For colocalization experiments, islets were enzymatically digested into single cells with a trypsin-like enzyme (12605-01; TrypLE selleck catalog Express; Invitrogen, Carlsbad, CA) and cytocentrifuged onto glass slides. In situ hybridization was performed using the Quantigene ViewRNA technique, based on multiple oligonucleotide probes and branched-DNA signal amplification technology, according to the manufacturer’s instructions (Affymetrix, Santa Clara, CA). The probe set used was designed to hybridize the H1N1/A/New Caledonia/20/99 virus (GenBank sequence “type”:”entrez-nucleotide”,”attrs”:”text”:”DQ508858.1″,”term_id”:”94483620″,”term_text”:”DQ508858.1″DQ508858.1). Due to sequence homology in the region covered by the probes, the same set also recognized the H3N2 virus RNA, as confirmed in pilot experiments.
To identify cell types within islets, the following Quantigene probes were used: insulin for beta cells (INS gene; NCBI reference sequence “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_000207″,”term_id”:”109148525″,”term_text”:”NM_000207″NM_000207), alpha-amylase 1 for exocrine cells (AMY1A gene; NCBI reference sequence “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_004038″,”term_id”:”57014084″,”term_text”:”NM_004038″NM_004038), and cytokeratin 19 for duct cells (KRT19 gene; NCBI reference sequence “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_002276″,”term_id”:”131412244″,”term_text”:”NM_002276″NM_002276). Quantification of cells positive for each probe was performed within 8 randomly chosen fields using the IN Cell Investigator software (GE Healthcare United Kingdom Ltd.
). Determination of insulin secretion in infected islets. Aliquots of 100 islet equivalents (uninfected or infected with H1N1/A/New Caledonia/20/99 and H3N2/A/Wisconsin/67/05) per column were loaded onto Sephadex G-10 columns with medium at low glucose concentration (2 mM) and preincubated at 37��C for 1 h. After preincubation, the islets were exposed to sequential 1-h incubations at low (2 mM) and high (20 mM) glucose concentrations. Supernatants were collected with protease inhibitor cocktail (Roche Biochemicals, Indianapolis, IN) and stored at ?80��C at the end of each incubation. The insulin content was determined with an insulin enzyme-linked immunoassay kit (Mercodia AB, Uppsala, Sweden) following manufacturer’s instructions.
Insulin secretion indices were calculated as the ratio between the insulin concentration at the end of high-glucose incubation and the insulin concentration at the end of low-glucose incubation. Cytokine expression profile. The capability of H1N1 and H3N2 viruses to induce cytokine expression in human pancreatic islets was measured using multiplex bead-based assays Cilengitide based on xMAP technology (Bio-Plex; Bio-Rad Laboratories, Hercules, CA). The parallel wells of pancreatic islets were infected with viruses or were mock infected.