Phenotype profile CD68⁺ F4/80⁻ cells in the lungs following pancreatitis.

<p>Flow cytometry analysis of CCR2, CD11c (M1) and CD206 (M2) activation markers of lung macrophages gated for FSC/SSC (R1) and CD68⁺F4/80⁻. Significant enrichment of the number of CCR2⁺ CD11c⁻ (A) and CD206⁺ CD11c⁻ (B) cells in the lungs following pancreatitis (ligated) compared to sham controls. Bars show mean ±SEM, n = 8 per group. *<i>P</i><0.05 versus sham control, by two-tailed Student <i>t</i>-test.</p

Acute pancreatitis induces changes in local pancreatic and lung chemokine levels.

<p>Tissue homogenates from pancreas (A, C) and lungs (B, D) were evaluated by ELISA for CXCL1 (A, B) and CCL2 (C, D) levels at the indicated time points. Both CXCL1 and CCL2 levels were significantly elevated in the pancreas (A) and lungs (B) 9, 24 and 48 h after pancreatitis induction compared to sham controls. Bars show mean ± SEM, n = 10 per group. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 versus sham control, by two-tailed Student <i>t</i>-test.</p

Changes in pancreatic and lung F4/80⁺ macrophage number during acute pancreatitis.

<p>Representative photomicrographs of pancreas (A) and lung (C) sections stained for F4/80 as indicator of macrophage infiltration (brown) 24 h after pancreatitis induction (original magnification, ×20). Quantification of F4/80⁺ cells showed a significantly increased enrichment in the pancreas from 9 h following pancreatitis compared to sham controls (B). No significant difference was observed in the number of lung F4/80⁺ cells between pancreatitis and sham control groups (D). Bars show mean ± SEM, n = 10 per group. **<i>P</i><0.01, ***<i>P</i><0.001, by two-tailed Student <i>t</i>-test.</p

Morphological and physiological changes associated with acute pancreatitis and associated acute lung injury.

<p>Representative H&E images of pancreas (A, B) and lungs (C, D) 24 hours after induction of pancreatitis through BPD ligation (A, C) are depicted. Pronounced edema and necrosis observed in the pancreas after pancreatitis induction (B) compared to sham control (A). Lung section shows thickening of alveolar septa, hemorrhage and edema after pancreatitis induction (D) compared to sham control (C). Original magnification, 20×. Mice experienced a significant weight loss 24 and 48 h after induction of pancreatitis through BPD ligation compared to sham control (E). Data expressed as mean ± SEM, n = 10 per group. *<i>P</i><0.05, ***<i>P</i><0.001 versus sham control, by two-tailed Student <i>t</i>-test.</p

Changes in pancreatic and lung neutrophil number during acute pancreatitis.

<p>Representative photomicrographs of pancreas (A) and lung (C) tissue stained for NIMP-R14, showing infiltration of neutrophils (brown) 24 h after induction of pancreatitis (original magnification, ×20). The number of NIMP-R14⁺ neutrophils in the pancreas (B) and lungs (D) were significantly enriched following pancreatitis induction (ligated) compared to sham control. Lung MPO activity was significantly increased following acute pancreatitis compared to sham controls at all time points evaluated (Fig. E). Bars show mean ± SEM, n = 10 per group. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 versus sham control, by two-tailed Student <i>t</i>-test.</p

Changes in lung macrophage sub-populations during acute pancreatitis.

<p>Single cell preparations of the right lung were evaluated by flow cytometry. Dot plots from one representative experiment of sham control (A) and 24 h post pancreatitis induction (B) showing the gating strategy. Significant modulations in the percentage of R1 (C) and R2 (D) gated populations following acute pancreatitis compared to sham operated animals. Representative profiles of CD68 and F4/80 expressing cells in the R1 population of sham (D) and ligated (E) mice after 24 h are shown. A significant enrichment in the total number of R1 gated CD68⁺ F4/80⁻ cells in the right lung 9 h (F) and 24 h (G) after pancreatitis induction compared to sham controls. CD68⁺ cells were increased significantly in the immunohistochemical staining of the lung sections in the acute pancreatitis compared to sham at 9, 24 and 48 h. , n = 8 per group. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 versus control, by two-tailed Student <i>t</i>-test.</p

Quantitative Assessment of Urea In-Solution Lys-C/Trypsin Digestions Reveals Superior Performance at Room Temperature over Traditional Proteolysis at 37 °C

Urea-containing buffer solutions are generally used in proteomic studies to aid protein denaturation and solubilization during cell and tissue lysis. It is well-known, however, that urea can lead to carbamylation of peptides and proteins and, subsequently, incomplete digestion of proteins. By the use of cells and tissues that had been lysed with urea, different solution digestion strategies were quantitatively assessed. In comparison with traditional proteolysis at 37 °C, urea in-solution digestion performed at room temperature improved peptide and protein identification and quantitation and had a minimum impact on miscleavage rates. Furthermore, the signal intensities and the number of carbamylated and pyroglutamic acid-modified peptides decreased. Overall, this led to a reduction in the negative effects often observed for such modifications. Data are available via ProteomeXchange with identifier PXD009426

Immunofluorescence staining for ICAM-1 and PECAM-1 in the pancreas.

<p><b>A)</b> The expression of ICAM-1 in vascular endothelial cells in the pancreas increased after induction of severe acute pancreatitis (SAP) compared to the sham group; findings significantly attenuated by regional arterial infusion (RAI) with Asprin-Triggered Lipoxin A₄ (ATL). <b>C)</b> Fluorescence intensity for ICAM-1. The results are expressed as means and SD. The mean fluorescence intensity of ICAM-1 decreased significantly in the ATL group both at 12 and 24 h (<i>P</i><0.05 vs. SAP). <b>B)</b> The expression of PECAM-1 in vascular endothelial cells in the pancreas increased significantly after induction of SAP compared to the sham group, attenuated by RAI with ATL. <b>D)</b> Bar graph demonstrating fluorescence intensity for PECAM-1. The results are expressed as means and SD. The mean fluorescence intensity of PECAM-1 decreased significantly in the ATL group both at 12 and 24 h (<i>P</i><0.05). * <i>P</i><0.05 and ** <i>P</i><0.01, ATL vs. the SAP group.</p

Pathological scores of the pancreas and kidneys following CRAI with 5-Fu and Octreotide (Oct).

<p>Three treatment groups have lower pancreatic pathologic scores than SAP group. Combination using of 5-Fu and Oct significantly improved the pancreatic pathologic scores as compared to the single use of 5-Fu or Oct. Both Oct treatment groups demonstrated lower pathologic scores than the 5-Fu group. No significant pathologic changes were found in the sham group. *: vs. SAP group, P<0.05; ☆: vs. SAP group, P<0.01; $: 5-FU group vs. Oct group, P<0.05;$$: 5-FU group vs. Oct group, P<0.01; #: 5-FU group or Oct group vs. 5-FU + Oct group, P<0.05; ##: 5-FU group or Oct group vs. 5-FU + Oct group, P<0.01.</p

CRAI with 5-Fu and Octreotide (Oct) increased the ratio of TXB₂ to 6-k-PGF_1α.

<p>No significant difference of TXB₂ was found among the 4 experimental groups. However, 6-k-PGF1α significantly increased in all 3 treatment groups. So the ratio of TXB2 to 6-k-PGF1α was significantly lower in the treatment groups as compared to the SAP group. The sham group had normal TXB2, 6-k-PGF1α, and ratio of TXB2 to 6-k-PGF1α. *: vs. SAP group, P<0.05; ☆: vs. SAP group, P<0.01; $: 5-FU group vs. Oct group, P<0.05;$$: 5-FU group vs. Oct group, P<0.01; #: 5-FU group or Oct group vs. 5-FU + Oct group, P<0.05; ##: 5-FU group or Oct group vs. 5-FU + Oct group, P<0.01.</p