Supplementary MaterialsESM Strategies: (PDF 96 kb) 125_2014_3416_MOESM1_ESM

Supplementary MaterialsESM Strategies: (PDF 96 kb) 125_2014_3416_MOESM1_ESM. mice had been treated with AAV8 vectors expressing the international antigen luciferase. Islet regeneration and irritation was noticed at 3, 6, 10 and 22?weeks post-AAV delivery. LEADS TO youthful C57BL/6J mice, sturdy mobile and humoral defense replies had been created towards antigen-expressing beta cells, leading to reduced beta cell mass. This is accompanied by beta cell mass replenishment, along with improved proliferation of insulin-positive cells, recruitment of nestin/Compact disc34-positive endothelial cells, displacement of alpha mobilisation and cells of cytoplasmic neurogenin 3-positive cells. Mice with recovering beta cells showed reduced or regular fasting blood sugar amounts and faster blood sugar clearance than settings. Although aged mice proven similar reactions to the procedure, they exhibited significant islet skin damage and fluctuations in blood sugar amounts primarily, indicating that beta cell regeneration can be slower in aged mice. Conclusions/interpretation Our hit-and-run, beta cell-targeted antigen manifestation system has an possibility to monitor the effect of single-round immunological beta cell damage in pets with diverse hereditary backgrounds or ageing position. Electronic supplementary materials The web version of the content (doi:10.1007/s00125-014-3416-4) contains peer-reviewed but unedited supplementary materials, which is open to authorised users. ELISpot Mouse Arranged (BD Pharmingen, NORTH PARK, CA, USA). Splenocytes had been added to duplicate wells at a density of 0.1??106, 0.5??106 or 1??106 cells per well along with DMEM-10 with or without 2.0?g/ml ovalbumin peptide or 2.0?g/ml firefly luciferase epitope peptide. The ovalbumin T cell-reactive peptide sequence (SIINFEKL) [17] and firefly luciferase T cell-reactive epitope (LMYRFEEEL) [18] were synthesised by GenScript (Piscataway, New Jersey, USA). Immunostaining All immunostaining was conducted as previously described [16]. Antibodies and the concentrations used for immunocytochemistry are described in the ESM Methods. Insulitis scoring The insulitis score was determined by following established criteria [19]. Three 7?m thick whole head-to-tail pancreatic sections (each 200?m in depth) were collected per animal and co-stained with anti-insulin and anti-cluster of differentiation 45 (CD45) antibodies, with DAPI labelling. Insulin- and glucagon-positive area analysis Pancreatic sections were prepared and the insulin-positive area was quantified by using the formula: Percentage insulin-positive area?=?insulin-positive area/total tissue area??100 [16]. Mouse pancreatic RNA extraction Pancreases were isolated and three tissue sections (~20?mg) were immediately processed using an RNeasy Plus Mini Kit (Qiagen, Limburg, Netherlands). RT-PCR and quantitative PCR One microgram of total RNA was used to synthesise cDNA (EcoDry Premix, Clontech Laboratories, Mountain View, California, USA). Quantitative PCR was conducted using SYBR green-based expression analysis in QuantiTect Primer Assays (Qiagen). Firefly luciferase expression was determined using SYBR green quantitative PCR with primers based on a 140?bp segment of the luciferase gene: Forward FFLuc_qPCR_F, 5-GCTATTCTGATTACACCCGAGG-3; Reverse FFLuc_qPCR_R 5-TCCTCTGACACATAATTCGCC-3. Sample size and statistical analysis Groups were compared by unpaired Students test, and data are expressed as means??SEM. Significance was set at gene (Fig.?1a, ESM Fig.?1a, b). Mice were i.p. injected with the AAV8 vectors, and luciferase expression was monitored 2?weeks post infection (p.i.). AAV vectors containing mIP2 [15] exhibited potent, pancreas-restricted luciferase expression (Fig.?1b). When the mIP2-AAV8 vector was used to deliver the EmGFP gene (Fig.?1a), i.p. administration of AAV8 vectors (2??1011 genome copies/mouse) resulted in selective EmGFP expression in insulin-positive beta cells (Fig.?1c), demonstrating beta cell-specific transgene expression via the mIP2-AAV8 vector system. When EmGFP transduction efficiency was assessed from 15 random islets, the proportion of EmGFP-positive islet mass reached up to 66% (average 47.8%), relative to the insulin-positive area (and was comparable between treated and untreated mice. In contrast, Dovitinib lactate expression Dovitinib lactate of and transcripts in inflamed pancreas at 3?weeks p.i. (ESM Fig.?8b). Recruitment Dovitinib lactate of CD34-positive cells was evident at the same time Dovitinib lactate point (ESM Fig.?8c). Indeed, many NES-positive cells in and around damaged islets were also positive for CD34 and endoglin (CD105; ESM Fig.?8d), suggesting that NES/CD34 double-positive cells are vascular endothelial cells. Some NES-positive cells did not express CD34 or CD105 (ESM Fig.?8d). Similar to the biased distribution of NGN3- and glucagon-positive cells during islet recovery, more NES-positive cells COG3 were found between recovering islets and ducts at 10?weeks p.i. (ESM Fig.?8e), although the NES-positive cells were distinct from glucagon- and NGN3-expressing cells (ESM Fig.?8e,.