal performance of transplanted islets, named auxiliary technologies. These include control of inflammation, apoptosis, angiogenesis, and the intra-capsular environment. The review highlights that curing diabetes with a functional bio-artificial pancreas requires optimizing all of these aspects, and that significant advances have already been made in many of them

Pengaruh proporsi tepung labu kuning dan tepung terigu terhadap sifat fisikokimia dan organoleptik cake kukus

Labu kuning kaya akan β-karoten, gula, dan serat. Upaya diversifikasi pengolahan labu kuning adalah tepung labu kuning. Tepung labu kuning dapat dimanfaatkan dalam pembuatan cake kukus sebagai bahan pensubstitusi tepung terigu. Penggunaan tepung labu kuning diharapkan dapat meningkatkan kadar β-karoten dalam cake kukus. Penelitian ini bertujuan untuk menentukan proporsi tepung labu kuning dan tepung terigu yang tepat untuk menghasilkan cake kukus dengan sifat fisikokimia dan organoleptik yang dapat diterima oleh konsumen. Rancangan penelitian yang digunakan adalah Rancangan Acak Kelompok dengan satu faktor, yaitu faktor proporsi tepung labu kuning dan tepung terigu dengan lima level, yaitu: 0%:100%; 10%:90%; 20%:80%; 30%:70%; 40%:60%, yang diulang sebanyak lima kali. Parameter pengujian meliputi volume spesifik, firmness, dan organoleptik kesukaan warna, kemudahan digigit, moistness, dan rasa. Data dianalisis dengan ANOVA (Analysis of Variance) pada α = 5% untuk mengetahui ada tidaknya pengaruh proporsi tepung labu kuning dan tepung terigu terhadap sifat fisikokimia dan organoleptik cake kukus. Apabila terdapat pengaruh nyata maka dilanjutkan dengan uji DMRT (Duncan's Multiple Range Test) pada α = 5%. Penentuan perlakuan terbaik dilakukan dengan uji pembobotan. Cake kukus dengan perlakuan terbaik diuji kadar β-karoten. Hasil penelitian menunjukkan bahwa perlakuan proporsi tepung labu kuning : tepung terigu memberikan pengaruh nyata (α = 5%) terhadap volume spesifik, firmness, dan tingkat kesukaan terhadap warna, kemudahan digigit, moistness, dan rasa. Semakin besar proporsi tepung labu kuning, semakin kecil nilai volume spesifik, firmness, dan kesukaan warna cake, namun semakin besar nilai kesukaan kemudahan digigit, moistness, dan rasa cake. Proporsi tepung labu kuning : tepung terigu 20%:80% merupakan perlakuan terbaik dengan kadar β-karoten sebesar 8,569 µg β- karoten/g cake kukus

Long-term viability and function of transplanted islets macroencapsulated at high density are achieved by enhanced oxygen supply

Transplantation of encapsulated islets can cure diabetes without immunosuppression, but oxygen supply limitations can cause failure. We investigated a retrievable macroencapsulation device wherein islets are encapsulated in a planar alginate slab and supplied with exogenous oxygen from a replenishable gas chamber. Translation to clinically-useful devices entails reduction of device size by increasing islet surface density, which requires increased gas chamber pO Here we show that islet surface density can be substantially increased safely by increasing gas chamber pO to a supraphysiological level that maintains all islets viable and functional. These levels were determined from measurements of pO profiles in islet-alginate slabs. Encapsulated islets implanted with surface density as high as 4,800 islet equivalents/cm in diabetic rats maintained normoglycemia for more than 7 months and provided near-normal intravenous glucose tolerance tests. Nearly 90% of the original viable tissue was recovered after device explantation. Damaged islets failed after progressively shorter times. The required values of gas chamber pO were predictable from a mathematical model of oxygen consumption and diffusion in the device. These results demonstrate feasibility of developing retrievable macroencapsulated devices small enough for clinical use and provide a firm basis for design of devices for testing in large animals and humans

Extended Microbiological Characterization of Göttingen Minipigs in the Context of Xenotransplantation: Detection and Vertical Transmission of Hepatitis E Virus

Xenotransplantation has been proposed as a solution to the shortage of suitable human donors. Pigs are currently favoured as donor animals for xenotransplantation of cells, including islet cells, or organs. To reduce the xenotransplantation-associated risk of infection of the recipient the pig donor should be carefully characterised. Göttingen minipigs from Ellegaard are often used for biomedical research and are regularly tested by their vendor for the presence of numerous bacteria, fungi, viruses and parasites. However, screening for some pathogens transmittable to humans had not been performed.The presence of microorganisms was examined in Göttingen Minipigs by PCR methods. Since zoonotic transmission of porcine hepatitis E virus HEV to humans has been demonstrated, extended search for HEV was considered as a priority. RNA from sera, islet and other cells from 40 minipigs were examined for HEV using different real-time reverse transcription (RT)-PCRs, among them two newly established. In addition, sera were examined by Western blot analysis using two recombinant capsid proteins of HEV as antigens. HEV RNA was not detected in pigs older than one year including gilts, but it was detected in the sera of three of ten animals younger than 1 year. Furthermore, HEV was also detected in the sera of three sows six days after delivery and their offspring, indicating vertical transmission of the virus. PCR amplicons were cloned, sequenced and the viruses were found to belong to the HEV genotype (gt) 3/4. Anti-HEV immunoglobulins G were detected in one sow and maternal antibodies in her six day old piglet. Since Göttingen minipigs were negative for many xenotransplantation-relevant microorganisms, they can now be classified as safe. HEV may be eliminated from the Ellegaard herd by selection of negative animals and/or by treatment of the animals

Transplantation of bovine adrenocortical cells encapsulated in alginate.

Current treatment options for adrenal insufficiency are limited to corticosteroid replacement therapies. However, hormone therapy does not replicate circadian rhythms and has unpleasant side effects especially due to the failure to restore normal function of the hypothalamic–pituitary–adrenal (HPA) axis. Adrenal cell transplantation and the restoration of HPA axis function would be a feasible and useful therapeutic strategy for patients with adrenal insufficiency. We created a bioartificial adrenal with 3D cell culture conditions by encapsulation of bovine adrenocortical cells (BACs) in alginate (enBACs). We found that, compared with BACs in monolayer culture, encapsulation in alginate significantly increased the life span of BACs. Encapsulation also improved significantly both the capacity of adrenal cells for stable, long-term basal hormone release as well as the response to pituitary adrenocorticotropic hormone (ACTH) and hypothalamic luteinizing hormone-releasing hormone (LHRH) agonist, [D-Trp6]LHRH. The enBACs were transplanted into adrenalectomized, immunodeficient, and immunocompetent rats. Animals received enBACs intraperitoneally, under the kidney capsule (free cells or cells encapsulated in alginate slabs) or s.c. enclosed in oxygenating and immunoisolating βAir devices. Graft function was confirmed by the presence of cortisol in the plasma of rats. Both types of grafted encapsulated cells, explanted after 21–25 d, preserved their morphology and functional response to ACTH stimulation. In conclusion, transplantation of a bioartificial adrenal with xenogeneic cells may be a treatment option for patients with adrenocortical insufficiency and other stress-related disorders. Furthermore, this model provides a microenvironment that ensures 3D cell–cell interactions as a unique tool to investigate new insights into cell biology, differentiation, tissue organization, and homeostasis

Islet cell transplantation from Göttingen minipigs to cynomolgus monkeys: analysis of virus safety

BACKGROUND Xenotransplantation using pig cells, tissues or organs may be associated with the transmission of porcine zoonotic micro-organisms. Hepatitis E virus (HEV), porcine cytomegalovirus (PCMV) and porcine endogenous retroviruses (PERVs) are potentially zoonotic micro-organisms which do not show clinical symptoms in pigs and which are due to the low expression level difficult to detect. Göttingen Minipigs (GöMP) are often used for biomedical investigations and they are well characterized concerning the presence of numerous bacteria, fungi, viruses and parasites and therefore may be used for islet cell transplantation. METHODS Islet cells derived from three GöMP were transplanted into four healthy, non-diabetic cynomolgus monkeys using a macroencapsulation device. PCR, nested PCR, real-time PCR, real-time RT-PCR and Western blot analyses were used to estimate the presence of PERV, PCMV and HEV in the donors and recipients. RESULTS Using sensitive detection methods, no HEV was found in the donor pigs and in the pig islet cell preparations. Antibodies against PERV, PCMV and HEV were not found in all cynomolgus monkeys with exception of one monkey showing an immune response against HEV. Using real-time PCR, no PCMV and HEV were found in the sera of all monkeys. CONCLUSION Although the donor islet cells and the recipients were negative for HEV using PCR and Western blot analysis, in one recipient, antibodies against HEV were found, indicating infection in a single case. All recipients were negative for antibodies against PERV, and all were negative for PCMV, indicating absence of infection. As HEV was not detected in the donor pig before transplantation, a more complex and regular screening of the animals using highly sensitive methods is required to avoid virus transmission

HEV genome, primers and probes, recombinant proteins and real-time RT-PCR parameters.

<p>(A) Schematic presentation of the HEV genome with three open reading frames (ORF 1–3). UTR—untranslated region. The numbers of nucleotides are given from the first nucleotide of ORF 2. Cap- cap structure, Poly (A)—poly A sequence. PCR primers (black arrows) and probes (red arrows) used in the real-time RT-PCR methods “A”, “J”, “M1”,and “M2” for the detection of HEV ORF2. Primers for methods M1 and M2 are given in brackets as 1 or 2, respectively. Numbers are given as in A. ^*Method “A” was established by Adlhoch et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139893#pone.0139893.ref041" target="_blank">41</a>]; **Method “J” was established by Jothikumar et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139893#pone.0139893.ref059" target="_blank">59</a>]. (B) Parameters of the used real-time RT-PCR methods. Standard curves and PCR efficiencies are shown. One reference plasmid was used for the real-time RT-PCR method “A” and another, for the other three methods. (C) HEV ORF 2 is coding for the 660 aa long capsid glycoprotein. Putative glycosylation sites are marked in green, a low probability glycosylation site is marked in grey. Positions of the asparagine are numbered. The immunodominant region (IDR) is given in yellow. The signal peptide is shown as a blue arrow. (D) Recombinant proteins (Prospec and GT3) used as antigens in Western blot analysis are shown. GT3 contains the entire immunodominant region (IDR). The Prospec antigen contains glutathion-S-transferase (GST) on the N-terminus. GT3 contains a 6His-tag on the N-terminus. The fragments corresponding to the ORF2 are given in green. The numbers started from the first amino acids of the capsid protein. (E) Comparative analysis of the recombinant antigens by SDS-PAGE is shown on the right. 500 ng of proteins were loaded on the gel, the gel was stained with Coomassie brilliant blue. Lane 1 –Prospec, lane 2 –GT3, M–Size markers.</p