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1.
Transplant Proc ; 42(6): 2027-31, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20692399

RESUMO

The general consensus among transplant centers is that a cold ischemia time (CIT) beyond 8 hours results in reduced yields and quality of human islets. We sought to optimize the isolation process and enzymes for pancreata with extended CIT. We processed 16 extended CIT pancreata (13.2 +/- 0.7 hours). Donors averaged 50.8 +/- 2.6 (standard error of the mean) years old with a body mass index of 28.6 +/- 1.5. Glands were shipped in cold organ preservation solution without oxygenated perfluorocarbon. Isolations were performed under a protocol optimized for digestion with the new cGMP collagenase from Roche. Purification used continuous Euroficoll/University of Wisconsin gradients. Islets were cultured in two types of Prodo cGMP islet culture media and/or in Miami 1A media. Glucose-stimulated insulin secretion assays were performed after 8 to 16 days of culture. Prepurification yield averaged 415 +/- 41 KIEQ postpurification, 359 +/- 29 KIEQ (purification loss 13.5%); and postculture 317 +/- 27 KIEQ (culture loss 11.7%). Our process liberated an average of 4278 IEQ/g of pancreas (97 +/- 5 g). Most islets were recovered in the purest fraction (purity 79.7% +/- 1.9%). Culture loss in our enhanced culture media was 11.7%. After 2 to 3 days in culture, viability was 92% +/- 1%. Islets exhibited compactness and dithizone staining. Glucose-stimulated insulin secretion assays performed after 3 to 23 days in our PIM(R) media resulted in a stimulation index of 6.8 +/- 1.7 (G50 to G350). We concluded that our human islet isolation process permitted the recovery of large numbers of high-quality human islets from extended CIT pancreata and that our cGMP islet culture media was superior to the current standard CMRL-based media.


Assuntos
Ilhotas Pancreáticas/citologia , Adenosina , Adulto , Idoso , Alopurinol , Causas de Morte , Técnicas de Cultura de Células/métodos , Separação Celular/métodos , Meios de Cultura , GMP Cíclico/farmacologia , Feminino , Glucose/farmacologia , Glutationa , Humanos , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Masculino , Pessoa de Meia-Idade , Soluções para Preservação de Órgãos , Tamanho do Órgão , Pâncreas/anatomia & histologia , Rafinose , Obtenção de Tecidos e Órgãos/métodos
2.
Trop Anim Health Prod ; 31(3): 135-41, 1999 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-10445249

RESUMO

Mass vaccination with reduced dose 1/50 Rev 1 strain live vaccine (1-2 10(9) colony forming units), administered subcutaneously, over a four and a half year period reduced the prevalence of Brucella melitensis in Kuwait's small ruminant population from 5.8% in 1993 to 2.02% in 1997. Serological test results using the Rose Bengal Plate Test, Rivanol Agglutination Test and Complement Fixation showed no evidence of persistence of positive serology in animals nine or more months after vaccination. Questionnaires and post-vaccination flock inspections found that the effects on gestation (abortions) were minimal--and not proven to be due to the vaccine. The conclusion from these findings is that mass vaccination with reduced dose Rev 1 administered by the subcutaneous route is a practical field strategy for control of Brucella melitensis.


Assuntos
Vacina contra Brucelose , Brucella melitensis/imunologia , Brucelose/veterinária , Doenças das Cabras/prevenção & controle , Doenças dos Ovinos/prevenção & controle , Vacinação/veterinária , Aborto Animal/epidemiologia , Testes de Aglutinação/veterinária , Animais , Anti-Infecciosos Locais/química , Vacina contra Brucelose/administração & dosagem , Brucelose/epidemiologia , Brucelose/prevenção & controle , Testes de Fixação de Complemento/veterinária , Etacridina/química , Feminino , Corantes Fluorescentes/química , Doenças das Cabras/epidemiologia , Cabras , Injeções Subcutâneas/veterinária , Kuweit/epidemiologia , Gravidez , Rosa Bengala/química , Estudos Soroepidemiológicos , Ovinos , Doenças dos Ovinos/epidemiologia , Inquéritos e Questionários , Vacinas Atenuadas/administração & dosagem
6.
Ann N Y Acad Sci ; 831: 332-43, 1997 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-9616725

RESUMO

Functional porcine islets, free of known pathogens, can serve as a source of insulin producing cells for the treatment of experimentally induced insulin dependent Diabetes Mellitus. Porcine islets can be conformally coated (microencapsulated) with a covalently linked, stable permselective membrane while maintaining islet viability and function. The PEG conformal coating is immunoprotective in a discordant xenograft animal model (porcine islets to rat).


Assuntos
Diabetes Mellitus Tipo 1/cirurgia , Transplante das Ilhotas Pancreáticas/métodos , Pâncreas Artificial , Polietilenoglicóis , Imunologia de Transplantes , Animais , Materiais Biocompatíveis , Cápsulas , Transplante das Ilhotas Pancreáticas/imunologia , Membranas Artificiais , Fotoquímica , Ratos , Suínos
7.
Artif Organs ; 20(8): 878-82, 1996 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-8853799

RESUMO

Previously we established human peripheral blood lymphocyte-reconstituted severe combined immunodeficiency (SCID) (hu-PBL-SCID) mice as a model for human islet allograft rejection. The function of xenografted hu-PBL was confirmed to reject human alloislets in hu-PBL-SCID mice. In this study, we modified this model as a porcine islet xenograft to study porcine islet rejection in humans. Chimeric mice were used as the recipients of porcine islets to reveal the mechanisms of xenograft rejection in humans. SCID mice were reconstituted with 30 x 10(6) of hu-PBL initially, and 10 x 10(6) of antihuman CD3-primed PBL was injected intraperitoneally 2 days later as a booster. An additional booster injection provided greater possibility (86.7%, n = 15) of chimera establishment as well as a higher human immunoglobulin concentration in SCID mice than the single injection group. In an in vitro assay, sera from hu-PBL-SCID mice were found to recognize porcine islets by FACS staining. In an in vivo study, immunofluorescent analysis of a frozen section showed that human immunoglobulins adhered to the xenografted porcine islet under the kidney capsule of hu-PBL-SCID mice. Although no mouse immunoglobulins were detected on sections, mouse complement (C3) was shown to adhere to the xenografted porcine islet. Thus, hu-PBL-SCID mice provide a useful model for investigating the real-life situation of porcine islet xenograft rejection in humans.


Assuntos
Transplante de Células , Rejeição de Enxerto/imunologia , Imunodeficiência Combinada Severa/imunologia , Animais , Anticorpos Monoclonais/imunologia , Adesão Celular/imunologia , Complemento C3/imunologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Fluoresceína-5-Isotiocianato/química , Humanos , Técnicas Imunoenzimáticas , Imunoglobulina M/metabolismo , Ilhotas Pancreáticas/citologia , Rim/metabolismo , Camundongos , Suínos , Transplante Heterólogo/imunologia
9.
Transplantation ; 61(7): 1100-2, 1996 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-8623192

RESUMO

Purified porcine islets were prepared by collagenase digestion and density gradient purification, and transplanted under the kidney capsule of C57B/B6 mice with streptozotocin-induced diabetes which were receiving varying temporary immunosuppressive therapies. Islets that had been cultured for 1 day at 37 degree C were rejected after : 9+/-0.1 (mean+/-SE) days in control mice: 14+/-3 days in mice receiving mouse antilymphocyte serum (MLS) plus porcine antilymphocyte serum (PLS) on day of transplant (day 0); 43+/-6 days in mice treated for 1 week with anti-CD4 antibody (aCD4); 36+/-4 days in mice given aCD4 for 1 week plus PLS on days 0 and 7; 47+/-3 days in mice treated with aCD4 for 1 week plus MLS and PLS on day 21. Porcine islet survival in these latter three groups was significantly (P<0.01) and similarly longer than in the control and MLS plus PLS groups. Then, we transplanted islets that had been either cultured at 24 degrees C for 7 days or cryopreserved into 7-day aCD4-treated mice, to evaluate whether low temperature culture or the freezing-thawing procedure could affect survival. Neither 7-day, low temperature culture (mean survival time: 37+/-2 days) nor cryopreservation (mean survival time: 39+/-2 days) prolonged islets function further. Thus, the present study demonstrates that prolonged survival can be achieved with discordant porcine islet xenografts, and shows the greater efficacy of aCD4 treatment, which was not improved by additional immunosuppressive therapies we tested, nor by culture or cryopreservation of the islets.


Assuntos
Sobrevivência de Enxerto , Transplante das Ilhotas Pancreáticas , Transplante Heterólogo , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Soro Antilinfocitário/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Suínos
10.
Diabetes Care ; 19(3): 236-40, 1996 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-8742568

RESUMO

OBJECTIVE: The majority of islet transplant recipients remain insulin-requiring, although many have near-normal connecting peptide (CP) levels. Insulin resistance may be one possible cause of the continuing need for exogenous insulin in islet transplant recipients. To assess this, we have studied the insulin sensitivity index (S1) in one patient with near-normal CP levels after islet transplant who remained insulin-requiring. RESEARCH DESIGN AND METHODS: The islet transplant recipient is a 36-year-old woman with no residual CP who received a kidney transplant, followed 7 days later by an islet transplant. The islets were infused into the liver via the umbilical vein. Induction immunosuppression consisted of OKT3, prednisone, cyclosporin A, and azathioprine, with maintenance on the latter three. RESULTS: Maximum CP levels after a standardized Sustacal meal were 2.09, 1.18, 0.85, and 0.81 nmol/l at 1,6,18, and 24 months posttransplant, respectively. Insulin requirements at the same times were 0.27, 0.45, 0.49, and 0.62 U.kg(-1).d(-1), while S1 was 36.3, 53.3, and 13.2 min (-1).nmol(-1).ml at 6,18, and 24 months, respectively. This compares with S1 values of 43.3+/- 10.0 min (-1).nmol(-1).ml for normal subjects. CONCLUSIONS: This patient had near-normal S1 and CP levels, but she was unable to discontinue insulin therapy, suggesting that other factors are critical. Despite this, she maintained normal or near-normal glycated hemoglobins, indicating metabolic benefit from the islet transplant.


Assuntos
Peptídeo C/sangue , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/cirurgia , Insulina/uso terapêutico , Transplante das Ilhotas Pancreáticas , Transplante de Rim , Adulto , Glicemia/metabolismo , Nefropatias Diabéticas/cirurgia , Feminino , Seguimentos , Humanos , Terapia de Imunossupressão , Insulina/sangue
11.
Transplant Proc ; 27(6): 3400-2, 1995 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-8540018

RESUMO

1. BSA-containing solutions improve islet yields using standard collagenase digestion techniques. 2. The BSA effect on islet isolation is independent of source and lot of collagenase. 3. The BSA effect on islet isolation is not due solely to its colloid action, as HES failed to achieve the same level of improvement seen with albumin. 4. BSA can protect islets from warm ischemic injury, and the protective action appears to be unique to albumin, as HES was not as effective.


Assuntos
Separação Celular/métodos , Ilhotas Pancreáticas/citologia , Soroalbumina Bovina , Animais , Colagenases , Coloides , Estudos de Avaliação como Assunto , Técnicas In Vitro , Isquemia/prevenção & controle , Ilhotas Pancreáticas/irrigação sanguínea , Ilhotas Pancreáticas/lesões , Transplante das Ilhotas Pancreáticas , Soluções Isotônicas , Ratos , Ratos Endogâmicos Lew
14.
Acta Diabetol ; 32(2): 75-7, 1995 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-7579537

RESUMO

It is still a controversial question whether insulin suppresses its own secretion. We prepared pure human islets from three pancreases by collagenase digestion and density gradient purification. Aliquots of 200 islet equivalents (IE, 150-microns sized-islets) were sequentially perifused at 37 degrees C with 3.3 mmol/l glucose (3.3G, 40 min), 16.7 mmol/l glucose (16.7G, 30 min) and again 3.3G (30 min) after 24 h, 37 degrees C culture in CMRL 1066 medium with or without the addition of either 200 or 400 microU/ml human insulin in the incubation medium (6 replicates each). Insulin secretion was assessed by C-peptide (Cp) measurement in the perifusate. Without added insulin (C) and with 200 (Ins200) or 400 (Ins400) microU/ml added insulin, basal Cp release was 0.12 +/- 0.03, 0.14 +/- 0.02 and 0.14 +/- 0.04 ng/ml, respectively. At 16.7G, the first-phase secretion peak (expressed as Cp value) was significantly lower with Ins200 (0.47 +/- 0.13 ng/ml, P < 0.02) and Ins400 (0.68 +/- 0.15 ng/ml, P < 0.05) than C (0.83 +/- 0.15 ng/ml). The second-phase secretion peak was also significantly (P < 0.05) reduced with added insulin (Ins200: 0.47 +/- 0.08 ng/ml; Ins400: 0.45 +/- 0.07 ng/ml) than in its absence (C: 0.65 +/- 0.09 ng/ml). Accordingly, total Cp secretion was lower with Ins200 (10.6 +/- 2.3 ng/ml, P = 0.03) and Ins400 (11.8 +/- 2.3 ng/ml) than with C (16.0 +/- 2.2 ng/ml).(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Peptídeo C/metabolismo , Glucose/farmacologia , Insulina/metabolismo , Insulina/farmacologia , Ilhotas Pancreáticas/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Retroalimentação , Humanos , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Cinética , Perfusão , Fatores de Tempo
15.
Transplantation ; 59(6): 817-20, 1995 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-7701574

RESUMO

Streptozocin-induced diabetic nude mice (blood glucose 493 +/- 14 mg/dl) received aliquots of 2000 human islet equivalents (IE) under the kidney capsule and were then followed for up to 30 days with measurement of blood glucose concentration and body weight. Characterization of islet aliquots before the implantation included the assessment of the endocrine beta cell and nonbeta cell mass, estimated by point counting morphometry of immunostained sections. Islet transplantation was followed by a rapid decrease in blood glucose levels and by a progressive increase in body weight; 15 days after transplantation mean glycemic levels were 102 +/- 11 mg/dl and further decreased to 70 +/- 3 mg/dl at 30 days. Despite the progressive improvement in the glucose levels, the beta cell mass of the islet grafts significantly decreased over time from 2.63 +/- 0.2 mg, at the time of transplantation, to 1.16 +/- 0.1 and 0.86 +/- 0.1 mg 15 and 30 days, respectively, after transplantation. In contrast, the endocrine nonbeta cell mass remained stable from before the implantation to 30 days after. Therefore, the endocrine nonbeta cell/beta cell ratio increased from 14% at the time of transplantation, to 35% and 37%, 15 and 30 days, respectively, after transplantation. The rate of replication of the transplanted beta cells was similar in the grafts harvested at 15 and 30 days, with the percentage of beta cells positive for bromo-2' deoxyuridine (BrdU) incorporation being in the range of approximately 0.1% 6 hr after the BrdU injection. These data demonstrate that an important decrease in beta cell mass takes place immediately after islet transplantation--the most dramatic decrease occurring in the first 15 days and persisting even after revascularization has occurred. However, endocrine nonbeta cell mass remained stable indicating that engrafted nonbeta cells are less likely to die than beta cells. The finding that the nonbeta/beta cell ratio of a human islet graft can increase over time, raises questions about whether such a change in islet structure could have an influence upon function.


Assuntos
Morte Celular , Diabetes Mellitus Experimental/cirurgia , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas/fisiopatologia , Animais , Glicemia , Peso Corporal , Diabetes Mellitus Experimental/fisiopatologia , Sobrevivência de Enxerto , Humanos , Masculino , Camundongos , Camundongos Nus , Fatores de Tempo
17.
Eur Surg Res ; 27(3): 167-75, 1995.
Artigo em Inglês | MEDLINE | ID: mdl-7781654

RESUMO

This study evaluated the ability of cryopreservation to store large quantities of canine islets for transplantation studies. Islets were isolated by automated screen methods and purified by Euro-Ficoll gradients. After overnight 37 degrees C incubation, islets were equilibrated with 2 M dimethylsulfoxide, cryopreserved at a cooling rate of 0.25 degree C/min and subjected to long-term storage in liquid nitrogen. Six months later, some islets were thawed at a rate of 180 degrees C/min. The viability of cryothawed islets was determined in vitro by comparing islet count, total insulin content, morphology and perifusion studies in both control and cryothawed islets, and in vivo by transplantation into diabetic nude mice. The percentages of recovery of both islet count and total insulin content were 74.17 +/- 4.46 and 70.66 +/- 14.08, respectively. Islet morphology after cryothaw by light and electron microscopy revealed structurally intact islets with varying degrees of granulation. The results of perifusion indicated that there was no significant difference (p > 0.1) in both total stimulated insulin secretion and stimulation index response to high-dose glucose plus 3-isobutyl-1-methylxanthine and carbachol of isolated islets as compared to those of cryothawed islets. Transplantation into nude mice proved that grafted islets can successfully reverse diabetes. In conclusion, these findings indicate that the majority of purified canine islets can survive the frozen-thawed insult while maintaining their secretory function and permitting mass storage of canine islets for further transplant studies.


Assuntos
Criopreservação , Transplante das Ilhotas Pancreáticas , Preservação de Órgãos , Animais , Cães , Feminino , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante Heterólogo
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