On the Etiology of Type 1 Diabetes
2012; Elsevier BV; Volume: 181; Issue: 5 Linguagem: Inglês
10.1016/j.ajpath.2012.07.022
ISSN1525-2191
AutoresStella Korsgren, Ylva Molin, K. Salmela, Torbjörn Lundgren, Åsa Melhus, Olle Korsgren,
Tópico(s)Pancreatic function and diabetes
ResumoThe cause of type 1 diabetes (T1D) remains unknown; however, a decisive role for environmental factors is recognized. The increased incidence of T1D during the last decades, as well as regional differences, is paralleled by differences in the intestinal bacterial flora. A new animal model was established to test the hypothesis that bacteria entering the pancreatic ductal system could trigger β-cell destruction and to provide new insights to the immunopathology of the disease. Obtained findings were compared with those present in two patients dying at onset of T1D. Different bacterial species, present in the human duodenum, instilled into the ductal system of the pancreas in healthy rats rapidly induced cellular infiltration, consisting of mainly neutrophil polymorphonuclear cells and monocytes/macrophages, centered around the pancreatic ducts. Also, the islets of Langerhans attracted polymorphonuclear cells, possibly via release of IL-6, IL-8, and monocyte chemotactic protein 1. Small bleedings or large dilatations of the capillaries were frequently found within the islets, and several β-cells had severe hydropic degeneration (ie, swollen cytoplasm) but with preserved nuclei. A novel rat model for the initial events in T1D is presented, revealing marked similarities with the morphologic findings obtained in patients dying at onset of T1D and signifying a decisive role for bacteria in eliciting an adverse innate immunity response. The present findings support the hypothesis that T1D is an organ-specific inflammatory disease. The cause of type 1 diabetes (T1D) remains unknown; however, a decisive role for environmental factors is recognized. The increased incidence of T1D during the last decades, as well as regional differences, is paralleled by differences in the intestinal bacterial flora. A new animal model was established to test the hypothesis that bacteria entering the pancreatic ductal system could trigger β-cell destruction and to provide new insights to the immunopathology of the disease. Obtained findings were compared with those present in two patients dying at onset of T1D. Different bacterial species, present in the human duodenum, instilled into the ductal system of the pancreas in healthy rats rapidly induced cellular infiltration, consisting of mainly neutrophil polymorphonuclear cells and monocytes/macrophages, centered around the pancreatic ducts. Also, the islets of Langerhans attracted polymorphonuclear cells, possibly via release of IL-6, IL-8, and monocyte chemotactic protein 1. Small bleedings or large dilatations of the capillaries were frequently found within the islets, and several β-cells had severe hydropic degeneration (ie, swollen cytoplasm) but with preserved nuclei. A novel rat model for the initial events in T1D is presented, revealing marked similarities with the morphologic findings obtained in patients dying at onset of T1D and signifying a decisive role for bacteria in eliciting an adverse innate immunity response. The present findings support the hypothesis that T1D is an organ-specific inflammatory disease. Our understanding of the etiology of type 1 diabetes (T1D) remains limited and originates to a large extent from two animal models: the nonobese diabetic mouse and the BioBreeding-diabetes prone rat.1Roep B.O. Atkinson M. von Herrath M. Satisfaction (not) guaranteed: re-evaluating the use of animal models of type 1 diabetes.Nat Rev Immunol. 2004; 4: 989-997Crossref PubMed Scopus (158) Google Scholar In both models a progressive T-cell–mediated destruction of the β-cells occurs; however, the immunopathologic findings reveal limited similarities with the human disease.2Foulis A.K. Francis N.D. Farquharson M.A. Boylston A. Massive synchronous B-cell necrosis causing type 1 (insulin-dependent) diabetes–a unique histopathological case report.Diabetologia. 1988; 31: 46-50PubMed Google Scholar, 3Foulis A.K. Liddle C.N. Farquharson M.A. Richmond J.A. Weir R.S. The histopathology of the pancreas in type 1 (insulin-dependent) diabetes mellitus: a 25-year review of deaths in patients under 20 years of age in the United Kingdom.Diabetologia. 1986; 29: 267-274Crossref PubMed Scopus (393) Google Scholar, 4Gepts W. Pathologic anatomy of the pancreas in juvenile diabetes mellitus.Diabetes. 1965; 14: 619-633Crossref PubMed Scopus (979) Google Scholar, 5Lecompte P.M. Insulitis in early juvenile diabetes.AMA Arch Pathol. 1958; 66: 450-457PubMed Google Scholar In human pancreatic specimens, insulitis is discrete, affects only a few islets, and is heterogeneously distributed within the gland. In a recent meta-analysis, insulitis was reported in only 29% of patients with onset between 15 and 39 years of age and with a disease duration of <1 month.6In't Veld P. Insulitis in human type 1 diabetes: the quest for an elusive lesion.Islets. 2011; 3: 131-138Crossref PubMed Scopus (151) Google Scholar At the time of diagnosis, autoantibodies were only present in approximately 70% to 80% of affected patients.7Notkins A.L. Lernmark A. Autoimmune type 1 diabetes: resolved and unresolved issues.J Clin Invest. 2001; 108: 1247-1252Crossref PubMed Scopus (213) Google Scholar Likewise, attempts to prevent disease progression with immunosuppression8Staeva-Vieira T. Peakman M. von Herrath M. Translational mini-review series on type 1 diabetes: immune-based therapeutic approaches for type 1 diabetes.Clin Exp Immunol. 2007; 148: 17-31Crossref PubMed Scopus (123) Google Scholar, 9Pescovitz M.D. Greenbaum C.J. Krause-Steinrauf H. Becker D.J. Gitelman S.E. Goland R. Gottlieb P.A. Marks J.B. 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Treatment of patients with new onset Type 1 diabetes with a single course of anti-CD3 mAb Teplizumab preserves insulin production for up to 5 years.Clin Immunol. 2009; 132: 166-173Crossref PubMed Scopus (164) Google Scholar or immunointerventions12Wherrett D.K. Bundy B. Becker D.J. DiMeglio L.A. Gitelman S.E. Goland R. Gottlieb P.A. Greenbaum C.J. Herold K.C. Marks J.B. Monzavi R. Moran A. Orban T. Palmer J.P. Raskin P. Rodriguez H. Schatz D. Wilson D.M. Krischer J.P. Skyler J.S. Antigen-based therapy with glutamic acid decarboxylase (GAD) vaccine in patients with recent-onset type 1 diabetes: a randomised double-blind trial.Lancet. 2011; 378: 319-327Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 13Skyler J.S. Krischer J.P. Wolfsdorf J. Cowie C. Palmer J.P. Greenbaum C. Cuthbertson D. Rafkin-Mervis L.E. Chase H.P. Leschek E. 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Jaeger C. Shimura H. Kobayashi T. Distinct autoantibodies against exocrine pancreatic antigens in European patients with type 1 diabetes mellitus and non-alcoholic chronic pancreatitis.JOP. 2008; 9: 683-689PubMed Google Scholar, 16Panicot L. Mas E. Thivolet C. Lombardo D. Circulating antibodies against an exocrine pancreatic enzyme in type 1 diabetes.Diabetes. 1999; 48: 2316-2323Crossref PubMed Scopus (53) Google Scholar, 17Taniguchi T. Tanaka J. Seko S. Okazaki K. Okamoto M. Association of rapid-onset type 1 diabetes and clinical acute pancreatitis positive for autoantibodies to the exocrine pancreas.Diabetes Care. 2001; 24: 2156-2157Crossref PubMed Scopus (13) Google Scholar, 18Taniguchi T. Okazaki K. Okamoto M. Seko S. Tanaka J. Uchida K. Nagashima K. Kurose T. Yamada Y. Chiba T. Seino Y. High prevalence of autoantibodies against carbonic anhydrase II and lactoferrin in type 1 diabetes: concept of autoimmune exocrinopathy and endocrinopathy of the pancreas.Pancreas. 2003; 27: 26-30Crossref PubMed Scopus (65) Google Scholar Mild to moderate exocrine pancreatic insufficiency is an early event in T1D,19Creutzfeldt W. Gleichmann D. Otto J. Stockmann F. Maisonneuve P. Lankisch P.G. Follow-up of exocrine pancreatic function in type-1 diabetes mellitus.Digestion. 2005; 72: 71-75Crossref PubMed Scopus (44) Google Scholar and a substantial reduction (32%) in pancreatic volume is already present 3 to 4 months after onset.20Gaglia J.L. Guimaraes A.R. Harisinghani M. Turvey S.E. Jackson R. Benoist C. Mathis D. Weissleder R. Noninvasive imaging of pancreatic islet inflammation in type 1A diabetes patients.J Clin Invest. 2011; 121: 442-445Crossref PubMed Scopus (157) Google Scholar Also, in the classic report by Gepts,4Gepts W. Pathologic anatomy of the pancreas in juvenile diabetes mellitus.Diabetes. 1965; 14: 619-633Crossref PubMed Scopus (979) Google Scholar lesions of the acinar tissue were reported to occur frequently in patients with recent onset of T1D. The findings comprised mostly focal or diffuse lesions of acute pancreatitis with accumulation of leukocytes, often centered around the excretory canals.2Foulis A.K. Francis N.D. Farquharson M.A. Boylston A. Massive synchronous B-cell necrosis causing type 1 (insulin-dependent) diabetes–a unique histopathological case report.Diabetologia. 1988; 31: 46-50PubMed Google Scholar, 3Foulis A.K. Liddle C.N. Farquharson M.A. Richmond J.A. Weir R.S. The histopathology of the pancreas in type 1 (insulin-dependent) diabetes mellitus: a 25-year review of deaths in patients under 20 years of age in the United Kingdom.Diabetologia. 1986; 29: 267-274Crossref PubMed Scopus (393) Google Scholar, 4Gepts W. 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The low concordance rate for the development of T1D in identical twins and the current rapid increase in incidence of T1D argue against a decisive role for genetic factors. Notably, there is a close to sixfold gradient in the incidence of T1D between Russian and Finland Karelia, although the population is homogenous and the predisposing HLA genotypes are equally frequent.22Kondrashova A. Viskari H. Kulmala P. Romanov A. Ilonen J. Hyoty H. Knip M. Signs of beta-cell autoimmunity in nondiabetic schoolchildren: a comparison between Russian Karelia with a low incidence of type 1 diabetes and Finland with a high incidence rate.Diabetes Care. 2007; 30: 95-100Crossref PubMed Scopus (40) Google Scholar In addition, children born in Finland by immigrants from Somalia, a low incidence country for T1D, acquire the same risk for T1D as the native Finish population.23Oilinki T. Otonkoski T. Ilonen J. Knip M. Miettinen P. Prevalence and characteristics of diabetes among Somali children and adolescents living in Helsinki, Finland.Pediatr Diabetes. 2012; 13: 176-180Crossref PubMed Scopus (34) Google Scholar On the basis of these and similar observations, it is generally assumed that environmental factors may act as triggers of T1D. For decades different enteroviruses have been implicated in the pathogenesis of T1D24Yoon J.W. Austin M. Onodera T. Notkins A.L. Isolation of a virus from the pancreas of a child with diabetic ketoacidosis.N Engl J Med. 1979; 300: 1173-1179Crossref PubMed Scopus (784) Google Scholar; however, evidence of causality remains missing. Bacterial colonization of the infantile gut is influenced by environmental factors and has changed markedly in developed countries during the last decades.25Adlerberth I. Lindberg E. Aberg N. Hesselmar B. Saalman R. Strannegard I.L. Wold A.E. Reduced enterobacterial and increased staphylococcal colonization of the infantile bowel: an effect of hygienic lifestyle?.Pediatr Res. 2006; 59: 96-101Crossref PubMed Scopus (234) Google Scholar Interestingly, the increased incidence of T1D26Patterson C.C. Dahlquist G.G. Gyurus E. Green A. Soltesz G. Incidence trends for childhood type 1 diabetes in Europe during 1989–2003 and predicted new cases 2005–20: a multicentre prospective registration study.Lancet. 2009; 373: 2027-2033Abstract Full Text Full Text PDF PubMed Scopus (1374) Google Scholar and the difference in incidence of T1D in Sweden, Italy, and Africa26Patterson C.C. Dahlquist G.G. Gyurus E. Green A. Soltesz G. Incidence trends for childhood type 1 diabetes in Europe during 1989–2003 and predicted new cases 2005–20: a multicentre prospective registration study.Lancet. 2009; 373: 2027-2033Abstract Full Text Full Text PDF PubMed Scopus (1374) Google Scholar, 27Bizzarri C. Patera P.I. Arnaldi C. Petrucci S. Bitti M.L. 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The composition of the faecal microflora in breastfed and bottle fed infants from birth to eight weeks.Acta Paediatr Scand. 1985; 74: 45-51Crossref PubMed Scopus (159) Google Scholar, 31Lindberg E. Adlerberth I. Matricardi P. Bonanno C. Tripodi S. Panetta V. Hesselmar B. Saalman R. Aberg N. Wold A.E. Effect of lifestyle factors on Staphylococcus aureus gut colonization in Swedish and Italian infants.Clin Microbiol Infect. 2011; 17: 1209-1215Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 32Efuntoye M.O. Adetosoye A.I. Enterotoxigenicity and drug sensitivity of staphylococci from children aged five years and below with sporadic diarrhoea.East Afr Med J. 2003; 80: 656-659PubMed Google Scholar Bacteria entering the ductal system of the pancreas would be exposed to the pancreatic juice–containing substances, with marked antibacterial effects initiating release of bacterial components, such as lipopolysaccharide (LPS), lipoteichoic acid (LTA), and various toxins. Notably, these substances have been implicated in the etiology of neurogenerative diseases and neural cell death because they stimulate microglia to produce proinflammatory cytokines (IL-1b, IL-6, tumor necrosis factor-α), nitric oxide, and reactive oxygen species, causing significant cell death in neighboring neural cells.33Kinsner A. Pilotto V. Deininger S. Brown G.C. Coecke S. Hartung T. Bal-Price A. Inflammatory neurodegeneration induced by lipoteichoic acid from Staphylococcus aureus is mediated by glia activation, nitrosative and oxidative stress, and caspase activation.J Neurochem. 2005; 95: 1132-1143Crossref PubMed Scopus (73) Google Scholar The present study was conducted to establish an animal model for the initial events in T1D to test the hypothesis that bacteria entering into the ductal system of the pancreas could elicit an adverse innate immunity response. Different bacterial species present transiently or continuously in the human duodenum were instilled into the ductal system of the pancreas in healthy rats. To examine the clinical relevance of the experimental model, obtained findings were compared with those present in the pancreases of two patients dying at onset of T1D. All work involving human tissue was conducted according to the principles expressed in the Declaration of Helsinki and in the European Council's Convention on Human Rights and Biomedicine. Consent for organ donation (for clinical transplantation and for use in research) was obtained from the relatives of the deceased donors by the donor's physicians and documented in the medical records of the deceased patient. The study was approved by the Regional Ethics Committee in Uppsala, Sweden, according to the Act Concerning the Ethical Review of Research Involving Humans (2003:460; permit number Dnr 2009/043, 2009/371). The animal experiments were in accordance with the Swedish Animal Welfare Act (SFS 1988:534) and The Swedish Animal Welfare Ordinance (SFS 1988:539) both in agreement with directives 86/609/EEG and 2010/63/EU of the European Parliament and of the Council on the Protection of Animals Used for Scientific Purposes. The ethical application was approved by the Uppsala Laboratory Animal Ethical Committee (permit number C153/10). A total of 23 human pancreases were included in the study. Two pancreases were obtained at the onset of T1D (patients 1 and 2), and the remaining 21 were collected consecutively from multiorgan donors of similar age and without any known pancreatic disease in the time span between the two T1D patients. The latter donors were aged 29 to 40 years (mean ± SD age, 35.6 ± 0.7 years). A 29-year-old, previously healthy male (HLA-A24, HLA-B39, HLA-DR4) walked to the emergency department of the local hospital after 2 days with increased thirst and nausea. At arrival, patient was fully conscious, oriented (Glasgow Coma Scale score of 15), and vomiting. Laboratory test results were as follows: B-glucose, 46 mmol/L; B-pH 6.92; P-HCO3−, <3; B-hemoglobin, 187 g/L; and P-sodium, 125 mmol/L. Rehydration was initiated together with an i.v. bolus of short-acting insulin, 10 IU, which was repeated after 3 hours, at which time the B-glucose level was 31 mmol/L. Physical status deteriorated during transfer to another hospital. At admittance 5 hours after the initial contact, he was unconscious and suddenly stopped breathing. The patient was intubated and given norepinephrine. Laboratory tests revealed further derangements: P-Na, 168 mmol/L; P-potassium, 4 mmol/L; B-hemoglobin, 115 g/L: C-reactive protein, 69 mg/L; and P-amylase, 82 μkat/L. Computed tomography (CT) revealed massive brain edema and a suspected massive subarachnoid hemorrhage. The patient was referred to the Department of Neurosurgery, Helsinki University Hospital. The brain edema increased, and the patient developed total brain infarction. The death of the patient was considered the result of a series of complications associated with the recent onset of T1D. The patient was a 40-year-old, previously healthy male (HLA-A23/24, HLA-B27/44, HLA-DR4/7) with a history of increasing severe thirst and high diuresis during the past 3 weeks. The patient had, in addition, been treated with clindamycin for a sore throat and a skin infection but disrupted the treatment 2 days before admittance because of vomiting. The patient arrived at the hospital by ambulance after being found unconscious at work. The initial Glasgow Coma Scale rating was 12. The patient was paretic in the right arm and leg but could answer questions with yes or no. Laboratory tests revealed the following: B-glucose, 33 mmol/L; a B-pH 7.29; and base excess, −12.7 mmol/L. High levels of glucose and ketones were found in the urine. The patient required 73 IU of i.v. insulin during the following 16 hours to reach a B-glucose level <10 mmol/L. A CT scan of the brain showed no bleedings and the patient received thrombolysis for a suspected brain infarction without clinical improvement. Two days after admittance, the patient's state deteriorated and he became unconscious. New CT revealed a major infarction of an area corresponding to an occlusion of the left medial cerebral artery. The patient subsequently developed edema and total brain infarction. The death of this patient was also considered to be the result of a series of complications associated with his recent T1D debut. Healthy, male Wistar rats weighing 250 to 300 g (Taconic, Ry, Denmark) were used. To exclude strain-dependent reactions, Lewis and Sprague Dawley rats were also included in some experiments. Before the bacterial challenge, the animals were kept under standard laboratory conditions and given water and food ad libitum. The animals were anesthetized by intraperitoneal injection with thiobutabarbital (Research Biochemicals International, Natick, MA). At challenge, the common bile duct was exposed through a ventral midline incision after a blunt dissection. Via the common bile duct, 0.2 mL of a suspension was injected into the pancreas. Animals were kept anesthetized for a period of 1 to 5 hours, after which they were sacrificed by heart puncture. Control animals were either sacrificed without any prior treatment or inoculated with brain heart infusion (BHI) broth without bacteria. Plasma and serum samples were assayed for aspartate transaminase, alanine aminotransferase, alkaline phosphatase, antitrypsin, orosomucoid (α1-acid glycoprotein), haptoglobin, and C-reactive protein at the Department for Clinical Chemistry, Uppsala University Hospital. Biopsies were fixed in 4% paraformaldehyde and prepared for paraffin embedding. Four different bacterial species were used (Table 1). All strains were clinical isolates from the Department of Clinical Microbiology, Uppsala University Hospital, Uppsala, Sweden. The bacterial choice was based on a documented ability of these bacteria to translocate into the pancreas, liver, and/or gallbladder and to be leading causes of infections in these organs.34Flores C. Maguilnik I. Hadlich E. Goldani L.Z. Microbiology of choledochal bile in patients with choledocholithiasis admitted to a tertiary hospital.J Gastroenterol Hepatol. 2003; 18: 333-336Crossref PubMed Scopus (39) Google Scholar, 35Negm A.A. Schott A. Vonberg R.P. Weismueller T.J. Schneider A.S. Kubicka S. Strassburg C.P. Manns M.P. Suerbaum S. Wedemeyer J. Lankisch T.O. Routine bile collection for microbiological analysis during cholangiography and its impact on the management of cholangitis.Gastrointest Endosc. 2010; 72: 284-291Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 36Schmid S.W. Uhl W. Friess H. Malfertheiner P. Buchler M.W. The role of infection in acute pancreatitis.Gut. 1999; 45: 311-316Crossref PubMed Scopus (184) Google Scholar, 37Stelzmueller I. Berger N. Wiesmayr S. Eller M. Tabarelli W. Fille M. Margreiter R. Bonatti H. Group milleri streptococci: significant pathogens in solid organ recipients.Transpl Int. 2007; 20: 51-56Crossref PubMed Scopus (11) Google Scholar The S. aureus strain produced toxic shock syndrome toxin 1, the most common toxin harbored by S. aureus isolated from children,38Megevand C. Gervaix A. Heininger U. Berger C. Aebi C. Vaudaux B. Kind C. Gnehm H.P. Hitzler M. Renzi G. Schrenzel J. Francois P. Molecular epidemiology of the nasal colonization by methicillin-susceptible Staphylococcus aureus in Swiss children.Clin Microbiol Infect. 2010; 16: 1414-1420Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar to make it possible to study the effects of a potent exotoxin, in addition to the endotoxin of Escherichia coli.Table 1Bacteria and Culture ConditionsBacterial speciesStrainOriginCulture mediaGrowth phaseViable countE. faecalisB1008314BloodBHI brothLate stationary phase109 CFU/mLE. coliS0705198Peritoneal fluidBHI brothLate stationary phase109 CFU/mLE. faecalisB1008314BloodBHI broth with 70% CMRL-1066 and 5% FBSEarly stationary phase109 CFU/mLE. coliS0705198Peritoneal fluidBHI broth with 70% CMRL-1066 and 5% FBSEarly stationary phase109 CFU/mLS. aureus (preparation A)B0451020BloodBHI brothLate stationary phaseBacterial suspension sterilized through 0.45-μm filterS. aureus (preparation B)B0451020BloodBHI brothStationary phase50% of bacterial suspension sterilized through 0.45-μm filter and 50% heat inactivatedα-StreptococcusB1008638BloodTrypticase soy broth BBL with 10% inactivated horse serum and 5% Fildes enrichment BBLLate stationary phase109 CFU/mLFBS, fetal bovine serum. Open table in a new tab FBS, fetal bovine serum. The bacteria were stored at −70°C, and all cultures were initially inoculated from these frozen stocks onto blood or chocolate agar (Acumedia Manufacturers, Lansing, MI). The bacteria for pancreatic challenge were prepared by incubating at 37°C in the appropriate atmosphere for 4 to 5 hours (early stationary phase), 7 hours (stationary phase), or 15 to 20 hours (late stationary phase) according to growth curves. The α-hemolytic streptococci were grown in trypticase soy broth BBL (Becton Dickinson, Sparks, MD) supplemented with 10% inactivated horse serum (National Veterinary Institute, Uppsala, Sweden) and 5% Fildes enrichment BBL (Becton Dickinson). All other bacterial species were inoculated into BHI broth BBL with or without 70% CMRL-1066 (ICN Biomedicals, Costa Mesa, CA) and 5% fetal bovine serum (Invitrogen, Lidingö, Sweden). Heat-killed bacteria were boiled for 15 minutes. Viable counts of all inocula were performed before challenge. Consecutive 6-mm sections from the two patients with early T1D and the control donors were processed and labeled using a standard immunoperoxidase technique for paraffin section. With the exception of chromogranin A, glucagon, and insulin, all other antigens were unmasked by heat-induced epitope retrieval. Antibodies against chromogranin A (NeoMarkers, Thermo Fisher Scientific Inc, Fremont, CA) were used to identify islets. Antibodies against macrophages (CD68), granulocytes [myeloperoxidase (MPO)], and glucagon were from Dako (Glostrup, Denmark), whereas antibodies against insulin and CD3 were from BioGene (Kimbolton, UK) and Novocastra (Leica Microsystems AB, Stockholm, Sweden), respectively. Bound antibodies were visualized with Dako EnVision (Dako). Consecutive sections from rats were processed using the same technique. Antibodies against leukosialin (CD43, all leukocytes except B cells), macrophages (ED1/CD68 and ED2/CD163), and T cells (CD3 and CD8) were from AbD Serotec (Dusseldorf, Germany); antibodies against insulin and glucagon were from Dako. The mouse antibody against insulin was from Sigma-Aldrich (St. Louis, MO). Bound antibodies were visualized using Dako EnVision or EnVision DuoFLEX Doublestain System (both Dako) and diaminobenzidine-based substrate or 3-amino, 9-ethyl-carbazole (Dako). Sections were counterstained with hematoxylin and analyzed by light microscopy. Islets of Langerhans were isolated as described previously.39Goto M. Eich T.M. Felldin M. Foss A. Kallen R. Salmela K. Tibell A. Tufveson G. Fujimori K. Engkvist M. Korsgren O. Refinement of the automated method for human islet isolation and presentation of a closed system for in vitro islet culture.Transplantation. 2004; 78: 1367-1375Crossref PubMed Scopus (178) Google Scholar Islet preparations were of good quality but were made available for research because of a too low total islet number for clinical transplantation. Islets were kept in culture bags (Baxter Medical AB, Kista, Sweden) with 200 mL of CMRL-1066 supplemented with 10 mmol/L HEPES, 2 mmol/L l-glutamine, 50 μg/mL of gentamicin, 20 μg/mL of ciprofloxacin (Bayer Health Care AG, Leverkusen, Germany), 10 mmol/L nicotinamide, and 10% heat-inactivated human serum (Uppsala blood bank, Uppsala, Sweden) at 37°C in 5% CO2 and humidified air for 1 to 7 days. The culture medium was changed on day 1 and then every other day. Insulin secretion in response to glucose stimulation was assessed in a dynamic perfusion system. Twenty randomly handpicked islets were exposed to an initial baseline period with 1.7 mmol/L glucose for 36 minutes. It was followed by 16.7 mmol/L glucose for 42 minutes and a final period with 1.7 mmol/L glucose for 48 minutes. Fractions were collected at 6-minute intervals. The insulin concentration was determined by enzyme-linked immunosorbent ass
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