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Type 1 diabetes mellitus (T1DM) is characterized by C-peptide deficiency and elevated levels of pro-inflammatory cytokines. The aim of this study was to investigate the role of C-peptide in renal and inflammatory complications in streptozotocin (STZ)-diabetic mice model of T1DM with kidney disease. The study was performed in 8-week old male C57BL/6 mice. Two streptozotocin-diabetic groups (a T1DM animal model), after 4 weeks of diabetes, were treated with subcutaneous infusion of either vehicle (n = 12) or C-peptide (n = 11). Two non-diabetic groups (vehicle, n = 10; C-peptide, n = 9) were treated using the same protocol as described for the diabetic mice. The treatment with C-peptide in the diabetic group reduced the urinary levels of IL17 and TNFα, as well as IL4 and IL10 (p

Cell encapsulation coats cells with an artificial membrane to preserve their physical and functional integrity. Different approaches try to develop more functional and biocompatible materials to avoid cell loss after transplantation due to inflammatory reaction, one of the main causes for graft failure. In this study, the LN-Biodritin biomaterial, based on alginate, chondroitin sulfate, and laminin, previously developed by our group, was further improved by replacing laminin by polylaminin, an artificial laminin polymer with anti-inflammatory properties, generating the new biomaterial polyLN-Biodritin. Capsules containing polylaminin are stable, do not induce macrophage activation in vitro, and are also able to prevent macrophage activation by encapsulated human pancreatic islets in vitro, preserving their glucose-stimulated insulin secretion potential. In addition, when empty capsules containing polylaminin were implanted into immunocompetent mice, the inflammatory response towards the implant was attenuated, when compared with capsules without polylaminin. The results indicate that polylaminin incorporation leads to lower levels of pericapsular growth on the capsules surface, lower infiltration of cells into the peritoneal cavity, and lower production of proinflammatory cytokines, both at the implant site (interleukin-12p70 (IL-12p70), tumor necrosis factor-α (TNF-α), monocyte chemotactic protein-1 (MCP-1), and interferon-γ (IFN-γ)) and systemically (IL-12p70 and TNF-α). Therefore, polylaminin incorporation into the microcapsules polymer attenuates the host posttransplantation immune response against implanted microcapsules, being likely to favor maintenance of engrafted encapsulated cells.

Pancreatic islet microencapsulation constitutes an attractive therapy for type 1 diabetes mellitus; however, long-term β-cell function remains a major problem. Loss of extracellular matrix interactions during islet isolation dramatically affects β-cell viability. We have previously shown beneficial effects of laminin (LN) in human islet cultures. Herein, we investigated whether LN could improve the outcome of transplantation after islet microencapsulation in Biodritin, an alginate-based material. To test LN-Biodritin stability, microcapsules were subjected to different types of in vitro stress. Focusing on biocompatibility, empty microcapsules were coincubated with the RAW 264.7 macrophage cell line for up to 24 h, and empty beads were implanted IP in mice and retrieved for analyses after 7 and 30 days. Upon culturing for 48 h, mRNA, protein levels, and caspase 3 activity were evaluated in islets microencapsulated with LN-Biodritin. Mice rendered diabetic by streptozotocin injection were transplanted with microencapsulated islets, followed by assessment of body weight, glycemia, and graft function (evaluated by OGTT). Graft efficiency was observed upon microencapsulated islet explantation. The results obtained showed that LN-Biodritin microcapsules were as stable and biocompatible as Biodritin. Modulation of mRNA and protein levels suggested protection against apoptosis and islet stress. Mice transplanted with LN-Biodritin microencapsulated islets presented a better outcome at 198 days postsurgery. Graft explantation led animals to hyperglycemia. In conclusion, LN-Biodritin constitutes a very promising biomaterial for islet transplantation.

Transplantation of pancreatic islets is efficient in improving the metabolic control and quality of life and in preventing severe hypoglycemia in patients with brittle type 1 diabetes mellitus. More accurate methods to assess islet viability would be extremely useful in designing target interventions for islet cytoprotection and in reducing the number of islets required to achieve insulin independence. Here we report on an application of calorimetry to evaluate the metabolic response of pancreatic islets to glucose stimulation. A significant increase in metabolic heat was produced by islet samples when consecutively subjected to 2.8 and 16.3 mmol L −1 glucose. Under these glucose concentrations, 1000 islets released average heat values of 9.16 ± 0.71 mJ and 14.90 ± 1.21 mJ over 50 min, respectively. Additionally, the glucose stimulation indexes were 1.67 ± 0.30 for insulin, 1.72 ± 0.13 for heat and 2.91 ± 0.50 for lactate, raising the important possibility of substituting the secreted insulin index/ratio by the index/ratio of the heat released in the evaluation of Langerhans islets viability for transplantation. Altogether, our results demonstrate the applicability of calorimetry to assess the quality of isolated pancreatic islets and to study vital islet functions.

Background Microencapsulation of pancreatic islets with polymeric compounds constitutes an attractive alternative therapy for type 1 diabetes mellitus. The major limiting factor is the availability of a biocompatible and mechanically stable polymer. We investigated the potential of Biodritin, a novel polymer constituted of alginate and chondroitin sulfate, for islet microencapsulation. Methods Biodritin microcapsules were obtained using an air jet droplet generator and gelated with barium or calcium chloride. Microencapsulated rat insulinoma RINm5F cells were tested for viability using the [3-(4,5-dimetyl-thiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide] [MTT] colorimetric assay. Microencapsulated rat pancreatic islets were coincubated with macrophages derived from mouse peritoneal liquid to assess the immunomodulatory potential of the microcapsules, using quantitative real time-PCR (qPCR). Biodritin biocompatibility was demonstrated by subcutaneous injection of empty microcapsules into immunocompetent Wistar rats. Insulin secretion by microencapsulated human pancreatic islets was evaluated using an electrochemoluminescent assay. Microencapsulated human islets transplanted into chemically induced diabetic mice were monitored for reversal of hyperglycemia. Results The metabolic activity of microencapsulated RINm5F cells persisted for at least 15 days. Interleukin-1β expression by macrophages was observed during coculture with islets microencapsulated with Biodritin-CaCl2, but not with Biodritin-BaCl2. No statistical difference in glucose-stimulated insulin secretion was observed between nonencapsulated and microencapsulated islets. Upon microencapsulated islet transplantation, the blood glucose level of diabetic mice normalized; they remained euglycemic for at least 60 days, displaying normal oral glucose tolerance tests. Conclusion This study demonstrated that Biodritin can be used for islet microencapsulation and reversal of diabetes; however, further investigations are required to assess its potential for long-term transplantation.

Disruption of protein digestion in insects by specific endoprotease inhibitors is being regarded as an alternative to conventional insecticides for pest control. To optimize the effectiveness of this strategy, the understanding of the endoprotease diversity of the target insect is crucial. In this sense, a membrane-bound trypsin-like enzyme from the gut of Anticarsia gemmatalis fifth-instar larvae was purified. Non-soluble fraction of the gut extract was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and subjected to a p-aminobenzamidine affinity chromatography followed by anion-exchange chromatography. The yield of the purified enzyme was 11% with a purification factor of 143 and a final specific activity of 18.6 µM min. -1 mg -1 protein using N-α-benzoyl-L- Arg-p-nitroanilide (L-BApNA) as substrate. The purified sample showed a single band with proteolytic activity active and apparent molecular mass of 25 kDa on SDS-PAGE. Molecular mass determined by MALDI-TOF mass spectrometry was 28,632 ± 26 Da. Although the low recovery and the difficulties in purifying large enzyme amounts limited its further characterization, the results contribute for the understanding of the proteases present on A. gemmatalis gut, which are potential targets for natural or specifically designed protease inhibitors.

Many studies have evaluated whether there are characteristics related to pancreas donors and the islet isolation process that can influence pancreatic islet yield. However, this analysis has not yet been performed in Brazil, one of the world leaders in whole pancreas organ transplantation (WOPT), where pancreas allocation for pancreatic islet transplantation (PIT) has no officially defined criteria. Definition of parameters that would predict the outcome of islet isolation from local pancreas donors would be useful for defining allocation priority in Brazil.To analyze the relationship between multiple donor-related and islet isolation variables with the total number of isolated pancreatic islet equivalents (IEQ) in a brazilian sample of pancreas donors.Several variables were analyzed in 74 pancreata relative to the outcome of total IEQs obtained at the end of the process.In univariate analysis, body mass index (BMI) (p = 0.003), the presence of fatty infiltrates in the pancreas as observed during harvesting (p = 0.042) and pancreas digestion time (p = 0.046) were identified as variables related to a greater IEQ yield. In a multivariate analysis a statistically significant contribution to the variability of islet yield was found only for the BMI (p = 0.017). A ROC curve defined a BMI = 30 as a cut-off point, with pancreata from donors with BMI30 yielding more islets than donors with BMI30 (p0.001).These data reinforce the importance of the donor BMI as a defining parameter for successful islet isolation and establishes this variable as a potential pancreas allocation criterion in Brazil, where there is unequal competition for good quality organs between WOPT and PIT.

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.

Social and economical development is closely associated with technological innovation and a well-developed biotechnological industry. In the last few years, Brazil’s scientific production has been steadily increasing; however, the number of patents is lagging behind, with technological and translational research requiring governmental incentive and reinforcement. The Cell and Molecular Therapy Center (NUCEL) was created to develop activities in the translational research field, addressing concrete problems found in biomedical and veterinary areas and actively searching for solutions by employing a genetic engineering approach to generate cell lines over-expressing recombinant proteins to be transferred to local biotech companies, aiming at furthering the development of a national competence for local production of biopharmaceuticals of widespread use and of life-saving importance. To this end, mammalian cell engineering technologies were used to generate cell lines over-expressing several different recombinant proteins of biomedical and biotechnological interest, namely, recombinant human Amylin/IAPP for diabetes treatment, human FVIII and FIX clotting factors for hemophilia, human and bovine FSH for fertility and reproduction, and human bone repair proteins (BMPs). Expression of some of these proteins is also being sought with the baculovirus/insect cell system (BEVS) which, in many cases, is able to deliver high-yield production of recombinant proteins with biological activity comparable to that of mammalian systems, but in a much more cost-effective manner. Transfer of some of these recombinant products to local Biotech companies has been pursued by taking advantage of the Sao Paulo State Foundation (FAPESP) and Federal Government (FINEP, CNPq) incentives for joint Research Development and Innovation partnership projects.

The ethanol extract from Xyris pteygoblephara aerial parts was evaluated against five microorganism strains, by the microdilution and agar diffusion methods. Extract fractionation led to the isolation of three compounds, whose structures were assigned by spectrometric data (1D and 2D NMR, IR, MS and UV) as (3R,4R)-(-)-6-methoxy-3,4-dihydro-3-n-pentil-4-acethoxy-1H-2-benzopyran-1-one (1), moronic acid and quercetin. The absolute configuration of 1 was defined by circular dichroism spectroscopy and comparison with data reported for other dihydroisocoumarins. Assay of 1 (100 microg/disc) by the agar diffusion method against clinical isolates of the dermatophytes Epidermophyton floccosum (inhibition zone, mm+/-s.d.: 4.5+/-0.8), Trichophyton mentagrophytes (4.8+/-0.4) and Trichophyton rubrum (10.2+/-0.8) revealed similar inhibition zones to the positive control amphotericin B (32 microg/disc; 5.0+/-0.2; 5.0+/-0.6 and 8.8+/-1.2, respectively). The result corroborates the ethnomedical use of Xyris species to treat dermatitis.

Aims NADPH oxidase (NOX) is a known source of superoxide anions in phagocytic and non-phagocytic cells. In this study, the presence of this enzyme in human pancreatic islets and the importance of NADPH oxidase in human β-cell function were investigated. Main methods and key findings In isolated human pancreatic islets, the expression of NADPH oxidase components was evidenced by real-time PCR (p22 PHOX , p47 PHOX and p67 PHOX ), Western blotting (p47 PHOX and p67 PHOX ) and immunohistochemistry (p47 PHOX , p67 PHOX and gp91 PHOX ). Immunohistochemistry experiments showed co-localization of p47 PHOX , p67 PHOX and gp91 PHOX (isoform 2 of NADPH oxidase—NOX2) with insulin secreting cells. Inhibition of NADPH oxidase activity impaired glucose metabolism and glucose-stimulated insulin secretion. Significance These findings demonstrate the presence of the main intrinsic components of NADPH oxidase comprising the NOX2 isoform in human pancreatic islets, whose activity also contributes to human β-cell function.

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-β 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- 1β 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 (