Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

University of Florida 

Early systemic treatment of nonobese diabetic mice with high doses of recombinant adeno-associated virus (rAAV) vector expressing murine IL-10 prevents type 1 diabetes. To determine the therapeutic parameters and immunological mechanisms underlying this observation, female nonobese diabetic mice at 4, 8, and 12 wk of age were given a single i.m. injection of rAAV-murine IL-10 (10(4), 10(6), 10(8), and 10(9) infectious units (IU)), rAAV-vector expressing truncated murine IL-10 fragment (10(9) IU), or saline. Transduction with rAAV-IL-10 at 10(9) IU completely prevented diabetes in all animals injected at all time points, including, surprisingly, 12-wk-old animals. Treatment with 10(8) IU provided no protection in the 12-wk-old injected mice, partial prevention in 8-wk-old mice, and full protection in all animals injected at 4 wk of age. All other treatment groups developed diabetes at a similar rate. The rAAV-IL-10 therapy attenuated pancreatic insulitis, decreased MHC II expression on CD11b+ cells, increased the population of CD11b+ cells, and modulated insulin autoantibody production. Interestingly, rAAV-IL-10 therapy dramatically increased the percentage of CD4+CD25+ regulatory T cells. Adoptive transfer studies suggest that rAAV-IL-10 treatment alters the capacity of splenocytes to impart type 1 diabetes in recipient animals. This study indicates the potential for immunomodulatory gene therapy to prevent autoimmune diseases, including type 1 diabetes, and implicates IL-10 as a molecule capable of increasing the percentages of regulatory cells in vivo.

Systemic overexpression of IL-10 induces CD4+CD25+ cell populations in vivo and ameliorates type 1 diabetes in nonobese diabetic mice in a dose-dependent fashion.

Gene transfer into pancreatic cells in vivo could be of immense therapeutic benefit in cases of type 1 diabetes (T1D) through the production of molecules capable of interrupting the progression of autoimmunity or promoting regeneration of insulin-secreting beta cells. We adapted a clinically relevant surgical technique (endoscopic retrograde cholangiopancreatography) to deliver rAAV encoding human alpha1-antitrypsin (approved gene symbol SERPINA1) to the pancreas of 3-week-old Fisher 344 rats and C57BL/6 mice. We compared natural as well as bioengineered serotypes of rAAV (rAAV1, rAAV2/Apo, rAAV8) as well as different promoters (chicken beta-actin, human insulin) for their expression in vivo. Rats injected with rAAV1 showed the highest hAAT expression (week 2, rAAV1/CB-AT, 579 +/- 457 ng/ml). In mice, rAAV8 vector delivered the highest serum concentration of hAAT (week 2, rAAV8/CB-AT, 19 +/- 6 microg/ml). The chicken beta-actin promoter provided the highest expression in both rodent experiments. Immunohistochemical staining indicated transduction primarily of pancreatic acinar cells with either the rAAV1/CB-AT vector in the rat or the rAAV8/CB-AT vector in the mouse. This study demonstrates that rAAV vectors can be designed to deliver therapeutic genes efficiently to the pancreas and achieve high levels of gene expression and may be useful in treating pancreatic disorders, including T1D.

Localized gene expression following administration of adeno-associated viral vectors via pancreatic ducts.

The cause of cystic fibrosis-related diabetes (CFRD) remains unknown, but cystic fibrosis transmembrane conductance regulator (CFTR) mutations contribute directly to multiple aspects of the cystic fibrosis phenotype. We hypothesized that susceptibility to islet dysfunction in cystic fibrosis is determined by the lack of functional CFTR. To address this, glycemia was assessed in CFTR null (CFTR(-/-)), C57BL/6J, and FVB/NJ mice after streptozotocin (STZ)-induced beta-cell injury. Fasting blood glucose levels were similar among age-matched non-STZ-administered animals, but they were significantly higher in CFTR(-/-) mice 4 weeks after STZ administration (288.4 +/- 97.4, 168.4 +/- 35.9, and 188.0 +/- 42.3 mg/dl for CFTR(-/-), C57BL/6J, and FVB/NJ, respectively; P < 0.05). After intraperitoneal glucose administration, elevated blood glucose levels were also observed in STZ-administered CFTR(-/-) mice. STZ reduced islets among all strains; however, only CFTR(-/-) mice demonstrated a negative correlation between islet number and fasting blood glucose (P = 0.02). To determine whether a second alteration associated with cystic fibrosis (i.e., airway inflammation) could impact glucose control, animals were challenged with Aspergillus fumigatus. The A. fumigatus-sensitized CFTR(-/-) mice demonstrated similar fasting and stimulated glucose responses in comparison to nonsensitized animals. These studies suggest metabolic derangements in CFRD originate from an islet dysfunction inherent to the CFTR(-/-) state.

Cystic fibrosis transmembrane conductance regulator deficiency exacerbates islet cell dysfunction after beta-cell injury.

&#x81EA;&#x8EAB;&#x514D;&#x75AB;&#x6027;&#x7834;&#x574F; &beta; &#x7EC6;&#x80DE;&#x7C7B;&#x578B; 1 &#x578B;&#x7CD6;&#x5C3F;&#x75C5;&#x7684;&#x80F0;&#x5C9B;&#x7D20;&#x751F;&#x4EA7;&#x548C;&#x8461;&#x8404;&#x7CD6;&#x7A33;&#x6001;&#x4E2D;&#x635F;&#x5931;&#x7684;&#x7ED3;&#x679C;&#x4E2D;&#x3002;&#x4E3A;&#x6B64;&#xFF0C;&#x7597;&#x6CD5;&#x80FD;&#x591F;&#x51CF;&#x8F7B;&#x8FD9;&#x4E00;&#x7834;&#x574F;&#x6027;&#x901A;&#x8FC7;&#x9002;&#x5F53;&#x7684;&#x514D;&#x75AB;&#x8BC6;&#x522B;&#x7684;&#x6062;&#x590D;&#x8FDB;&#x7A0B;&#x7684;&#x53D1;&#x5C55;&#x5B58;&#x5728;&#x7740;&#x5DE8;&#x5927;&#x7684;&#x5229;&#x76CA;&#x3002;&#x56E0;&#x6B64;&#xFF0C;&#x6211;&#x4EEC;&#x8C03;&#x67E5;&#x7403;&#x514D;&#x75AB;&#x6D88;&#x8017;&#x4EE3;&#x7406;&#x80F8;&#x817A;&#x86CB;&#x767D; (ATG) &#x7684;&#x80FD;&#x529B;&#x52A8;&#x5458;&#x5242;&#x7C92;&#x7EC6;&#x80DE;&#x96C6;&#x843D;&#x523A;&#x6FC0;&#x56E0;&#x5B50; &#xFF08;GCSF&#xFF09;&#xFF0C;&#x4EE5;&#x626D;&#x8F6C;&#x80A5;&#x80D6;&#x578B;&#x7CD6;&#x5C3F;&#x75C5; (&#x70B9;&#x5934;) &#x5C0F;&#x9F20;&#x6A21;&#x578B;&#x7684; 1 &#x578B;&#x7CD6;&#x5C3F;&#x75C5;&#x660E;&#x663E;&#x9AD8;&#x8840;&#x7CD6;&#x3002;

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The autoimmune destruction of beta-cells in type 1 diabetes results in a loss of insulin production and glucose homeostasis. As such, an immense interest exists for the development of therapies capable of attenuating this destructive process through restoration of proper immune recognition. Therefore, we investigated the ability of the immune-depleting agent antithymocyte globulin (ATG), as well as the mobilization agent granulocyte colony-stimulating factor (GCSF), to reverse overt hyperglycemia in the nonobese diabetic (NOD) mouse model of type 1 diabetes.

Immune depletion with cellular mobilization imparts immunoregulation and reverses autoimmune diabetes in nonobese diabetic mice.

La destruction auto-immune des cellules b&#xEA;ta dans les r&#xE9;sultats de diab&#xE8;te de type 1 dans une perte de production d&#x26;#39;insuline et l&#x26;#39;hom&#xE9;ostasie du glucose. En tant que tel, un immense int&#xE9;r&#xEA;t existe pour le d&#xE9;veloppement de th&#xE9;rapies capables d&#x26;#39;att&#xE9;nuer ce processus destructeur &#xE0; travers la restauration de la reconnaissance immunitaire appropri&#xE9;e. Par cons&#xE9;quent, nous avons &#xE9;tudi&#xE9; la capacit&#xE9; de l&#x26;#39;immunoglobuline qui appauvrissent l&#x26;#39;agent antithymocyte (ATG), ainsi que l&#x26;#39;agent de mobilisation de granulocytes colony-stimulating factor (G-CSF), pour inverser l&#x26;#39;hyperglyc&#xE9;mie chez le diab&#xE9;tique manifeste non ob&#xE8;ses (NOD) mod&#xE8;le murin de diab&#xE8;te de type 1 .

D&#xE9;ficit immunitaire cellulaire avec la mobilisation Donne Immunor&#xE9;gulation et Inverse diab&#xE8;te auto-immun chez des souris diab&#xE9;tiques non ob&#xE8;ses

Die autoimmune Zerst&ouml;rung der Beta-Zellen in Typ 1-Diabetes f&uuml;hrt zu einem Verlust der Insulin-Produktion und der Glukose-Hom&ouml;ostase. So existiert ein immenses Interesse f&uuml;r die Entwicklung von Therapien kann diesen zerst&ouml;rerischen Prozess durch Wiederherstellung der richtige immun Anerkennung abgeschw&auml;cht. Daher untersuchten wir die F&auml;higkeit des immunen abbauende Agent antithymocyte Globulin (ATG), sowie der Mobilisierung Agent Granulozyten-Kolonie-stimulierende Faktor (GCSF), offene Hyperglyk&auml;mie im nonobese Diabetische (NOD) Mausmodell des Typ 1 Diabetes r&uuml;ckg&auml;ngig zu machen.

Immun Raubbau mit zellul&auml;ren Mobilisierung Immunoregulation vermittelt und kehrt autoimmune Diabetes nonobese diabetischen M&auml;usen.

La distruzione autoimmune delle cellule beta in tipo 1 diabete determina una perdita dell&#39;omeostasi produzione e glucosio insulina. Come tale, un immenso interesse esiste per lo sviluppo di terapie in grado di attenuare questo processo distruttivo attraverso il ripristino del corretto riconoscimento immunitario. Pertanto, abbiamo studiato la possibilit&agrave; dell&#39;esaurimento immunitario agente antithymocyte globulina (ATG), cos&igrave; come la mobilitazione agente granulocyte colony-stimulating factor (GCSF), di invertire l&#39;iperglicemia conclamata in nonobese diabetico modello del mouse (NOD) del diabete di tipo 1.

Esaurimento immunitario con mobilizzazione cellulare conferisce immunoregolazione e inverte il diabete autoimmune in nonobese topi diabetici.

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&#xC778;&#xC290;&#xB9B0; &#xC0DD;&#xC0B0;&#xACFC; &#xD3EC;&#xB3C4; &#xB2F9; &#xD56D;&#xC0C1;&#xC131;&#xC758; &#xC190;&#xC2E4;&#xC5D0; &#xD0C0;&#xC785; 1 &#xB2F9;&#xB1E8;&#xBCD1; &#xACB0;&#xACFC;&#xC5D0;&#xC11C; &#xBCA0;&#xD0C0; &#xC138;&#xD3EC;&#xC758; &#xC790;&#xAC00; &#xBA74;&#xC5ED; &#xD30C;&#xAD34;. &#xC774;&#xC640; &#xAC19;&#xC774; &#xC5C4;&#xCCAD;&#xB09C; &#xAD00;&#xC2EC; &#xACF5;&#xAC04;&#xC740; &#xC801;&#xC808; &#xD55C; &#xBA74;&#xC5ED; &#xC778;&#xC2DD;&#xC758; &#xBCF5;&#xC6D0;&#xC744; &#xD1B5;&#xD574; &#xD30C;&#xAD34;&#xC801;&#xC778; &#xACFC;&#xC815;&#xC774; &#xC218; &#xCE58;&#xB8CC;&#xC758; &#xAC1C;&#xBC1C;&#xC5D0; &#xB300; &#xD55C; &#xC874;&#xC7AC; &#xD569;&#xB2C8;&#xB2E4;. &#xB530;&#xB77C;&#xC11C; &#xC6B0;&#xB9AC; &#xB3D9;&#xC6D0; &#xC5D0;&#xC774;&#xC804;&#xD2B8; granulocyte &#xC2DD;&#xBBFC;&#xC9C0; &#xC790;&#xADF9; &#xC694;&#xC18C; (GCSF), &#xB9AC;&#xBC84;&#xC2A4; &#xD0C0;&#xC785;-1 &#xB2F9;&#xB1E8;&#xBCD1;&#xC758; nonobese &#xB2F9;&#xB1E8;&#xBCD1; (&#xB044; &#xB355;) &#xB9C8;&#xC6B0;&#xC2A4; &#xBAA8;&#xB378;&#xC5D0;&#xC11C; &#xBA85;&#xBC31;&#xD55C; &#xACE0; &#xD608;&#xB2F9; &#xBFD0;&#xB9CC; &#xC544;&#xB2C8;&#xB77C;&#xB294; &#xBA74;&#xC5ED; &#xC5C6;&#xC560;&#xACE0; &#xC5D0;&#xC774;&#xC804;&#xD2B8; antithymocyte &#xAE00;&#xB85C;&#xBD88;&#xB9B0; (ATG)&#xC758; &#xB2A5;&#xB825;&#xC744; &#xC870;&#xC0AC;.

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La destrucci&#xF3;n autoinmune de las c&#xE9;lulas beta en los resultados de la diabetes tipo 1 en una p&#xE9;rdida de la producci&#xF3;n de insulina y la homeostasis de la glucosa. Como tal, un gran inter&#xE9;s existe para el desarrollo de terapias capaces de atenuar este proceso destructivo a trav&#xE9;s de la restauraci&#xF3;n de reconocimiento inmune apropiada. Por lo tanto, se determin&#xF3; la capacidad de la globulina inmune de agotamiento del agente de antitimocito (ATG), as&#xED; como la movilizaci&#xF3;n de agentes de granulocitos factor estimulante de colonias (G-CSF), para revertir la hiperglucemia manifiesta en el paciente diab&#xE9;tico no obeso (NOD) modelo de rat&#xF3;n de la diabetes tipo 1 .

El agotamiento inmunol&#xF3;gico con la movilizaci&#xF3;n celular Imparte inmunorregulaci&#xF3;n y revierte la diabetes autoinmune en ratones diab&#xE9;ticos no obesos

Type 1 diabetes (T1D) is considered a disorder whose pathogenesis is autoimmune in origin, a notion drawn in large part from studies of human pancreata performed as far back as the 1960s. While studies of the genetics, epidemiology, and peripheral immunity in T1D have been subject to widespread analysis over the ensuing decades, efforts to understand the disorder through analysis of human pancreata have been far more limited. We have reviewed the published literature pertaining to the pathology of the human pancreas throughout all stages in the natural history of T1D. This effort uncovered a series of findings that challenge many dogmas ascribed to T1D and revealed data suggesting the marked heterogeneity in terms of its pathology. An improved understanding and appreciation for pancreatic pathology in T1D could lead to improved disease classification, an understanding of why the disorder occurs, and better therapies for disease prevention and management.

Martha L. Campbell-Thompson

Department of Pathology,
Immunology,
and Laboratory Medicine,
Department of Pathology, Immunology, and Laboratory Medicine

University of Florida

Collection Protocol for Human Pancreas

Staining Protocols for Human Pancreatic Islets

Martha L. Campbell-Thompson

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Pathology,Immunology,and Laboratory Medicine,Department of Pathology, Immunology, and Laboratory Medicine

University of Florida

Collection Protocol for Human Pancreas

Staining Protocols for Human Pancreatic Islets

Department of Pathology,
Immunology,
and Laboratory Medicine,
Department of Pathology, Immunology, and Laboratory Medicine