Additional file 1: of Pancreatoduodenectomy with or without prophylactic falciform ligament wrap around the gastroduodenal artery stump for prevention of pancreatectomy hemorrhage

SPIRIT 2013 checklist: recommended items to address in a clinical trial protocol and related documents. (DOC 127 kb

Real-time quantitative PCR analysis of CXCR5 and CXCL13.

<p>Real-time quantitative RT-PCR analysis of pancreatic tissues showing significantly higher levels of CXCL13 (A) and CXCR5 (B) mRNA in AIPC than in CP and PSC. RNA input was normalized to the average expression of the two housekeeping genes HPRT and cyclophilin B as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002539#s2" target="_blank"><i>Methods</i></a>. Y-axis indicates the relative amount of mRNA in a logarithmic scale for better representation. Box and whiskers blot with 10–90 percentile are shown. * indicates statistical significance (p≤0.05).</p

Histopathological features of AIPC.

<p>(A) Overview of pancreatic parenchyma with typical AIPC changes. In the center, a duct (*) with periductal mononuclear infiltration and fibrosis. Interlobular fibrosis is shown (arrows). (B) Interlobular duct with moderate periductal mononuclear infiltration, fibrosis and beginning stenosis of the lumen. (C) Higher magnification of an interlobular duct with early granulocytic epithelial lesion (GEL). Note the few neutrophils that infiltrate through the basal membrane into the epithelium (arrows). (D) Advanced GEL with abscess formation (*) in the lumen of an interlobular duct. (E) Lymphoplasmocytic inflammation of the wall of a small peripancreatic vein (*). Note the extension of the inflammatory infiltrate into the peripancreatic fat (arrows). (F) Lymphoplasmocytic inflammation of the wall of a small-sized intrapancreatic artery (*). (G) Involvement of the gallbladder in a case of AIPC. In the mucosa, a dense lymphoplasmacytic infiltrate below an almost intact epithelium. Note the thickening of the wall, due to the fibrosis and the extension of the inflammation into the deeper layers (arrows, inset). (H) Involvement of the gallbladder in a case of primary sclerosing cholangitis. Here, also a predominant lymphoplasmacytic inflammation with focal erosions of the epithelium and only superficial fibrosis. The deeper portions of the gallbladder wall are intact (inset).</p

Overview and comparison of the immunohistochemical analysis of the inflammatory infiltrate in AIPC, CP and PSC.

<p><b>AIPC total</b>: all AIPC cases are included; <b>AIPC intrapancreatic</b>: cases with no extension into the extrapancreatic biliary tract; <b>AIPC extrapancreatic</b>: cases with extension into the extrapancreatic biliary tree.</p><p>Data are expressed as mean number of positive cells/mm²±SEM.</p

Overview and comparison of the main clinical and laboratory findings in AIPC, CP, and PSC.

<p><b>DPPHR</b>: duodenum-preserving pancreatic head resection; <b>ppPD</b>: pylorus preserving pancreaticoduodenectomy; <b>PD</b>: pancreaticoduodenectomy; <b>O</b>: others; <b>AIPC total</b>: all AIPC cases are included; <b>AIPC intrapancreatic</b>: cases with no extension into the extrapancreatic biliary tract; <b>AIPC extrapancreatic</b>: cases with extension into the extrapancreatic biliary tract.</p>*<p>for these parameters, only AIPC total and CP groups are compared.</p>§<p>probably due to low sample number in the AIPC extrapancreatic group.</p><p>Data are expressed as mean±SEM, unless otherwise specified.</p

Immunohistochemical characterization of AIPC, CP and PSC.

<p>(A–H) Immunohistochemical characterization of the inflammatory infiltrate of AIPC. (A) CD20-positivity of B-lymphocytes in periductal localization and with tendency to form lymph follicles (arrow). (B) CD5-positive T-lymphocytes with diffuse arrangement around an interlobular duct. (C) Scattered CXCR5-positive cells (arrowheads) around the main pancreatic duct. (D) CXCL13-positive cells arranged in small clusters in the pancreatic tissue. (E) Numerous CD138-positive plasma cells in a diffuse arrangement in the pancreatic parenchyma. (F–G) IgG4-positive cells diffusely distributed in the peripancreatic adipose tissue (F) and in a periductal localization (G). (H) Isolated IgG4-positive cells (arrow) below the epithelium of the gallbladder in a case of AIPC with extrapancreatic biliary involvement. (I–K) Immunohistochemical characterization of the inflammatory infiltrate in CP. (I) A few CD138-positive plasma cells in a diffuse arrangement in pancreatic scar tissue. (J–K) IgG4-positive cells in large (J) and small (K) clusters in the pancreatic parenchyma. (L) Isolated IgG4-positive cells (arrow) in the gallbladder of a patient affected by PSC. (M–P) Immunohistochemical characterization of the stromal reaction in AIPC. (M) Low-power view of pancreatic tissue shows a weak (+) to moderate (++) interstitial staining for collagen I. (N) Moderate (++) to strong (+++) interlobular and periductal (inset) collagen V staining. (O) Strong (+++) diffuse and periductal (inset) α-smooth muscle actin (α-SMA) staining. (P) Diffuse interlobular and strong (+++) periductal (inset) positivity for Tenascin C. (Q–T) Immunohistochemical characterization of the stromal reaction in CP. (Q) Moderate (++) interlobular collagen I staining. (R) Very weak (−/+) interlobular collagen V staining. (S) Moderate (++) positivity for α-SMA in the interlobular connective tissue, without periductal arrangement (inset, here notice the extensive squamous cell metaplasia of the duct epithelium). (T) Moderate (++) staining for Tenascin C at the interface between the pancreatic lobules and the interlobular connective tissue.</p

Activation of PI3K/Akt but not Jak2/STAT5 signaling in PDAC cells exposed to Epo.

<p>(A) Phosphorylation status of Stat5 was assessed in serum-starved pancreatic tumor cells and hEpoR-transduced NIH/3T3 cells stimulated with 50 U/ml erythropoietin (Epo) for 10 min. Clear accumulation of pSTAT5 was observed in hEpoR-NIH/3T3 but not in mock-transduced NIH/3T3 cells or in PDAC cells, independent of the level of constitutive pSTAT5 activation. (B) Phosphorylation status of Akt was assessed in serum-starved (left panel) or non-starved (right panel) PANC-1 cells consequently stimulated with Epo at 0–50 U/ml for 15 min. Epo-enhanced pAkt phosporylation was detected only under conditions of serum starvation and could be specifically inhibited by anti-EpoR antibody (+Ab) or phosphatidylinositol-3-OH kinase (PI3K) inhibitor LY2940020 (+LY). (C) Accumulation of mRNA coding for soluble EpoR isoform (upper panel; primers: EpoR_S5/6, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023151#pone-0023151-t002" target="_blank">table 2</a>) as compared to mRNAs coding for full-length isoforms (two middle panels) and further related to expression of ß-actin (lower panel). 3′-end-based detection of EpoR mRNA was performed with antisense primers binding after (EpoR_FL1/2) or prior to a stop codon (EpoR_FL3/4) as visualized by a hEpoR plasmid carrying only the coding sequence for a full-length isoform.</p

Overview of EpoR RT-PCR primers [24].

<p>Overview of EpoR RT-PCR primers <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023151#pone.0023151-Arcasoy2" target="_blank">[24]</a>.</p

Ectopic sources of Epo in tissues of patients with pancreatic diseases.

<p>(A) Remnants of Epo-producing islets in degrading CP-parenchyma. (B) Epo-negativity of tumor cells in primary pancreatic lesion, except for sporadic focal staining observed in intracellular vacuoles of tumor cells (C, arrows and inset). Peripheral capillaries (C, arrowheads) and <i>vasa vasorum</i> (D, arrowheads) of bigger vessels frequently demonstrated Epo positivity in PDAC and CP. (E, F) In liver, cytoplasmic staining of hepatocytes was strong in areas directly bordering Epo-negative metastatic tumor cells and inflammatory infiltrates, but faded away distally, thus pointing to the spatially regulated <i>de novo</i> synthesis and creation of multiple Epo-rich niches. Insets in A, D and E depict negative (isotype IgG) controls; insets in C and F show high-magnification (×630) images of staining patterns in intracellular vacuoles of tumor cells and cytoplasm in hepatocytes.</p

Detection of EpoR-positive tumor cells in PDAC pancreata.

<p>(A) Staining of erythroid precursor cells in human bone marrow with anti-EpoR 3D10 antibody (inset, ×400) and (B) loss of staining if the anti-EpoR antibody has been pre-incubated with soluble EpoR (sol-EpoR). (C, E) Varying intensity and focal character of EpoR-immunopositivity of tumor cells in PDAC tissues (arrows) and (D) blocking of EpoR signal with sol-EpoR. (F) Sporadic EpoR-immunopositivity of non-malignant structures within a PDAC sample and blocking of EpoR signal with sol-EpoR (inset).</p