PRSS2 regulates EMT and metastasis via MMP-9 in gastric cancer.

Serine protease 2 (PRSS2) is upregulated in gastric cancer tissues, correlates with poor prognosis and promotes migration and invasion of gastric cancer cells. However, the exact mechanism by which PRSS2 promotes metastasis in gastric cancer is unclear. We examined serum PRSS2 levels in healthy controls and gastric cancer patients by enzyme linked immunosorbent assay (ELISA) and analyzed the correlation between PRSS2 serum level with the clinicopathological characteristics of gastric cancer patients and matrix metalloproteinase-9 (MMP-9) expression. A lentiviral MMP-9 overexpression vector was constructed and used to transfect gastric cancer cells with stable silencing of PRSS2, and migration, invasion and epithelial-mesenchymal transition (EMT) of gastric cancer cells were examined. High serum PRSS2 levels were detected in gastric cancer patients and associated with lymphatic metastasis and TNM stage. Serum PRSS2 was positively correlated with serum MMP-9 level. PRSS2 silencing inhibited EMT, and knock-down of PRSS2 partially abrogated cell metastasis and EMT caused by overexpression of MMP-9. These results suggest that PRSS2 promotes the migration and invasion of gastric cancer cells through EMT induction by MMP-9. Our findings suggest that PRSS2 may be a potential early diagnostic marker and therapeutic target of gastric cancer.

High expression of serine protease 2 (PRSS2) associated with invasion, metastasis, and proliferation in gastric cancer.

BACKGROUND: Accumulating evidence indicates that the occurrence and development of tumors are related to the activation of oncogenes and the inactivation of tumor suppressor genes caused by epigenetic mechanisms. However, the function of serine protease 2 (PRSS2) in gastric cancer (GC) is still unknown. Our study aimed to find a regulation network involved in GC. METHODS: The mRNA data (GSE158662 and GSE194261) of GC and normal tissues were downloaded from the Gene Expression Omnibus (GEO) dataset. Differential expression analysis was performed using R software, and Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was conducted by using Xiantao software. Besides, we used Quantitative Real-time PCR (qPCR) to verify our conclusions. After gene knockdown, cell migration and CCK-8 experiment were carried out to detect the effect of gene on cell proliferation and invasion. RESULTS: Totally, 412 differentially expressed genes (DEGs) were identified from GSE158662 and 94 DEGs were identified from GSE196261. Km-plot database results indicated that PRSS2 exhibited high diagnosis worth for GC. Gene functional annotation enrichment analysis revealed that these hub mRNAs were mainly taken part in the process of tumorigenesis and development. Besides, vitro experiments showed that down-regulation of PRSS2 gene reduced the proliferation and invasion ability of GC cells. CONCLUSIONS: Our results indicated that PRSS2 may play vital roles in the carcinogenesis and progression of GC and can be potential biomarkers for patients with GC.

Double deficiency of cathepsin B and L in the mouse pancreas alters trypsin activity without affecting acute pancreatitis severity.

BACKGROUND: Premature intracellular trypsinogen activation has long been considered a key initiator of acute pancreatitis (AP). Cathepsin B (CTSB) activates trypsinogen, while cathepsin L (CTSL) inactivates trypsin(ogen), and both proteins play a role in the onset of AP. METHODS: AP was induced by 7 hourly intraperitoneal injections of cerulein (50 µg/kg) in wild-type and pancreas-specific conditional Ctsb knockout (CtsbΔpan), Ctsl knockout (CtslΔpan), and Ctsb;Ctsl double-knockout (CtsbΔpan;CtslΔpan) mice. Pancreatic samples were collected and analyzed by histology, immunohistochemistry, real-time PCR, and immunoblots. Trypsin activity was measured in pancreatic homogenates. Peripheral blood was collected, and serum amylase activity was measured. RESULTS: Double deletion of Ctsb and Cstl did not affect pancreatic development or mouse growth. After 7 times cerulein injections, double Ctsb and Ctsl deficiency in mouse pancreases increased trypsin activity to the same extent as that in Ctsl-deficient mice, while Ctsb deficiency decreased trypsin activity but did not affect the severity of AP. CtsbΔpan;CtslΔpan mice had comparable serum amylase activity and histopathological changes and displayed similar levels of proinflammatory cytokines, apoptosis, and autophagy activity compared with wild-type, CtsbΔpan, and CtslΔpan mice. CONCLUSION: Double deletion of Ctsb and Ctsl in the mouse pancreas altered intrapancreatic trypsin activity but did not affect disease severity and inflammatory response after cerulein-induced AP.

AXL and MERTK receptor tyrosine kinases inhibition protects against pancreatic necrosis via selectively limiting CXCL2-related neutrophil infiltration.

BACKGROUND: Acute pancreatitis (AP) was initiated within pancreatic parenchymal cells and sustained by uncontrolled inflammatory responses. AXL and MERTK receptor tyrosine kinases play a crucial role in negatively regulating the innate immunity. Therefore, this study aimed to investigate the role and underlying mechanism of AXL and MERTK in AP. METHODS: Experimental AP was induced by ten hourly intraperitoneal administration of caerulein in global, hematopoietic- and pancreas-specific Axl and Mertk deficient mice. Pancreatitis severity was assessed biochemically and histologically. Pancreatic transcriptomics and pancreatic infiltrating immune cells were profiled. Some mice were given R428, an antagonist of AXL and MERTK. AXL and MERTK in peripheral leukocytes were measured by flow cytometry. FINDINGS: The levels of AXL and MERTK in pancreatic tissue and pancreatic CD45+ cells were dynamically altered at 6 h and 12 h after the 1st injection of caerulein. Global and hematopoietic-specific, but not pancreas-specific deletion of Axl and Mertk protected against pancreatic necrosis and trypsinogen activation. Pancreatic transcriptomic analysis revealed that differentially expressed gene signatures were mainly related to metabolic and inflammatory pathways. Furthermore, deletion or inhibition of Axl and Mertk selectively inhibited pancreatic neutrophil infiltration, which was primarily related to CXCL2 secreted by pro-inflammatory macrophages. Increased levels of MERTK in peripheral leukocytes were correlated with more severe form of AP. INTERPRETATION: Our findings reveal that specific AXL/MERTK antagonist may be a novel and potential early treatment for AP and the levels of MERTK in peripheral leukocytes may be a promising biomarker for predicting pancreatic severity in patients with AP. FUNDING: National Natural Science Foundation of China, Shanghai Natural Science Foundation, a Shanghai Young Talent Award and a Shanghai Young Orient Scholar Award. RESEARCH IN CONTEXT: Evidence before this study Acute pancreatitis (AP) is a common inflammatory disorder of the exocrine pancreas, the severity of which was determined by the extent of pancreatic necrosis, with no targeted therapy. AP was initiated by signals within pancreatic parenchymal cells and sustained by uncontrolled innate immune responses. One of the three crucial regulatory roles for AXL and MERTK is to negatively regulate innate immune responses. Added value of this study Global and hematopoietic-, but not pancreas-specific Axl and Mertk deficiency protected against pancreatitis, primarily pancreatic necrosis. Deletion of Axl and Mertk selectively inhibited pancreatic neutrophil infiltration that was related to CXCL2 secreted by pro-inflammatory macrophages. AXL and MERTK antagonist similarly reduced pancreatitis severity via limiting CXCL2-mediated pancreatic neutrophil infiltration. Higher levels of MERTK, but not AXL in peripheral leukocytes were correlated with more severe form of acute pancreatitis. Implications of all the available evidence A specific AXL/MERTK antagonist may be a novel and potential early treatment for AP. The level of MERTK on peripheral leukocytes may be a promising biomarker for predicting disease severity in patients with AP.

Effects of tRNA-derived fragments and microRNAs regulatory network on pancreatic acinar intracellular trypsinogen activation.

Acute pancreatitis (AP) is a common gastrointestinal disease with substantial morbidity and mortality. Pancreatic acinar intracellular trypsinogen activation (PAITA) is an important event in the early stage of AP. The present study aimed to investigate the effects of tRNA-derived fragments (tRFs) and the microRNA regulatory network on pancreatic acinar intracellular trypsinogen activation (PAITA) and identify novel key targets in AP. Taurolithocholic acid 3-sulfate (TLC-S)-treated AR42J cells were used to establish a PAITA model. Twenty differentially expressed tRFs and 35 DE microRNAs were identified in PAITA through gene sequencing. Based on these genes, we established the tRF-mRNA and microRNA-mRNA regulatory networks by using bioinformatics methods. The networks revealed 29 hub microRNAs (e.g., Let-7 family, miR-21-3p.) and 19 hub tRFs (e.g., tRF3-Thr-AGT, i-tRF-Met-CAT) in PAITA. GO analysis showed that the functions of the two networks were similar and mainly enriched in RNA splicing, mRNA processing, and so on. tRF3-Thr-AGT, targeting Btg2, Cd44, Zbp1, etc., was significantly decreased in PAITA. Moreover, the trypsinogen activation level was increased significantly in the tRF3-Thr-AGT deficiency groups, but rescued by tRF3-Thr-AGT mimics. The results revealed that downregulated tRF3-Thr-AGT was involved in PAITA. This study provides potential novel targets for researching the underlying mechanisms of AP.

A mutant equipped with a regenerated disulphide for the missing His loop of a serine protease zymogen in the horseshoe crab coagulation cascade.

The lipopolysaccharide (LPS)-triggered coagulation cascade in horseshoe crabs is composed of three zymogens belonging to the trypsinogen family: prochelicerase C, prochelicerase B (proB) and the proclotting enzyme (proCE). Trypsinogen-family members contain three conserved disulphides located around the active site. While it is known that proB evolutionarily lost one of the disulphides, the His-loop disulphide, the roles of the missing His-loop disulphide in proB remain unknown. Here, we prepared a proB mutant, named proB-murasame, equipped with a regenerated His-loop disulphide. The activation rate by upstream α-chelicerase C for proB-murasame was indistinguishable from that for wild-type (WT) proB. The resulting protease chelicerase B-murasame exhibited an 8-fold higher kcat value for downstream proCE than WT chelicerase B, whereas the Km value of chelicerase B-murasame was equivalent to that of WT chelicerase B. WT serpins-1, -2 and -3, identified as scavengers for the cascade, had no reactivity against WT chelicerase B, whereas chelicerase B-murasame was inhibited by WT serpin-2, suggesting that WT chelicerae B may trigger as-yet-unsolved phenomena after performing its duty in the cascade. The reconstituted LPS-triggered cascade containing proB-murasame exhibited ∼5-fold higher CE production than that containing WT proB. ProB-murasame might be used as a high value-adding reagent for LPS detection.

Trypsinogen and chymotrypsinogen: the mysterious hyper-reactivity of selected cysteines is still present after their divergent evolution.

An enigmatic and never described hyper-reactivity of most of the cysteines resident in the reduced, molten globule-like intermediate of a few proteins has been recently discovered. In particular, all ten cysteines of chymotrypsinogen showed hundred times increased reactivity against hydrophobic reagents. A single cysteine (Cys1) was also found thousand times more reactive toward GSSG, making speculate that a single glutathionylation could represent the primordial event of its oxidative folding. In the present study, we compare these kinetic properties with those present in trypsinogen taken in its reduced, molten globule-like intermediate and identify the origin of these unusual properties. Despite the divergent evolution of these two proteins, the different amount of disulfides and the very different 3D localization of three disulfides, their hyper-reactivity toward hydrophobic thiol reagents and disulfides is very similar. Mass spectrometry identifies two cysteines in trypsinogen, Cys148 and Cys197, 800 times more reactive toward GSSG than an unperturbed protein cysteine. These results point toward a stringent and accurate preservation of these peculiar kinetic properties during a divergent evolution suggesting some important role, which at the present can only be hypothesized. Similar extraordinary hyper-reactivity has been found also in albumin, ribonuclease, and lysozyme confirming that it cannot be considered a kinetic singularity of a single protein. Interestingly, the very flexible and fluctuating structures like those typical of the molten globule status prove capable of enabling sophisticated actions typical of enzymes such as binding to GSSG with relevant specificity and high affinity (KD  = 0.4 mm) and accelerating the reaction of its cysteines by thousands of times.

Mouse model suggests limited role for human mesotrypsin in pancreatitis.

Mesotrypsin is a low-abundance human trypsin isoform with a unique evolutionary mutation that conferred resistance to trypsin inhibitors and restricted substrate specificity. Mesotrypsin degrades the serine protease inhibitor Kazal type 1 (SPINK1) and thereby might increase risk for pancreatitis. Here, we report a mouse model designed to test the role of mesotrypsin in pancreatitis. We introduced the human mesotrypsin evolutionary signature mutation into mouse cationic trypsinogen (isoform T7), resulting in a Gly to Arg change at the corresponding position 199. In biochemical experiments using purified proteins, the p.G199R T7 mutant recapitulated all salient features of human mesotrypsin. T7G199R mice developed normally with no spontaneous pancreatitis or other obvious phenotypic changes. Cerulein-induced acute pancreatitis in C57BL/6N and T7G199R mice showed similar severity with respect to inflammatory parameters and acinar cell necrosis while plasma amylase activity was higher in T7G199R mice. Neither SPINK1 degradation nor elevated intrapancreatic trypsin activation was apparent in T7G199R mice. The results indicate that in T7G199R mice the newly created mesotrypsin-like activity has no significant impact on cerulein-induced pancreatitis. The observations suggest that human mesotrypsin is unimportant for pancreatitis; a notion that is consistent with published human genetic studies.

Pancreatic Inflammation and Proenzyme Activation Are Associated With Clinically Relevant Postoperative Pancreatic Fistulas After Pancreas Resection.

OBJECTIVE: We investigated the activation of pancreatic proenzymes and signs of peripancreatic inflammation in patients with clinically relevant postoperative pancreatic fistulas (POPFs). SUMMARY BACKGROUND DATA: An increase of systemic amylase concentration was associated with POPFs. This suggested parallels in the pathomechanisms between the development of POPFs and pancreatitis. METHODS: Trypsinogen, procathepsin B, and IL-6 concentrations as well as cathepsin B, myeloperoxidase and trypsin activities were determined throughout the first 7 postoperative days in drain fluids of 128 consecutive patients after pancreas resection. Histology and immunohistochemistry were performed in pancreatic specimens after total pancreatectomy due to complications and after placing experimental pancreatic sutures in the pancreatic tail of C57/Bl6 mice. RESULTS: Trypsin activity, cathepsin B activity and myeloperoxidase activity on the first postoperative day were elevated and predictive for clinically relevant pancreatic fistulas. Drain fluid stabilized trypsin activity and prevented the activation of the cascade of digestive enzymes. Leukocytes were the source of cathepsin B in drain fluid. Findings differed between fistulas after distal pancreatectomy and pancreatoduodenectomy. Immunohistochemistry of the pancreatic remnant revealed an inflammatory infiltrate expressing cathepsin B, independent of the presence of pancreatic fistulas. The infiltrate could be reproduced experimentally by sutures placed in the pancreatic tail of C57/Bl6 mice. CONCLUSIONS: Trypsinogen activation, increased cathepsin B activity and inflammation around the pancreato-enteric anastomosis on post operative day 1 are associated with subsequent clinically relevant POPFs after pancreatoduodenectomy. The parenchymal damage seems to be induced by placing sutures in the pancreatic parenchyma during pancreatic surgery.

Roles of Autophagy and Pancreatic Secretory Trypsin Inhibitor in Trypsinogen Activation in Acute Pancreatitis.

The focus of the review is on roles of autophagy and pancreatic secretory trypsin inhibitor (PSTI), an endogenous trypsin inhibitor, in trypsinogen activation in acute pancreatitis. Acute pancreatitis is a disease in which tissues in and around the pancreas are autodigested by pancreatic digestive enzymes. This reaction is triggered by the intrapancreatic activation of trypsinogen. Autophagy causes trypsinogen and cathepsin B, a trypsinogen activator, to colocalize within the autolysosomes. Consequently, if the resultant trypsin activity exceeds the inhibitory activity of PSTI, the pancreatic digestive enzymes are activated, and they cause autodigestion of the acinar cells. Thus, autophagy and PSTI play important roles in the development and suppression of acute pancreatitis, respectively.

A model-based economic evaluation of four newborn screening strategies for cystic fibrosis in Flanders, Belgium.

Objectives: The most cost-effective newborn screening strategy for cystic fibrosis (CF) for Flanders, Belgium, is unknown. The aim of this study was to assess the cost-effectiveness of four existing newborn screening strategies for CF: IRT-DNA (immunoreactive trypsinogen, cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation analysis), IRT-PAP (pancreatitis-associated protein), IRT-PAP-DNA, and IRT-PAP-DNA-EGA (extended CFTR gene analysis).Methods: Using data from published literature, the cost-effectiveness of the screening strategies was calculated for a hypothetical cohort of 65,606 newborns in Flanders, Belgium. A healthcare payer perspective was used, and the direct medical costs associated with screening were taken into account. The robustness of the model outcomes was assessed in sensitivity analyses.Results: The IRT-PAP strategy was the most cost-effective strategy in terms of costs per CF case detected (€9314 per CF case detected). The IRT-DNA strategy was more costly (€13,966 per CF case detected), but with an expected sensitivity of 93.4% also the most effective strategy, and was expected to detect 2.2 more cases of CF than the IRT-PAP strategy. The incremental cost-effectiveness ratio of IRT-DNA vs. IRT-PAP was €54,180/extra CF case detected. The IRT-PAP-DNA strategy and the IRT-PAP-DNA-EGA strategy were both strongly dominated by the IRT-PAP strategy.Conclusion: The IRT-PAP strategy was the most cost-effective strategy in terms of costs per CF case detected. However, the strategy did not fulfil the European Cystic Fibrosis Society guidelines for sensitivity and positive predictive value. Therefore, the more costly and more effective IRT-DNA strategy may be the most appropriate newborn screening strategy for Flanders.

Pancreatic (pro)enzymes treatment suppresses BXPC-3 pancreatic Cancer Stem Cell subpopulation and impairs tumour engrafting.

Cancer stem cells (CSCs) subpopulation within the tumour is responsible for metastasis and cancer relapse. Here we investigate in vitro and in vivo the effects of a pancreatic (pro)enzyme mixture composed of Chymotrypsinogen and Trypsinogen (PRP) on CSCs derived from a human pancreatic cell line, BxPC3. Exposure of pancreatic CSCs spheres to PRP resulted in a significant decrease of ALDEFLUOR and specific pancreatic CSC markers (CD 326, CD 44 and CxCR4) signal tested by flow cytometry, further CSCs markers expression was also analyzed by western and immunofluorescence assays. PRP also inhibits primary and secondary sphere formation. Three RT2 Profiler PCR Arrays were used to study gene expression regulation after PRP treatment and resulted in, (i) epithelial-mesenchymal transition (EMT) inhibition; (ii) CSCs related genes suppression; (iii) enhanced expression of tumour suppressor genes; (iv) downregulation of migration and metastasis genes and (v) regulation of MAP Kinase Signalling Pathway. Finally, in vivo anti-tumor xenograft studies demonstrated high anti-tumour efficacy of PRP against tumours induced by BxPC3 human pancreatic CSCs. PRP impaired engrafting of pancreatic CSC's tumours in nude mice and displayed an antigrowth effect toward initiated xenografts. We concluded that (pro)enzymes treatment is a valuable strategy to suppress the CSC population in solid pancreatic tumours.

Pancreatitis-Induced Depletion of Syntaxin 2 Promotes Autophagy and Increases Basolateral Exocytosis.

BACKGROUND & AIMS: Pancreatic acinar cells are polarized epithelial cells that store enzymes required for digestion as inactive zymogens, tightly packed at the cell apex. Stimulation of acinar cells causes the zymogen granules to fuse with the apical membrane, and the cells undergo exocytosis to release proteases into the intestinal lumen. Autophagy maintains homeostasis of pancreatic acini. Syntaxin 2 (STX2), an abundant soluble N-ethyl maleimide sensitive factor attachment protein receptor in pancreatic acini, has been reported to mediate apical exocytosis. Using human pancreatic tissues and STX2-knockout (KO) mice, we investigated the functions of STX2 in zymogen granule-mediated exocytosis and autophagy. METHODS: We obtained pancreatic tissues from 5 patients undergoing surgery for pancreatic cancer and prepared 80-µm slices; tissues were exposed to supramaximal cholecystokinin octapeptide (CCK-8) or ethanol and a low concentration of CCK-8 and analyzed by immunoblot and immunofluorescence analyses. STX2-KO mice and syntaxin 2+/+ C57BL6 mice (controls) were given intraperitoneal injections of supramaximal caerulein (a CCK-8 analogue) or fed ethanol and then given a low dose of caerulein to induce acute pancreatitis, or saline (controls); pancreata were isolated and analyzed by histology and immunohistochemistry. Acini were isolated from mice, incubated with CCK-8, and analyzed by immunofluorescence microscopy or used in immunoprecipitation experiments. Exocytosis was quantified using live-cell exocytosis and Ca2+ imaging analyses and based on formation of exocytotic soluble N-ethyl maleimide sensitive factor attachment protein receptor complexes. Dysregulations in autophagy were identified using markers, electron and immunofluorescence microscopy, and protease activation assays. RESULTS: Human pancreatic tissues and dispersed pancreatic acini from control mice exposed to CCK-8 or ethanol plus CCK-8 were depleted of STX2. STX2-KO developed more severe pancreatitis after administration of supramaximal caerulein or a 6-week ethanol diet compared with control. Acini from STX2-KO mice had increased apical exocytosis after exposure to CCK-8, as well as increased basolateral exocytosis, which led to ectopic release of proteases. These increases in apical and basolateral exocytosis required increased formation of fusogenic soluble N-ethyl maleimide sensitive factor attachment protein receptor complexes, mediated by STX3 and STX4. STX2 bound ATG16L1 and prevented it from binding clathrin. Deletion of STX2 from acini increased binding of AT16L1 to clathrin, increasing formation of pre-autophagosomes and inducing autophagy. Induction of autophagy promoted the CCK-8-induced increase in autolysosome formation and the activation of trypsinogen. CONCLUSIONS: In studies of human pancreatic tissues and pancreata from STX2-KO and control mice, we found STX2 to block STX3- and STX4-mediated fusion of zymogen granules with the plasma membrane and exocytosis and prevent binding of ATG16L1 to clathrin, which contributes to induction of autophagy. Exposure of pancreatic tissues to CCK-8 or ethanol depletes acinar cells of STX2, increasing basolateral exocytosis and promoting autophagy induction, leading to activation of trypsinogen.

Role of the c-Jun N-terminal kinase signaling pathway in the activation of trypsinogen in rat pancreatic acinar cells.

Bile acid causes trypsinogen activation in pancreatic acinar cells through a complex process. Additional research is required to further elucidate which signaling pathways affect trypsinogen activation when activated. the changes in the whole­genome expression profile of AR42J cells under the effect of taurolithocholic acid 3­sulfate (TLC­S) were investigated. Furthermore, gene groups that may play a regulatory role were analyzed using the modular approach of biological networks. The aim of the present study was to improve our understanding of the changes in TLC­S­stimulated AR42J cells through a genetic functional modular analysis. whole­genome expression profile chip arrays were applied to detect genes that were differentially expressed in pancreatic acinar AR42J cells treated with TLC­S for 20 min. Based on the human protein reference database, a protein­protein interaction network was obtained, which was then processed by CFinder software to derive 14 modules. Among these 14 modules, the gene ontology biological processes enrichment analysis identified two as modules of interest. Kyoto encyclopedia of genes and genomes map analysis revealed that MAP2K4, MAPK8 and FLNA are part of the c-Jun N-terminal kinase (JNK) pathway. The JNK signaling pathway is involved in regulating trypsinogen activation in rat pancreatic AR42J cells. Next, a regulatory network of seven kinase inhibitors was constructed. SP600125 is an ATP­competitive, efficient, selective and reversible inhibitor of JNK. the results were verified by four sets of experiments and demonstrated that trypsinogen activation is mediated by the JNK signaling pathway in the pathogenesis of acute pancreatitis (AP). The present study provided a useful reference for better understanding the pathogenesis of AP and identifying new targets to regulate trypsinogen activation, in addition to providing valuable information for the treatment of AP.

Cathepsin B-Mediated Activation of Trypsinogen in Endocytosing Macrophages Increases Severity of Pancreatitis in Mice.

BACKGROUND & AIMS: Acute pancreatitis is characterized by premature intracellular activation of digestive proteases within pancreatic acini and a consecutive systemic inflammatory response. We investigated how these processes interact during severe pancreatitis in mice. METHODS: Pancreatitis was induced in C57Bl/6 wild-type (control), cathepsin B (CTSB)-knockout, and cathepsin L-knockout mice by partial pancreatic duct ligation with supramaximal caerulein injection, or by repetitive supramaximal caerulein injections alone. Immune cells that infiltrated the pancreas were characterized by immunofluorescence detection of Ly6g, CD206, and CD68. Macrophages were isolated from bone marrow and incubated with bovine trypsinogen or isolated acinar cells; the macrophages were then transferred into pancreatitis control or cathepsin-knockout mice. Activities of proteases and nuclear factor (NF)-κB were determined using fluorogenic substrates and trypsin activity was blocked by nafamostat. Cytokine levels were measured using a cytometric bead array. We performed immunohistochemical analyses to detect trypsinogen, CD206, and CD68 in human chronic pancreatitis (n = 13) and acute necrotizing pancreatitis (n = 15) specimens. RESULTS: Macrophages were the predominant immune cell population that migrated into the pancreas during induction of pancreatitis in control mice. CD68-positive macrophages were found to phagocytose acinar cell components, including zymogen-containing vesicles, in pancreata from mice with pancreatitis, as well as human necrotic pancreatic tissues. Trypsinogen became activated in macrophages cultured with purified trypsinogen or co-cultured with pancreatic acini and in pancreata of mice with pancreatitis; trypsinogen activation required macrophage endocytosis and expression and activity of CTSB, and was sensitive to pH. Activation of trypsinogen in macrophages resulted in translocation of NF-kB and production of inflammatory cytokines; mice without trypsinogen activation (CTSB-knockout mice) in macrophages developed less severe pancreatitis compared with control mice. Transfer of macrophage from control mice to CTSB-knockout mice increased the severity of pancreatitis. Inhibition of trypsin activity in macrophages prevented translocation of NF-κB and production of inflammatory cytokines. CONCLUSIONS: Studying pancreatitis in mice, we found activation of digestive proteases to occur not only in acinar cells but also in macrophages that infiltrate pancreatic tissue. Activation of the proteases in macrophage occurs during endocytosis of zymogen-containing vesicles, and depends on pH and CTSB. This process involves macrophage activation via NF-κB-translocation, and contributes to systemic inflammation and severity of pancreatitis.

Interferon-γ Aggravated L-Arginine-Induced Acute Pancreatitis in Sprague-Dawley Rats and Its Possible Mechanism: Trypsinogen Activation and Autophagy Up-regulation.

OBJECTIVES: It has been confirmed that the initiation of acute pancreatitis (AP) involves intracellular trypsinogen activation and local cytokines release during its early stage. The former is related to autophagic disorder, and the latter is resulting from nuclear factor-κB activation. Although great efforts have been exerted, there is still nonspecific treatment currently. Recent data showed that immunomodulatory therapy is always promising. However, the effects of interferon-γ (IFN-γ) on AP are controversial. This study is designed to elucidate the effects of IFN-γ on AP severity and explore its impacts on the major mechanisms of AP. METHODS: Sprague-Dawley rats were used to establish AP model by intraperitoneal injection of 20% L-arginine (4 g/kg) twice with an interval of 1 hour. The effects of IFN-γ on the severity of AP, trypsinogen activation peptide, and tumor necrosis factor α, Interleukin-1, Interleukin-6 levels, and autophagy activity were detected. RESULTS: Compared with AP rats without IFN-γ administration, AP rats with IFN-γ administration had more severe pathological changes in pancreata, greater levels of trypsinogen activation concomitant with autophagy up-regulation, and higher levels of cytokine release. CONCLUSIONS: Interferon-γ aggravated L-arginine-induced AP in Sprague-Dawley rats and led to intracellular trypsinogen activation and inflammatory response. The former may be related to autophagy up-regulation.

Differential gene expression profiling of endometrium during the mid-luteal phase of the estrous cycle between a repeat breeder (RB) and non-RB cows.

BACKGROUND: Repeat breeding directly affects reproductive efficiency in cattle due to an increase in services per conception and calving interval. This study aimed to investigate whether changes in endometrial gene expression profile are involved in repeat breeding in cows. Differential gene expression profiles of the endometrium were investigated during the mid-luteal phase of the estrous cycle between repeat breeder (RB) and non-RB cows using microarray analysis. METHODS: The caruncular (CAR) and intercaruncular (ICAR) endometrium of both ipsilateral and contralateral uterine horns to the corpus luteum were collected from RB (inseminated at least three times but not pregnant) and non-RB cows on Day 15 of the estrous cycle (4 cows/group). Global gene expression profiles of these endometrial samples were analyzed with a 15 K custom-made oligo-microarray for cattle. Immunohistochemistry was performed to investigate the cellular localization of proteins of three identified transcripts in the endometrium. RESULTS: Microarray analysis revealed that 405 and 397 genes were differentially expressed in the CAR and ICAR of the ipsilateral uterine horn of RB, respectively when compared with non-RB cows. In the contralateral uterine horn, 443 and 257 differentially expressed genes were identified in the CAR and ICAR of RB, respectively when compared with non-RB cows. Gene ontology analysis revealed that genes involved in development and morphogenesis were mainly up-regulated in the CAR of RB cows. In the ICAR of both the ipsilateral and contralateral uterine horns, genes related to the metabolic process were predominantly enriched in the RB cows when compared with non-RB cows. In the analysis of the whole uterus (combining the data above four endometrial compartments), RB cows showed up-regulation of 37 genes including PRSS2, GSTA3 and PIPOX and down-regulation of 39 genes including CHGA, KRT35 and THBS4 when compared with non-RB cows. Immunohistochemistry revealed that CHGA, GSTA3 and PRSS2 proteins were localized in luminal and glandular epithelial cells and stroma of the endometrium. CONCLUSION: The present study showed that endometrial gene expression profiles are different between RB and non-RB cows. The identified candidate endometrial genes and functions in each endometrial compartment may contribute to bovine reproductive performance.

Sweat chloride and immunoreactive trypsinogen in infants carrying two CFTR mutations and not affected by cystic fibrosis.

Newborns with raised immunotrypsinogen levels who have non-pathological sweat chloride values and carry two cystic fibrosis transmembrane regulator (CFTR) mutations of which at least one is not acknowledged to be cystic fibrosis (CF)-causing are at risk of developing clinical manifestations consistent with CFTR-related disorders or even CF. It is not known whether newborns with similar genotypes and normal immunoreactive trypsinogen (IRT) may share the same risk. This study found that newborns with these characteristics and normal IRT have lower sweat chloride values than those with raised IRT (p=0.007).

Pancreatic Acinar Cells Employ miRNAs as Mediators of Intercellular Communication to Participate in the Regulation of Pancreatitis-Associated Macrophage Activation.

Macrophage activation plays an important role in the inflammatory response in acute pancreatitis. In the present study, the activation of AR42J pancreatic acinar cells was induced by taurolithocholate treatment. The results showed that the culture medium from the activated AR42J cells significantly enhanced NFκB activation in the macrophages compared to that without taurolithocholate treatment. Additionally, the precipitates obtained from ultracentrifugation of the culture media that were rich in exosomes were markedly more potent in activating macrophages compared with the supernatant fraction lacking exosomes. The results indicated that the mediators carried by the exosomes played important roles in macrophage activation. Exosomal miRNAs were extracted and examined using microarrays. A total of 115 differentially expressed miRNAs were identified, and 30 showed upregulated expression, while 85 displayed downregulated expression. Target genes of the differentially expressed miRNAs were predicted using TargetScan, MiRanda, and PicTar software programs. The putative target genes were subjected to KEGG functional analysis. The functions of the target genes were primarily enriched in MAPK pathways. Specifically, the target genes regulated macrophage activation through the TRAF6-TAB2-TAK1-NIK/IKK-NFκB pathway. As the mediators of signal transduction, miRNAs and their predicted target mRNAs regulate every step in the MAPK pathway.

Hashimoto's thyroiditis could be secondary to vitiligo: the possibility of antigen crossover and oxidative stress between the two diseases.

Autoimmune thyroid diseases (AITDs) are often accompanied by vitiligo, and the sera of patients with vitiligo often demonstrate increased frequencies of thyroid autoantibodies. In this study, we investigated the expression of melanocyte-associated antigens in tissues from patients with Hashimoto's thyroiditis (HT) without vitiligo using immunohistochemistry. Tissues of HT without vitiligo, as well as normal thyroid tissues, were both negative for the expression of NKI/beteb, gp100, tyrosinase-related protein 1 (TRP1), HMB-45 and S100, whereas they were positive for the expression of tyrosinase-related protein 2 (TRP2), lysosome-associated membrane protein 1 (LAMP1) and CD69. Tyrosinase (TYR) was only detected in tissues of HT, and levels of LAMP1 and CD69 were higher in tissues of HT than in normal thyroid tissues (p < 0.005). These results suggest the possibility of antigen crossover and oxidative stress between vitiligo and HT that might represent an immunological basis for secondary HT associated with vitiligo.