Advances of the novel immunosuppressant brasilicardin A / 生物工程学报

Brasilicardin A (BraA) is a natural diterpene glycoside isolated from the pathogenic actinomycete Nocardia brasiliensis IFM 0406 with highly potent immunosuppressive activity (IC50=0.057 μg/mL). BraA potently inhibits the uptake of amino acids that are substrates for amino acid transport system L of T cells, which is different from the existing clinical immunosuppressants. BraA is more potent in a mouse mixed lymphocyte reaction and less toxic against various human cell lines compared with the known clinical immunosuppressants, such as cyclosporin A, ascomycin and tacrolimus. Therefore, BraA attracted more attention as a new promising immunosuppressant. However, the development of this promising immunosuppressant as drug for medical use is so far hindered because BraA has the unusual and synthetically challenging skeleton and shows the low-yield production in the natural pathogenic producer. This review introduces the molecular structure of BraA, its activity, mechanism of action, chemical synthesis of BraA analogs, heterologous expression of gene cluster, and an application of combining microbial and chemical synthesis for production of BraA, with the aim to facilitate the efficient production of BraA and its analogs.

Cyclosporine A impairs bone repair in critical defects filled with different osteoconductive bone substitutes

Abstract The aim of this study was to assess the influence of cyclosporine administration on the repair of critical-sized calvaria defects (CSDs) in rat calvaria filled with diverse biomaterials. Sixty animals were divided into two groups: the control (CTR) group (saline solution) and the cyclosporine (CCP) group (cyclosporine, 10 mg/kg/day). These medications were administered daily by gavage, beginning 15 days before the surgical procedure and lasting until the day the animals were euthanized. A CSD (5 mm Ø) was made in the calvaria of each animal, which was allocated to one of 3 subgroups, according to the biomaterial used to fill the defect: coagulum (COA), deproteinized bovine bone (DBB), or biphasic calcium phosphate ceramics of hydroxyapatite and β-phosphate tricalcium (HA/TCP). Euthanasia of the animals was performed 15 and 60 days after the surgical procedure (n = 5 animals/period/subgroup). Bone repair (formation) assessment was performed through microtomography and histometry, while the analyses of the expression of the BMP2, Osteocalcin, and TGFβ1 proteins were performed using immunohistochemistry. The CSDs not filled with biomaterials demonstrated lower bone formation in the CCP group. At 15 days, less bone formation was observed in the CSDs filled with DBB, a smaller volume of mineralized tissue was observed in the CSDs filled with HA/TCP, and the expression levels of BMP2 and osteocalcin were lower in the CCP group compared to the CTR group. The use of cyclosporine impaired bone repair in CSD, and this effect can be partially explained by the suppression of BMP2 and osteocalcin expression.

Cyclosporine A impairs bone repair in critical defects filled with different osteoconductive bone substitutes

Abstract The aim of this study was to assess the influence of cyclosporine administration on the repair of critical-sized calvaria defects (CSDs) in rat calvaria filled with diverse biomaterials. Sixty animals were divided into two groups: the control (CTR) group (saline solution) and the cyclosporine (CCP) group (cyclosporine, 10 mg/kg/day). These medications were administered daily by gavage, beginning 15 days before the surgical procedure and lasting until the day the animals were euthanized. A CSD (5 mm Ø) was made in the calvaria of each animal, which was allocated to one of 3 subgroups, according to the biomaterial used to fill the defect: coagulum (COA), deproteinized bovine bone (DBB), or biphasic calcium phosphate ceramics of hydroxyapatite and β-phosphate tricalcium (HA/TCP). Euthanasia of the animals was performed 15 and 60 days after the surgical procedure (n = 5 animals/period/subgroup). Bone repair (formation) assessment was performed through microtomography and histometry, while the analyses of the expression of the BMP2, Osteocalcin, and TGFβ1 proteins were performed using immunohistochemistry. The CSDs not filled with biomaterials demonstrated lower bone formation in the CCP group. At 15 days, less bone formation was observed in the CSDs filled with DBB, a smaller volume of mineralized tissue was observed in the CSDs filled with HA/TCP, and the expression levels of BMP2 and osteocalcin were lower in the CCP group compared to the CTR group. The use of cyclosporine impaired bone repair in CSD, and this effect can be partially explained by the suppression of BMP2 and osteocalcin expression.

Effects of benzalkonium chloride and cyclosporine applied topically to rabbit conjunctiva: a histomorphometric study / Efeitos do uso tópico do conservante cloreto de benzalcônio e ciclosporina na conjuntiva de coelhos: estudo histomorfométrico

ABSTRACT Purpose: Chronic instillation of benzalkonium chloride, a preservative, has inflammatory effects on the ocular surface. However, addition of the anti-inflammatory agent cyclosporine to a therapeutic protocol may mitigate these effects. This study compared the toxic effects of a 0.1% benzalkonium chloride solution and the possible protective effect of 0.05% cyclosporine when applied topically to the rabbit conjunctiva. Methods: Fifteen age- and weight-matched, female New Zealand white rabbits were categorized into three groups and treated for 30 consecutive days. Group 1, 2, and 3 - benzalkonium chloride received 0.1% every 24 h, 0.05% cyclosporine every 6 h, and both treatments, respectively. In each rabbit, the left eye was subjected to treatment and the right eye was a control. The rabbits were euthanized at after the experiment. Goblet cells and blood vessels were then enumerated in conjunctival tissues stained with periodic acid-Schiff and hematoxylin-eosin, respectively. Differences between treated and untreated eyes and between groups were compared using the t-test and analysis of variance. Results: Benzalkonium chloride treatment, with and without cyclosporine, significantly reduced (p≤0.05) in the number of goblet cells in treatment eyes compared with that in respective control eyes. Alternatively, adding cyclosporine to benzalkonium chloride did not prevent the loss of conjunctival goblet cells, and a significant reduction in the number of goblet cells was noted. Benzalkonium chloride-induced significant increase in the number of new blood vessels was mitigated significantly by the addition of cyclosporine. Conclusion: This study demonstrated the magnitude of conjunctival injury caused by chronic instillation of benzalkonium chloride. Although cyclosporine did not mitigate the effects on goblet cells, its addition minimized inflammatory angiogenesis induced by benzalkonium chloride.

RESUMO Objetivo: A instilação crônica de cloreto de benzal­cônio, um conservante, tem efeitos inflamatórios na superfície ocular. No entanto, a adição do agente anti-inflamatório ciclosporina a um protocolo terapêutico pode atenuar esses efeitos. Este estudo comparou os efeitos tóxicos de uma solução de cloreto de benzalcônio a 0,1% e o possível efeito protetor de ciclosporina a 0,05% quando aplicado topicamente à conjuntiva de coelho. Métodos: Quinze coelhos fêmeas brancos da raça Nova Zelândia, pareados por idade e peso, foram categorizados em três grupos e tratados por 30 dias consecutivos. Os grupos 1, 2 e 3 - receberam cloreto de benzalcônio 0,1% a cada 24h, ciclosporina a 0,005% a cada 6h e ambos os tratamentos, respectivamente. Em cada coelho, o olho esquerdo foi submetido a tratamento e o olho direito foi controle. Os coelhos foram submetidos à eutanásia após o experimento. Células caliciformes e vasos sanguíneos foram então enumerados em tecidos conjuntivais corados com ácido periódico-Schiff e hematoxilina-eosina, respectivamente. As diferenças entre os olhos tratados e não tratados e entre os grupos foram comparadas usando o teste t e análise de variância. Resultados: O tratamento com cloreto de benzalcônio, com e sem ciclosporina, reduziu significativamente (p£0,05) o número de células caliciformes nos olhos tratados em comparação com os olhos controle correspondentes. Alternativamente, a adição de ciclosporina ao cloreto de benzalcônio não impediu a perda de células caliciformes conjuntivais, e foi observada uma redução significativa no número de células caliciformes. O aumento significativo induzido pelo cloreto de benzalcônio no número de novos vasos sanguíneos foi significativamente mitigado pela adição da ciclosporina. Conclusão: Este estudo demonstrou a magnitude da lesão conjuntival resultante da instilação crônica de cloreto de benzalcônio. Embora a ciclosporina não tenha atenuado os efeitos nas células caliciformes, sua adição minimizou a angiogênese inflamatória induzida pelo cloreto de benzalcônio.

Effects of cyclosporin, nifedipine and phenytoin on gingival myofibroblast transdifferentiation in monkeys

Abstract Objective: Myofibroblasts have been associated with the development of several pathologic fibrotic conditions. This longitudinal study aims to assess the proliferative and antiapoptotic effects of cyclosporin, nifedipine and phenytoin on gingival connective tissue cells of nonhuman primate, as well as to analyze a possible role of myofibroblasts in gingival overgrowth. Materials and Methods: Gingival samples from the right superior canine area were obtained from 12 male monkeys ( Sapajus spp ) to comprise the control group. After one week, the animals were randomly assigned to three groups, which received daily oral doses of cyclosporin, nifedipine or phenytoin for 120 days. Gingival samples were collected from the left superior canine area of two animals of each group at 52 and 120 days. Histological sections were stained with hematoxylin and eosin, and immunoreacted against α-SMA, Ki- 67 and bcl-2. Results: α-SMA immunoreaction was negative in the control and experimental groups. Similarly, no difference between groups concerning immunostaining against Ki-67 and bcl-2 was observed in connective tissue cells. Conclusion: Based on this methodology, it may be concluded that gingival overgrowths induced by cyclosporin, nifedipine and phenytoin are not associated with neither myofibroblast transdifferentiation, proliferation nor apoptosis of gingival connective cells in monkeys.

Effects of cyclosporine on ischemia-reperfusion injuries in rat kidneys. An experimental model

Abstract Purpose To assess Cyclosporine A (CsA) therapy at an intraperitoneal dose of 15 mg.kg -1 in a rodent model of non-septic renal ischemia. Methods Twenty male Wistar rats were randomized to receive CsA therapy or none therapy before undergoing 30 minutes of renal ischemia followed by reperfusion. Additionally, 10 rats were randomized to undergo the same surgical procedure of the aforementioned animals with neither ischemia nor CsA therapy. Twelve hours after kidney ischemia, the left kidneys were evaluated for histological injury according to Park's criteria. Serum creatinine (Cr), urea nitrogen (Ur) and sodium levels were obtained at different times of the experimental protocol. Results Rodents in the CsA group showed negative results (p<0.05) in serum variables (Cr: 0.41±0.05mg/dL vs . 4.17±1.25mg/dL; Ur: 40.90±3.98mg/dL vs . 187.70±22.93mg/dL) even the non CsA or control group (Cr: 0.35±0.07mg/dL vs . 3.80±1.20mg/dL; Ur: 40.10±4.70mg/dL vs . 184.50±49.80mg/dL). The negative results were also verified in histological evaluation, CsA group had 50% in the very severe grade of lesion, 10% in the severe and 40% in the moderate to severe whereas the control group had 90% in the very severe grade. Conclusion CsA was incapable of preventing the deleterious effects of ischemia-reperfusion injury in rat kidneys.

Effects of combined oral doxycycline and topical cyclosporine treatment on ocular signs, symptoms, and tear film parameters in rosacea patients / Efeitos da doxiciclina oral combinada e tratamento tópico com ciclosporina nos sinais e sintomas oculares e em parâmetros do filme lacrimal em pacientes com rosácea ocular

ABSTRACT Purpose: This study reports the effects of combined use of oral doxycycline and topical cyclosporine on ocular signs, symptoms, and tear film parameters in rosacea patients. Methods: Fifty-four right eyes of 54 patients were included in this study. All patients underwent full ophthalmologic examination-including best corrected visual acuity measurement, slit-lamp anterior segment and fundus examination, tear film break-up time, and Schirmer test-before treatment and six months post-treatment. Patients were divided into two treatment groups. The first group was treated with oral doxycycline 100 mg twice daily for the first month and once daily for the following two months. The second group received topical 0.05% cyclosporine emulsion drops twice daily for six months in addition to the oral doxycycline treatment regimen. All patients received preservati ve-free artificial tear drops, warm compress, eyelash cleaning, and topical corticosteroid drops three times daily for one month. Results: A significant improvement in ocular signs and symptoms was recorded for all patients in groups 1 and 2 after treatment. There was not a significant difference in terms of itching, burning, meibomian gland inspissation, corneal neovascularization, and conjunctival hyperemia score changes between groups 1 and 2. The increases in Schirmer test and break-up time scores were significantly higher in group 2 than in group 1. Conclusions: Our results support the finding that topical cyclosporine in addition to the standard regimen improves tear function, as shown by Schirmer test and break-up time scores, in ocular rosacea patients.

RESUMO Objetivo: Este estudo relata os efeitos do uso combinado de doxiciclina oral e ciclosporina tópica sobre sinais e sintomas oculares e sobre parâmetros do filme lacrimal em pacientes com rosácea. Métodos: Cinquenta e quatro olhos direitos de 54 pacientes foram incluídos no estudo. Todos os pacientes foram submetidos a exame oftalmológico completo - incluindo a melhor medida da acuidade visual corrigida, segmento anterior em lâmpada de fenda e exame de fundo de olho, tempo de ruptura do filme lacrimal e teste de Schirmer - antes do tratamento e após seis meses de tratamento. O primeiro grupo foi tratado com doxiciclina oral 100 mg duas vezes ao dia no primeiro mês e uma vez ao dia nos dois meses seguintes. O segundo grupo recebeu gotas tópicas de emulsão de ciclosporina a 0,05% duas vezes ao dia por seis meses, além do tratamento com doxiciclina por via oral. Todos os pacientes receberam gotas de lágrima artificial sem conservantes, compressas mormas, limpeza de cílios e gotas de corticosteróide tópico três vezes ao dia durante um mês. Resultados: Uma melhora significativa nos sinais e sintomas oculares foi registrada para todos os pacientes do grupo 1 e 2 após o tratamento. Não houve diferença significativa em termos de prurido, queimação, inspeção da glândula meibomiana, neovascularização da cór nea e alterações na pontuação da hiperemia conjuntival entre os grupos 1 e 2. O teste de Schirmer e o aumento do tempo de ruptura no grupo 2 foram significativamente maiores do que no grupo 1. Conclusões: Os autores concluíram que os resultados apoiam a descoberta de que a ciclosporina tópica, além do tratamento padrão, melhora a função lacrimal como demonstrado pelo teste de Schirmer e o tempo de ruptura em pacientes com rosácea ocular.

Cyclosporine A attenuates apoptosis and necrosis after ischemia-reperfusion-induced renal injury in transiently hyperglycemic rats

Abstract Purpose: To investigate the effects of cyclosporine A on renal ischemia-reperfusion injury during transient hyperglycemia in rats. Methods: In a model of ischemia-reperfusion-induced renal injury and transiently induced hyperglycemia by intraperitoneal injection of glucose, 2.5 g.kg-1, Wistar rats were anesthetized with either isoflurane or propofol and received intravenous cyclosporine A, 5 mg.kg-1, five minutes before reperfusion. Comparison groups were isoflurane and propofol sham groups and isoflurane and propofol ischemia-reperfusion-induced renal injury. Renal tubular cell viability was quantitatively assessed by flow cytometry after cell culture and classified as early apoptosis, necrotic cells, and intact cells. Results: Early apoptosis was significantly higher in isoflurane and propofol anesthetized animals subjected to renal ischemia-reperfusion injury when compared to both cyclosporine A treated and sham groups. Necrosis percentage was significantly higher in propofol-anesthetized animals subjected to renal ischemia-reperfusion injury. The percentage of intact cells was lower in both, isoflurane and propofol anesthetized animals subjected to renal ischemia-reperfusion injury. Conclusion: In a model of ischemia-reperfusion-induced renal injury, cyclosporine A, 5 m.kg-1, administered five minutes before renal reperfusion in rats with acute-induced hyperglycemia under either isoflurano or propofol anesthesia, attenuated early apoptosis and preserved viability in renal tubular cells, regardless of the anesthetic used.

Preclinical evaluation of nephroprotective potential of a probiotic formulation LOBUN on Cyclosporine-A induced renal dysfunction in Wistar rats

ABSTRACT The aim of present study was to evaluate the nephroprotective effect of probiotic formulation LOBUN on Cyclosporine A (CsA) induced renal dysfunction in Wistar rats. CsA (20 mg/kg body weight s.c) was administered for 15 days to cause renal dysfunction in Wistar rats. The probiotic formulation LOBUN was administered with the dose of 500 mg/kg body weight (p.o) for twice (TGI) and thrice a day (TGII). The samples were analyzed for the parameters like blood urine nitrogen (BUN), serum creatinine, serum uric acid, total serum protein and urine proteins, urine potassium, urine sodium. The renal functional and histopathological studies revealed that the oral administration of probiotic formulation LOBUN has provided appreciable renoprotection and possibly alleviated the symptoms of Chronic Kidney Disease (CKD) at the dose of 500 mg/kg body weight administered thrice a day and also the results were supported by histopathological findings.

Effect of cyclosporine on liver regeneration in partial hepatectomized rats

PURPOSE: To evaluate the influence of the cyclosporine in liver regeneration in rats submitted to an experimental model of 70% hepatectomy. METHODS: Forty male rats were randomly divided in four subgroups (C.24h, C.7d, E.24h, E.7d), according to the drug used and the day of sacrifice (24 hours and 7 days). Cyclosporine (10mg/Kg/day) was given to the study subgroup and 1 ml of 0.9% sodium chloride was to the control subgroup. Resection of left lateral lobe and median lobe performing 70% of liver mass. During the animals' death, KWON formula was applied. Counting of mitotic figures and percentage of positive nucleus with PCNA and Ki-67 were evaluated. RESULTS: In the 2nd, 4th PO and death days, E.7d lose more weight than C.7d. Regarding to the KWON formula, the C.7d regenerated more than the C.24h and the same with the E.7d. Comparing between the groups, only E7d subgroup was statistically significant compared with C.7d, showing the stimulating effect of cyclosporine in liver regeneration. Immunohistochemestry had significant results between the study subgroups. The mitotic index revealed statistical differences in the control subgroups. CONCLUSION: Cyclosporine, in spite of being an immunosuppressive drug, has a positive effect in liver regeneration, although reduce the animal's body weight. .

Diseño de una formulación de ciclosporina A solución oral con alto grado de dispersión / Design of a formulation of highly dispersed cyclosporine A oral solution

La ciclosporina A (CsA) por sus propiedades inmunosupresoras es empleada en el transplante de órganos sólidos, médula ósea, así como para determinados estadíos de ciertas enfermedades autoinmunes. En Cuba, se comercializa una solución oral que presenta problemas de absorción gastrointestinal. Objetivos: lograr una microemulsión preconcentrada conteniendo ciclosporina A con alto grado de dispersión, igual o mayor que la formulación comercial. Métodos: se realizó un estudio de porcentajes de los emulgentes y el balance hidrófilo lipófilo (HLB), en los intervalos que permitieran obtener sistemas automicroemulsionables a través de un diseño factorial multinivel. Se empleó el porciento de transmitancia como variable de respuesta en la determinación del grado de dispersión de la formulación. También se realizó la evaluación de las características organolépticas de la formulación seleccionada, el estudio reológico, el conteo microbiano y la efectividad antimicrobiana. Resultados: los factores estudiados tienen influencia significativa sobre el porcentaje de transmitancia, la formulación con mayor grado de dispersión se logró empleando un 60 por ciento de emulgentes junto a un HLB de 11,5 con valores de transmitancia de 95,03 por ciento, significativamente mayor que el del producto líder en formato de cápsulas blandas que fue de 84,6 por ciento. Organolépticamente el producto cumple con las características de líquido transparente, brillante y sin partículas en suspensión, con comportamiento Newtoniano desde el punto de vista reológico. El conteo de bacterias y hongos cumplió con los requisitos establecidos en la Farmacopea de los Estados Unidos para soluciones orales. Conclusiones: se logró una microemulsión preconcentrada de CsA en solución bebible con alto grado de dispersión, cumpliendo con los requisitos físicos y microbiológicos establecidos para este tipo de forma farmacéutica

Cyclosporine A (CsA), due to its immunosuppressive properties, is used in the transplantation of solid organs, bone marrow, and in some stages of certain autoimmune diseases. In Cuba, the Cyclosporine A oral solution causes gastrointestinal absorption problems. Objectives: to achieve a pre-concentrated micro-emulsion containing highly dispersed cyclosporine A that may be equal to or higher than the one in the commercial formulation. Methods: study of percentages of emulsifiers and of hydrophilic lipophilic balance (HLB), at some intervals that allow obtaining self-emulsifying systems through a multilevel factorial design. The percent transmittance was used as the response variable in determining the formulation dispersion degree. The evaluation of the organoleptic characteristics of the selected formulation, the rheological study, the microbial count and the antimicrobial effectiveness estimation were all performed. Results: the studied factors have a significant influence on the transmittance percentage, the formulation with the highest degree of dispersion was achieved when using 60 percent of emulsifiers and an HLB of 11.5, being transmittance values of 95.03 percent, which were significantly higher than those of the leading product in the form of soft capsules, equal to 84.6 percent. Organoleptically speaking, the product meets the characteristics of a clear brilliant fluid without suspended particles and Newtonian behavior from the rheological point of view. The bacteria and fungi count met requirements of U.S. Pharmacopoeia for oral solutions. Conclusions: a pre-concentrate highly dispersed CsA microemulsion was achieved in a drinkable solution meeting the physical requirements and microbiological criteria established for this type of dosage form

Soporte nutricional en el niño con enfermedad de injerto contra huésped / Nutritional support in children with graft versus host disease

La enfermedad injerto contra huésped, generalmente ocurre luego del trasplante de médula ósea alogénico.Puede ser aguda, en dos fases, aferente y eferente; y crónica, que semeja una enfermedad autoinmune. Lasmanifestaciones clínicas más frecuentes son enterocólicas. La prevención es la clave del tratamiento. Su evolución depende de la severidad de las lesiones. El soporte nutricional comprende el aporte adecuado deenergía, macro y micronutrimentos y la interacción fármaco nutrimento. Los beneficios de la inmunonutrición incluyen la disminución del riesgo de infección, el menor tiempo de estancia hospitalaria, y en cuidados intensivos.

Graft versus host disease, usually occurs after bone marrow trasnplantation, allgeneic. It may be acute, intwo phases, afferent and efferent, and chronic, which resembles an autoinmune disease. The most frequentclinical manifestations are enterocolic. Prevention is the key to treatment. Its evolution depends on the severity of injuries. Nutritional support includes adequate intake of energy, macro and micronutrients and nutrient drug interaction. Immunonutrition benefits include reduced risk of infecion, shorter hospital stay, and intensive care.

Pancreatic islet allograft in spleen with immunossuppression with cyclosporine. Experimental model in dogs / Alotransplante de ilhotas pancreáticas no baço com imunossupressão com ciclosporina. Modelo experimental em cães

PURPOSE: To study the functional behavior of the allograft with immunosuppression of pancreatic islets in the spleen. METHODS: Five groups of 10 Mongrel dogs were used: Group A (control) underwent biochemical tests; Group B underwent total pancreatectomy; Group C underwent total pancreatectomy and pancreatic islet autotransplant in the spleen; Group D underwent pancreatic islet allograft in the spleen without immunosuppressive therapy; Group E underwent pancreatic islet allograft in the spleen and immunosuppression with cyclosporine. All of the animals with grafts received pancreatic islets prepared by the mechanical-enzymatic method - stationary collagenase digestion and purification with dextran discontinuous density gradient, implanted in the spleen. RESULTS: The animals with autotransplant and those with allografts with immunosuppression that became normoglycemic showed altered results of intravenous tolerance glucose (p < 0.001) and peripheral and splenic vein plasmatic insulin levels were significantly lower (p < 0.001) in animals that had allografts with immunosuppression than in those with just autotransplants. CONCLUSIONS: In the animals with immunosupression with cyclosporine subjected to allograft of pancreatic islets prepared with the mechanical-enzymatic preparation method (stationary collagenase digestion and purification with dextran discontinuous density gradient), the production of insulin is decreased and the response to intravenous glucose is altered.

OBJETIVO: Avaliar o comportamento funcional do alotransplante com imunossupressão de ilhotas pancreáticas no baço. MÉTODOS: Foram utilizados cinco grupos de 10 cães mestiços: grupo A (controle) submetido aos exames bioquímicos; grupo B, submetido à pancreatectomia total; grupo C (autotransplante) submetido à pancreatectomia total e autotransplantação de ilhotas pancreáticas no baço; grupo D, submetido à alotransplantação de ilhotas pancreáticas no baço sem terapia imunossupressiva; grupo E, submetido à alotransplantação de ilhotas no baço e imunossupressão com ciclosporina. Todos os animais transplantados receberam ilhotas pancreáticas isoladas pelo método mecânico-enzimático, digestão estacionária com colagenase e purificação com gradiente de densidade descontínua de dextran e foram implantadas no baço. RESULTADOS: Animais autotransplantados e alotransplantados com imunossupressão que se tornaram normoglicêmicos apresentaram testes de tolerância à glicose intravenosa alterados (p<0,001) e o nível de insulina plasmática periférica e na veia esplênica foram significantemente menores (p<0,001) nos animais alotransplantados com imunossupressão em relação aos autotransplantados. CONCLUSÃO: Nos animais submetidos ao alotransplante de ilhotas pancreáticas com imunossupressão com ciclosporina e preparadas pelo método mecânico-enzimático, digestão estacionária com colagenase e purificação com gradiente de densidade descontínua de dextran, a produção de insulina está diminuída e a resposta à sobrecarga de glicose intravenosa alterada.

Cyclosporin A Inhibits Albumin Synthesis in Huh7 Cells

BACKGROUND/AIMS: Hypoalbuminemia occurs frequently in renal transplant recipients immediately after renal transplantation. We studied the regulation of hepatic albumin synthesis by cyclosporin A (CsA) in Huh7 cells. METHODS: Huh7 cells were incubated with various concentrations of CsA for 4, 8, 16, and 24 hours. Albumin was measured in Huh7 cell-conditioned medium by sandwich enzyme-linked immunosorbent assay and Western blot. Albumin mRNA expression was analyzed by Northern blotting in CsA-treated cells. RESULTS: CsA (10(-7)-10(-4) M) inhibited albumin synthesis in Huh7 cells in a dose- dependent manner. A Western blot analysis for albumin in the conditioned medium released from CsA-treated (10(-7)-10(-5) M) cells also showed significant inhibition of albumin synthesis in a dose-dependent manner. Vehicle (olive oil) did not affect albumin synthesis. In contrast, a Northern blot analysis revealed no inhibition of albumin mRNA expression by CsA at any time point from 1-24 hours, indicating that the inhibition of albumin synthesis occurred at the translational level. CONCLUSIONS: Our results suggest that inhibition of hepatic albumin synthesis by high dose CsA contributes to the hypoalbuminemia in renal transplant recipients.

Electron microscopic evaluation of the secretory mechanisms of renin from juxtaglomerular cells / Evaluación con microscopía electrónica de los mecanismos secretorios de renina desde las células juxtaglomerulares

Although the structure and the functions of juxtaglomerular cells (JG) have been well defined, there is still a controversy about the secretory mechanisms of renin from these cells. It has been assumed that exocytosis is the main secretory mechanism in these cells in many studies, while others suggest that secretion occurs in a quite different way in these cells. There are several studies suggesting that diacrine secretion, which is very difficult to visualize, might be the other mechanism for secretion of renin. This study is an attempt to find the answers of these questions by identifying the fine structural features of the secretory granules in juxtaglomerular cells. Cyclosporin A (CyA) has been used in the current experimental study since it has already been reported that this drug increases the number of JG cells and stimulates secretion of Renin. Twelve female Sprague-Dawley rats had daily intraperitoneal injections of CyA for ten weeks. Tissue specimens from the kidneys of these animals were examined by electron microscopy. Fine structural characteristics of the secretory granules of juxtaglomerular cells have been examined. Considerable amount of granules, which goes to the exocytotic process, have been observed. Additionally, several cells, which their granules had been secreting their contents in a different way, were found. This was interpreted as the secretion type of diacrine secretion. In conclusion, this in vivo study presents morphologic evidences demonstrating that both exocytosis and diacrine secretion might occur in JG cells. We also had a chance to observe secretory granule probably exhibiting "diacrine secretion", which is very difficult to visualize, at electron microscope level for the first time. This report also provides morphologic proof which shows that these two distinct secretory mechanisms might occur simultaneously in the same juxtaglomerular cell.

Aunque la estructura y las funciones de las células yuxtaglomerulares (JG) han sido bien definidas, todavía existe controversia acerca de los mecanismos de secreción de renina en estas células. Se ha supuesto, en muchos estudios, que la exocitosis es el principal mecanismo de secreción de estas células, mientras que otros autores sugieren que la secreción se produce de una manera muy diferente en estas células. Hay varios estudios que plantean que la secreción diacrina, que es muy difícil de visualizar, podría ser otro mecanismo para la secreción de renina. Este estudio tiene como objetivo encontrar las respuestas a estas interrogantes mediante la identificación de las características estructurales de la secreción de gránulos en las células yuxtaglomerulares. Ciclosporina A (CyA) se ha utilizado en el estudio experimental actual, debido a que se ha informado que este medicamento aumenta el número de células JG y estimula la secreción de renina. Doce ratas hembras Sprague-Dawley fueron diariamente inyectadas por vía intraperitoneal, con CyA durante diez semanas. Las muestras de tejido renal de estos animales fueron examinadas a través de microscopía electrónica. Detalladas características estructurales han sido examinadas en los gránulos secretores de las células yuxtaglomerulares. Se ha observado una cantidad considerable de gránulos, que va con el proceso de exocitosis. Además, se encontaron células que habían secretado el contenido de sus gránulos de manera diferente. Esto fue interpretado como secreción de tipo diacrina. En conclusión, este estudio in vivo presenta evidencias morfológicas que demuestran que tanto la exocitosis y la secreción diacrina podría ocurrir en células JG. También tuvimos la oportunidad de observar probables gránulos secretores, que mostrarían "la secreción diacrina", que es muy difícil de visualizar, a nivel de microscopía electrónica. Este informe también proporciona la prueba morfológica que demuestra que estos dos mecanismos...

Inmunosupresíón en niños con trasplante hepático / Immunosuppression in children with liver transplantation

La inmunosupresión en niños con trasplante hepático, ha evolucionado con dos momentos clave: la disponibilidad de los inhibidores de calcineurina ciclosporina y tacrolimus. La inmunosupresión primaria se diseña sobre la base de un inhibidor de calcineurina como fármaco principal. Los esteroides se incluyen en la pauta de inmunosupresión primaria en la mayoría de los centros. Las pautas habituales a largo plazo consisten en ciclosporina o tacrolimus, en monoterapia a niveles inferiores a los deseados en el periodo precoz postrasplante, o en combinación con dosis bajas de esteroide. Los inhibidor e s de c a l c ineur ina induc en vasoconstricción arterial aguda y crónica que causa nefrotoxicidad, con disminución del filtrado glomerular y tubulopatía. Los niveles ensangre de ciclosporina o de tacrolimus se determinan para evaluar el estado de inmunosupresión. La edad de adolescente y adulto joven es una etapa de riesgo para el injerto por ser frecuente la omisión accidental de dosis de medicación inmunosupresora, una irregularidad que es difícil de evaluar en su extensión a pesar de una buena relación médicopaciente y frecuentes chequeos. El rechazo tiene una incidencia entre el 30 y 50% de los pacientes, entre los días 5 y 30 postrasplante.

Immunosuppression in children with liver transplantation has evolved with two key moments: the availability of calcineurin inhibitors, cyclosporine and tacrolimus. The primary immunosuppression is designed on the basis of a calcineurin inhibitor as primary drug. Steroids are included in the pr imary immunosuppression regimen in most schools. The long-term normal patterns consist of cyclosporine or tacrolimus as monotherapy to lower than desired levels in the early period aftertransplantation, or in combination with low dose steroid. Calcineurin inhibitors induce arterial vasoconstriction causing acute and chronic nephrotoxicity, with reduced glomerular filtration and tubular. Blood levels of cyclosporine or tacrolimus are determined to assess the state of immunosuppression. The age of adolescence and young adulthood is a time of risk to the graft by the accidental omission to be frequent doses ofimmunosuppressive medication, an irregularitywhich is difficult to assess its extent in spite of a good doctor-patient relationship and frequentcheckups. The rejection has an incidence between 30 and 50% of patients, between 5 and 30 aftertransplantation.

Cyclosporin A-induced gingival overgrowth is not associated with myofibroblast transdifferentiation

Cyclosporin A (CyA) induces gingival overgrowth via its stimulatory effects on expression of transforming growth factor-beta1 (TGF-â1) and collagen. It is not known whether CyA has a direct effect on gingival fibroblasts or induces its effect indirectly via stimulation of myofibroblast transdifferentiation. The present study was undertaken to examine the in vivo and in vitro effect of CyA on myofibroblast transdifferentiation. Rats were treated for 60 days with a daily subcutaneous injection of CyA, and the gingival overgrowth tissue was analyzed by immunohistochemistry. In vitro, fibroblasts from normal gingiva (NG) were cultured in the presence of different concentrations of CyA, and subjected to semi-quantitative reverse transcriptase-polymerase chain reaction and western blot. Although CyA treatment stimulated TGF-â1 expression by NG fibroblasts, it lacked to induce expression and production of isoform á of smooth muscle actin (á-SMA), the specific myofibroblast marker. The expression levels of connective tissue growth factor (CTGF), which has been considered a key molecule to promote the transdifferentiation of myofibroblasts via TGF-â1 activation, were unaffected by CyA. Our results demonstrate that CyA-induced gingival overgrowth is not associated with activation of myofibroblast transdifferentiation, since CyA is not capable to increase CTGF expression.

Cyclosporin A-treated Dendritic Cells may affect the outcome of organ transplantation by decreasing CD4+CD25+ regulatory T cell proliferation

One of the mechanisms for generation of tolerance involves immature dendritic cells (DCs) and a subpopulation of regulatory CD4+ CD25+ T lymphocytes (T REG). The purpose of this work was to analyze how Cyclosporine A (CsA), a widely used immunosuppressive drug, may affect T REG proliferation. Purified and activated murine DCs obtained from bone marrow precursors differentiated with rGMCSF were co-cultured with purified CFSE-labeled T REG from OTII mice, and their phenotype and proliferation analyzed by flow cytometry. Our data indicate that DCs differentiated in the presence of CsA show an altered phenotype, with a lower expression of MHC-II and a lower activating capacity. Additionally, these CsA-treated DCs show decreased production of IL-2 and IL-12 and increased IL-10 secretion when stimulated with LPS, indicating an effect on the polarization of the immune response. Interestingly, CsA-treated DCs show an anti-tolerogenic effect since they reduce the proliferation of T REG cells from 72 to 47 percent. Further inhibition to a 24 percent of T REG proliferation was obtained as a direct effect of CsA on T REG. In conclusion, the anti-tolerogenic effect of CsA should be considered in the planning of immunosuppression in the context of clinical transplantation.

Is mycophenolate more than just an immunosuppressant?--An overview.

The development of immunosuppressant compounds, such as cyclosporine and tacrolimus was crucial to the success of transplant surgery and for treatment of autoimmune diseases. However, immunosuppressant therapy may increase the concentrations of reactive oxygen species (ROS), inducing oxidative damage such as an increased vascular damage. The major source of ROS in the vascular endothelial cells is NADPH oxidase. The subunit structure and function of this enzyme complex in vascular cells differs from that in phagocytic leucocytes. The enzyme subunits Nox1, Nox2 and Nox4 are only found in vascular cells. The GTP-dependent protein subunit Rac 1 needs to be activated for this enzyme to function. Inhibiting this protein subunit should reduce NADPH oxidase-induced oxidative stress. In the cardiovascular system, oxidative stress is observed as hypertension, hypertrophy, fibrosis, conduction abnormalities and endothelial dysfunction, as well as cardiac allograft vasculopathy in transplant patients. In contrast to cyclosporine and tacrolimus, the immunosuppressant mycophenolate inhibits the Rac 1 subunit thus inhibiting NADPH oxidase in the vasculature. This may reduce oxidative stress, prevent the development of cardiac allograft vasculopathy, decrease the deterioration of vascular function and improve cardiovascular function chronically in transplant patients. This overview discusses whether this antioxidant immunosuppressive property could translate into a more general protective role for mycophenolate in the prevention of cardiovascular disease.