Then, 250 ��l of BD FACS lysing solution 1�� (BD Biosciences, San Jose, CA, USA) was added, the cells were incubated for 10 minutes, and the stained cells were washed, resuspended, and analyzed for three-color immunofluorescence by flow cytometry (FACS Aria, Becton Dickinson, Franklin Lakes, NJ, USA). Cells with CD14high expression were selected from a side scatter vs CD14/FITC dot plot. Temsirolimus mTOR inhibitor From this gated region, cells expressing TREM-1/PE or HLA-DR/PerCPCy5 were selected, using isotype controls as reference, and MFI or percentage values of the selected cells were taken. At least 5000 events in the CD14high region were analyzed. Data analysis was performed using FACS Diva software version 4.1 (Becton Dickinson, Franklin Lakes, NJ, USA).

Soluble TREM-1 and cytokine quantificationThe concentration of soluble TREM-1 was measured in previously aliquoted serum samples using an ELISA kit (R&D Systems Minneapolis, MN, USA), according to the manufacturer’s protocol. The concentrations of IL-6 and IL-10 were measured in previously aliquoted serum samples using ELISA kits (BD Biosciences Pharmingen, San Jose, CA, USA), according to the manufacturers’ protocols.Statistical analysisData are represented on box and whiskers graphs, which depict median and 5% to 95% percentiles. Data were analyzed by Kruskal-Wallis test with Dunn’s post hoc test using GraphPad Prism version 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). Statistical significance was set at P < 0.05.ResultsDemographic data of patients and controlsTwenty-nine patients with AP were included in this study.

Their average age was 43 years (range, 17 to 79 years); 18 were women and 11 were men. Twenty-two patients had AP of biliary origin (75%), one patient had AP caused by alcohol consumption (4%), one patient had AP caused by hypertriglyceridemia (4%), and five patients had idiopathic AP (17%). Eighteen patients had mild AP (62%), and 11 patients had severe AP (38%). One patient with mild AP was discharged from the hospital due to complete resolution of AP before the third blood sample was taken.Five patients with severe AP died; one patient developed respiratory insufficiency and died two days after admission to the hospital; this patient died before the second blood sample had been collected. The other four patients with severe AP that died developed infections. One patient had pancreatic abscess caused by Enterococcus faecalis (diagnosed on day 23, deceased on day 41). Another patient with AP from biliary origin developed acute cholecystitis complicated with emphysematous cholecystitis 2 days after the onset Anacetrapib of AP, and during the emergency laparotomy, purulent material was found in abdominal cavity and pancreas inflammation was confirmed (deceased on day 7).