The role of interleukin-18 in pancreatitis and pancreatic cancer

Originally described as an interferon (IFN)-γ-inducing factor, interleukin (IL)-18 has been reported to be involved in Th1 and Th2 immune responses, as well as in activation of NK cells and macrophages. There is convincing evidence that IL-18 plays an important role in various pathologies (i.e. inflammatory diseases, cancer, chronic obstructive pulmonary disease, Crohn's disease and others). Recently, IL-18 has also been shown to execute specific effects in pancreatic diseases, including acute and chronic pancreatitis, as well as pancreatic cancer. The aim of this study was to give a profound review of recent data on the role of IL-18 and its potential as a therapeutic target in pancreatic diseases. The existing data on this topic are in part controversial and will be discussed in detail. Future studies should aim to confirm and clarify the role of IL-18 in pancreatic diseases and unravel their molecular mechanisms.     

Ascitic fluid and serum from rats with acute pancreatitis injure rat pancreatic tissues and alter the expression of heat shock protein 60

Acute pancreatitis (AP) is an inflammatory process in which cytokines and chemokines are involved. After onset, extrapancreatic stimuli can induce the expression of cytokines in pancreatic acinar cells, thereby amplifying this inflammatory loop. To further determine the role and mechanism of irritating agents in the pathogenesis of AP, rat pancreatic tissues were stimulated with ascitic fluid (APa) and serum (APs) from rats with AP or with lipopolysaccharide (LPS). In addition, the alteration of heat shock protein 60 (HSP60) expression was evaluated. Rat pancreas was removed and meticulously snipped to fragments. The snips were cultured for up to 48 h. During this period, the tissue viability as well as amylase and TNF-α levels in the supernatant and the HSP60 expression in the pancreatic tissue before and after stimulation by APa, APs, and LPS were assayed time-dependently. At different time-points during the culture, the viability and the amylase activity in the pancreatic tissue remained largely stable. After stimulation with APa, APs, or LPS for 1 h, the pancreatic tissues showed some damage, and this was followed by a sharp decrease in the viability accompanied by increased levels of amylase and TNF-α in the culture medium 2 or 4 h after stimulation (p < 0.05). In contrast, both the HSP60 mRNA and protein levels had a relatively high expression in the freshly prepared tissue fragments (0 h). As the culturing period was extended, the expression of HSP60 mRNA decreased only slightly; at the same time, the HSP60 protein levels decreased over a prolonged culture time, significantly so from 12 through 48 h (p < 0.05). After stimulation with APs, APa, or LPS, both the expression of HSP60 mRNA and protein in the tissue fragments increased slightly at 1 h and decreased significantly thereafter at 2 and 4 h (p < 0.05). APa, APs, or LPS induce injuries on isolated pancreatic tissues, accompanied by an altered HSP60 expression pattern in a time-dependent manner.     

Alteration of Cpn60 expression in pancreatic tissue of rats with acute pancreatitis

The expression of heat-shock protein 60 (also known as chaperonin 60, Cpn60) in experimental acute pancreatitis (AP) is considered to play an active role in the prevention of abnormal enzyme accumulation and activation in pancreatic acinar cells. However, there are controversial results in the literature regarding the relationship between the abnormality of Cpn60 expression and AP onset and development. The purpose of this study was to investigate the alternations of Cpn60 expression and the relationship between the abnormal expression of Cpn60 and AP progression in rat severe acute pancreatitis (SAP) models. In this report, we induced SAP in Sprague–Dawley (SD) rats by reverse injection of sodium deoxycholate into the pancreatic duct, and examined the dynamic changes of Cpn60 expression in pancreatic tissues from different time points and at different levels with techniques of real-time PCR, western blotting, and immunohistochemistry. At 1 h after SAP induction, the expression of Cpn60 mRNA in the AP pancreatic tissues was higher than those in the sham-operation group and normal control group, but decreased sharply as the time period was extended, and there was a significant difference between 1 h and 10 h after SAP induction (p < 0.05). In the AP process, Cpn60 protein expression showed transient elevation as well, and the increased protein expression occurred predominantly in affected, but not totally destroyed, pancreatic acinar cells. As AP progressed, the pancreatic tissues were seriously damaged, leading to a decreased overall Cpn60 protein expression. Our results show a complex pattern of Cpn60 expression in pancreatic tissues of SAP rats, and the causality between the damage of pancreatic tissues and the decrease of Cpn60 level needs to be investigated further. Xue-Li Li and Kun Li contributed equally to this work.     

Environmental factors, health-related habits, and serum selenium levels in cancer patients and healthy controls

Previous studies conducted in Yugoslavia indicated that the concentration of selenium in soil, food items, and serum of the population is very low. The aim of the study was to investigate the possible relationship among environmental, health-related habits, nutrition, and selenium serum levels in cancer patients and the healthy population. The case-control study included a group of cancer patients and a matched group of healthy controls: 57 cancer patients and 41 healthy controls living in Stari Grad (an urban area of Belgrade), as well as 17 cancer patients and 13 healthy controls living in Barajevo (a rural community in the vicinity of Belgrade). The healthy controls were matched to cancer patients in sex and age; they were not blood related. The selenium serum levels were measured by atomic absorption spectrophotometry. Health-related habits and relevant dietary factors (“food frequency” method) that may influence the selenium serum levels were assessed by questionnaires. The differences in average values of selenium serum levels between the cancer patients and healthy controls were not significantly different, but both were below the lowest recorded in referential studies. A significant difference between the values obtained from urban and rural subgroups was noted. The most important factors that influenced the level of selenium included the residence place in the region with selenium deficiency (Barajevo), age, associated chronic diseases, and some dietary factors potentially related to the intake of selenium. The results obtained in this investigation pointed out that use of selenium supplementation in this area should be seriously considered. Deceased     

Targeted serum glycoproteomics for the discovery of lung cancer‐associated glycosylation disorders using lectin‐coupled ProteinChip arrays

To screen for glycoproteins showing aberrant sialylation patterns in sera of cancer patients and apply such information for biomarker identification, we performed SELDI‐TOF MS analysis coupled with lectin‐coupled ProteinChip arrays (Jacalin or SNA) using sera obtained from lung cancer patients and control individuals. Our approach consisted of three processes (i) removal of 14 abundant proteins in serum, (ii) enrichment of glycoproteins with lectin‐coupled ProteinChip arrays, and (iii) SELDI‐TOF MS analysis with acidic glycoprotein‐compatible matrix. We identified 41 protein peaks showing significant differences (p<0.05) in the peak levels between the cancer and control groups using the Jacalin‐ and SNA‐ProteinChips. Among them, we identified loss of Neu5Ac (α2,6) Gal/GalNAc structure in apolipoprotein C‐III (apoC‐III) in cancer patients through subsequent MALDI‐QIT‐TOF MS/MS. Furthermore, subsequent validation experiments using an additional set of 60 lung adenocarcinoma patients and 30 normal controls demonstrated that there is a higher frequency of serum apoC‐III with loss of α2,6‐linkage Neu5Ac residues in lung cancer patients compared to controls. Our results have demonstrated that lectin‐coupled ProteinChip technology allows the high‐throughput and specific recognition of cancer‐associated aberrant glycosylations, and implied a possibility of its applicability to studies on other diseases.     

Cross‐species analysis of nicotine‐induced proteomic alterations in pancreatic cells

Toxic compounds in tobacco, such as nicotine, may adversely affect pancreatic function. We aim to determine nicotine‐induced protein alterations in pancreatic cells, thereby revealing links between nicotine exposure and pancreatic disease. We compared the proteomic alterations induced by nicotine treatment in cultured pancreatic cells (mouse, rat, and human stellate cells and human duct cells) using MS‐based techniques, specifically SDS‐PAGE (gel) coupled with LC‐MS/MS and spectral counting. We identified thousands of proteins in pancreatic cells, hundreds of which were identified exclusively or in higher abundance in either nicotine‐treated or untreated cells. Interspecies comparisons of stellate cell proteins revealed several differentially abundant proteins (in nicotine treated versus untreated cells) common among the three species. Proteins appearing in all nicotine‐treated stellate cells include amyloid beta (A4), procollagen type VI alpha 1, integral membrane protein 2B, and toll‐interacting protein. Proteins that were differentially expressed upon nicotine treatment across cell lines were enriched in certain pathways, including nicotinic acetylcholine receptor, cytokine, and integrin signaling. At this analytical depth, we conclude that similar pathways are affected by nicotine, but alterations at the protein level among stellate cells of different species vary. Further interrogation of such pathways will lead to insights into the potential effect of nicotine on pancreatic cells at the biomolecular level and the extension of this concept to the effect of nicotine on pancreatic disease.     

Evaluation of changes in serum protein profiles during neoadjuvant chemotherapy in HER2‐positive breast cancer using an LC‐MALDI‐TOF/MS procedure

Comparison of protein profiles of sera acquired before and after preoperative chemotherapy for breast cancer may reveal tumor markers that could be used to monitor tumor response. In this study, we analyzed pre‐ and post‐chemotherapy protein profiles of sera from 39 HER2‐postive breast cancer patients (n=78 samples) who received 6 months of preoperative chemotherapy using LC‐MALDI‐TOF/MS technology. We detected qualitative and quantitative differences in pair‐wise comparison of pre‐ and post chemotherapy samples that were different in patients who achieved pathological complete response (pCR, n=21) compared with those with residual disease (n=18). We identified 2329 and 3152 peaks as differentially expressed in the pre‐chemotherapy samples of the responders and non‐responders. Comparison of matching pre‐ and post‐chemotherapy samples identified 34 (32 decreased, two increased) and 304 peaks (157 decreased, 147 increased) that significantly changed (p<0.01, false discovery rate ≤20%) after treatment in responders and non‐responders, respectively. The top 11 most significantly altered peptide peaks with the greatest change in intensity were positively identified. These corresponded to eight proteins including α‐2‐macroglobulin, complement 3, hemopexin, and serum amyloid P in the responder group and chains C and A of apolipoprotein A‐I, hemopexin precursor, complement C, and amyloid P component in the non‐responding groups. All proteins decreased after therapy, except chain C apolipoprotein A and hemopexin precursor that increased. These results suggest that changes in serum protein levels occur in response to chemotherapy and these changes partly appear different in patients who are highly sensitive to chemotherapy compared with those with lesser response.     

Micro‐ and macroheterogeneity of N‐glycosylation yields size and charge isoforms of human sex hormone binding globulin circulating in serum

Human sex hormone binding globulin (hSHBG) is a serum glycoprotein central to the transport and targeted delivery of sex hormones to steroid‐sensitive tissues. Several molecular mechanisms of action of hSHBG, including the function of its attached glycans remain unknown. Here, we perform a detailed site‐specific characterization of the N‐ and O‐linked glycosylation of serum‐derived hSHBG. MS‐driven glycoproteomics and glycomics combined with exoglycosidase treatment were used in a bottom‐up and top‐down manner to determine glycosylation sites, site‐specific occupancies and monosaccharide compositions, detailed glycan structures, and the higher level arrangement of glycans on intact hSHBG. It was found that serum‐derived hSHBG is N‐glycosylated at Asn³⁵¹ and Asn³⁶⁷ with average molar occupancies of 85.1 and 95.3%, respectively. Both sites are occupied by the same six sialylated and partly core fucosylated bi‐ and triantennary N‐Glycoforms with lactosamine‐type antennas of the form (±NeuAcα6)Galβ4GlcNAc. N‐Glycoforms of Asn³⁶⁷ were slightly more branched and core fucosylated than Asn³⁵¹ N‐glycoforms due probably to a more surface‐exposed glycosylation site. The N‐terminal Thr⁷ was fully occupied by the two O‐linked glycans NeuAcα3Galβ3(NeuAcα6)GalNAc (where NeuAc is N‐acetylneuraminic acid and GalNAc is N‐acetylgalactosamine) and NeuAcα3Galβ3GalNAc in a 1:6 molar ratio. Electrophoretic analysis of intact hSHBG revealed size and charge heterogeneity of the isoforms circulating in blood serum. Interestingly, the size and charge heterogeneity were shown to originate predominantly from differential Asn³⁵¹ glycan occupancies and N‐glycan sialylation that may modulate the hSHBG activity. To date, this work represents the most detailed structural map of the heterogeneous hSHBG glycosylation, which is a prerequisite for investigating the functional aspects of the hSHBG glycans.     

Intact protein profiling in breast cancer biomarker discovery: Protein identification issue and the solutions based on 3D protein separation,bottom‐up and top‐down mass spectrometry

Proteomics profiling of intact proteins based on MALDI‐TOF MS and derived platforms has been used in cancer biomarker discovery studies. This approach suffers from a number of limitations such as low resolution, low sensitivity, and that no knowledge is available on the identity of the respective proteins in the discovery mode. Nevertheless, it remains the most high‐throughput, untargeted mode of clinical proteomics studies to date. Here we compare key protein separation and MS techniques available for protein biomarker identification in this type of studies and define reasons of uncertainty in protein peak identity. As a result of critical data analysis, we consider 3D protein separation and identification workflows as optimal procedures. Subsequently, we present a new protocol based on 3D LC‐MS/MS with top‐down at high resolution that enabled the identification of HNRNP A2/B1 intact peptide as correlating with the estrogen receptor expression in breast cancer tissues. Additional development of this general concept toward next generation, top‐down based protein profiling at high resolution is discussed.     

Identification of blood-protein carriers of the CA 19-9 antigen and characterization of prevalence in pancreatic diseases

The current best serum marker for pancreatic cancer, CA 19-9, detects a carbohydrate antigen on multiple protein carriers. Better knowledge of the protein carriers of the CA 19-9 antigen in various disease states may lead to improved diagnostic tests. To identify proteins that carry the CA 19-9 antigen, we immunoprecipitated the CA 19-9 antigen from pooled sera and identified the associated proteins using MS. Among the high-confidence identifications, we confirmed the presence of the CA 19-9 antigen on Apolipoprotein B-100 by antibody arrays and Western blot and on kininogen, ARVCF, and Apolipoprotein E by antibody arrays. We characterized the frequency and levels of the CA 19-9 antigen on the four proteins across various patient groups (pancreatic cancer, pancreatitis, and healthy controls) using antibody arrays. Nearly, 10-25% of the subjects showed elevations of the antigen on each protein, but the elevations were not associated with disease state or total CA 19-9 levels. These results contribute to our knowledge of the carrier proteins of an important functional glycan and the rate at which the glycan is displayed. This work also demonstrates a strategy for using the complementary methods of MS and antibody microarrays to identify protein carriers of glycans and assess the diagnostic value of measuring glycans on individual proteins.     

Identification,prioritization, and evaluation of glycoproteins for aggressive prostate cancer using quantitative glycoproteomics and antibody‐based assays on tissue specimens

Prostate cancer is highly heterogeneous in nature; while the majority of cases are clinically insignificant, some cases are lethal. Currently, there are no reliable screening methods for aggressive prostate cancer. Since most established serum and urine biomarkers are glycoproteins secreted or leaked from the diseased tissue, the current study seeks to identify glycoprotein markers specific to aggressive prostate cancer using tissue specimens. With LC‐MS/MS glycoproteomic analysis, we identified 350 glycopeptides with 17 being altered in aggressive prostate cancer. ELISA assays were developed/purchased to evaluate four candidates, that is, cartilage oligomeric matrix protein (COMP), periostin, membrane primary amine oxidase (VAP‐1), and cathepsin L, in independent tissue sets. In agreement with the proteomic analysis, we found that COMP and periostin expressions were significantly increased in aggressive prostate tumors while VAP‐1 expression was significantly decreased in aggressive tumor. In addition, the expression of these proteins in prostate metastases also follows the same pattern observed in the proteomic analysis. This study provides a workflow for biomarker discovery, prioritization, and evaluation of aggressive prostate cancer markers using tissue specimens. Our data suggest that increase in COMP and periostin and decrease in VAP‐1 expression in the prostate may be associated with aggressive prostate cancer.     

Urgent endovascular ligature of a ruptured splenic artery pseudoaneurysm in a patient with acute pancreatitis: a case report

Introduction

We report on the successful endovascular treatment of a ruptured splenic artery pseudoaneurysm. Our patient had acute pancreatitis superimposed on chronic calcific pancreatitis and chronic renal impairment. Contrast-enhanced ultrasonography was used to assess post-embolization results.

Case presentation

Our patient was a 67-year-old white Caucasian man with recurrent pancreatitis. Computed tomography angiography showed a pancreatic pseudocyst with a ruptured pseudoaneurysm, which was successfully embolized using an endovascular percutaneous approach. At six months, persistent renal failure led to contrast-enhanced ultrasonography. This confirmed the absence of turbulent blood flow and extravasation of contrast medium in the pseudocyst.

Conclusion

Our experience with this case leads us to support the role of interventional radiology as a first-line treatment tool. Contrast-enhanced ultrasonography can be used to follow-up embolization procedures in patients with impaired renal function.     

双歧杆菌四联活菌片对急性胰腺炎患者血清内毒素和降钙素原水平的影响及疗效观察

目的探讨双歧杆菌四联活菌片对急性胰腺炎患者血清内毒素和降钙素原（PCT）水平的影响及疗效观察。方法将66例急性胰腺炎患者随机分为观察组和对照组。两组患者均予以禁食禁饮、持续胃肠减压、预防感染、抑酸、解痉镇痛、抑制胰酶分泌和纠正水电解质酸碱紊乱等常规治疗。观察组患者加用双歧杆菌四联活菌片1.5 g进行水化后自胃管灌注,夹管2 h,3次/d,对照组除不使用双歧杆菌四联活菌片外余治疗同观察组。观察两组患者治疗前后血清内毒素和PCT水平的变化,并比较其临床疗效。结果治疗7 d后,两组患者血清内毒素和PCT水平均有不同程度的下降（P〈0.05或P〈0.01）,且观察组下降值比对照组更明显（P〈0.05）;同时观察组临床总有效率为93.94%,明显高于对照组的75.76%（χ2=4.24,P〈0.05）。结论双歧杆菌四联活菌片辅助治疗急性胰腺炎具有较好疗效,能明显降低血清内毒素和PCT水平,减轻肠源性内毒素血症,对肠黏膜屏障功能具有良好的保护作用。     

慢性胰腺炎患者血清总胆固醇和血红蛋白水平与肠道菌群失调的相关性研究

目的 探讨慢性胰腺炎(CP)患者血清总胆固醇(TC)、血红蛋白(Hb)水平与肠道菌群失调的相关性。 方法 选取2014年1月至2019年1月在本院就诊的69例CP患者作为观察组,另选取同期行健康体检者120例作为对照组。记录两组性别、年龄、体质量指数(BMI)、收缩压、舒张压,采用日立7600全自动生化仪检测TC水平,采用CELL DYN1800全自动血细胞分析仪检测Hb水平,采用细菌培养法进行肠道菌群检查。比较两组基线资料、TC、Hb水平及肠道菌群数量差异,采用Spearman相关分析CP患者血清TC、Hb水平与肠道菌群数量的相关性。 结果 两组性别、年龄、BMI、收缩压、舒张压等差异均无统计学意义(P>0.05)。与对照组比较,观察组的血清TC水平较高(P结论 CP患者血清TC、Hb水平异常,且与患者肠道菌群失调密切相关,可为CP的临床治疗提供参考依据。     

Dietary Inflammatory Index,Alternative Healthy Eating Index-2010, Mediterranean Diet Score and the risk of pancreatic cancer

BackgroundPrevious studies of dietary patterns and pancreatic cancer risk have been inconclusive; we aimed to investigate the association of Mediterranean Diet Score (MDS), Alternative Healthy Eating Index-2010 (AHEI-2010), and Dietary Inflammatory Index (DII®) with risk of pancreatic cancer.MethodsWe used data from the Melbourne Collaborative Cohort Study including 33,690 men and women aged 40–69 years at recruitment in 1990–1994. A total of 258 incident cases of pancreatic cancer was identified over an average of 23.7 years of follow-up. Hazard ratios (HR) were estimated using Cox regression, with age as the underlying time metric, adjusting for potential confounders including sex, height, country of birth, education, socio-economic position, physical activity, energy intake, smoking status, pack-years smoking, years since quitting smoking, and alcohol intake.ResultsA healthier diet as assessed by the AHEI-2010 was associated with a lower risk of pancreatic cancer [HR_{Quartile4 vs Quartile1} = 0.58; 95%CI 0.40 – 0.85; p for trend 0.003]. Weaker but consistent evidence was observed for the other indexes [DII® HR_{Quartile4 vs Quartile1} = 1.30; 95%CI 0.82 – 2.06; p for trend 0.1], [MDS HR_{Category3 vs Category1} = 0.79; 95%CI 0.49 – 1.26; p for trend 0.06].ConclusionAdherence to a healthier diet, as assessed by the AHEI-2010, may reduce the risk of pancreatic cancer.     

A key role of neurokinin 1 receptors in acute pancreatitis and associated lung injury

Earlier studies have shown that mice deficient in NK1 receptors or its ligand, substance P, are protected against acute pancreatitis and associated lung injury. In the current study, the protective effect of NK1 receptor blockage against acute pancreatitis and associated lung injury was investigated, using a specific receptor antagonist, CP-96345. Acute pancreatitis was induced in mice by intraperitoneal (i.p.) injections of caerulein. Substance P levels in plasma, pancreas, and lungs were found to be elevated in a caerulein dose-dependent manner. Mice treated with CP-96345, either prophylactically, or therapeutically, were protected against acute pancreatitis and associated lung injury as evident by attenuation in plasma amylase, pancreatic and pulmonary myeloperoxidase activities, and histological evidence of pancreatic and pulmonary injuries. Pulmonary microvascular permeability was also reduced as a result of CP-96345 treatment. These results point to a key role of NK1 receptors in acute pancreatitis and associated lung injury.     

Anti‐inflammatory effects of melatonin in a rat model of caerulein‐induced acute pancreatitis

The purpose of our study was to evaluate the protective effect of melatonin in a rat model of caerulein‐induced acute pancreatitis. For the induction of experimental acute pancreatitis, four subcutaneous injections of caerulein (20 µg kg^–1 body weight) were given to Wistar rats at 2‐h intervals. Melatonin was injected intraperitoneally (25 mg kg^–1 body weight) 30 min before each caerulein injection. After 12 h, rats were sacrificed by decapitation. Blood and pancreas samples were collected and processed for serological and histopathological studies, respectively. Lipase, α‐amylase, corticosterone, total antioxidant power and cytokines interleukin (IL)‐1β, IL‐4 and tumour necrosis factor (TNF)‐α were determined using commercial kits. ANOVA and Tukey tests (P < 0.05) were performed for the statistical analysis of the results. Results showed that the administration of melatonin reduced histological damage induced by caerulein treatment as well as the hyperamylasemia and hyperlipidemia. Corticosterone and antioxidant total power were also reverted to basal activities. Furthermore, melatonin pre‐treatment reduced pro‐inflammatory cytokines IL‐1β and TNF‐α and increased the serum levels of anti‐inflammatory cytokine IL‐4. In conclusion, the findings suggest that the protective effect of melatonin in caerulein‐induced acute pancreatitis is mediated by the anti‐inflammatory ability of this indolamine. Thus, melatonin may have a protective effect against acute pancreatitis. Copyright © 2013 John Wiley & Sons, Ltd.     

Validation of reverse phase protein array for practical screening of potential biomarkers in serum and plasma: accurate detection of CA19-9 levels in pancreatic cancer

The current study analyzed reverse phase protein arrays (RPPA) as a means to experimentally validate biomarkers in blood samples. One microliter samples of sera (n = 71), and plasma (n = 78) were serially diluted and printed on NC-coated slides. CA19-9 levels from RPPA results were compared with identical patient samples as measured by ELISA. There was a strong correlation between RPPA and ELISA (r = 0.87) as determined by scatter plots. Sample reproducibility of CA19-9 levels was excellent (interslide correlation r = 0.88; intraslide correlation r = 0.83). The ability of RPPA to accurately distinguish CA19-9 levels between cancer and noncancer samples were determined using receiver operating characteristic curves and compared with ELISA. The AUC for RPPA and ELISA was comparable (0.87 and 0.86, respectively). When the mean CA19-9 levels of normal samples was used as a cutoff for RPPA and compared with the standard clinical ELISA cutoff, comparable specificities (71% for both) were observed. Notably, RPPA samples normalized to albumin showed increased sensitivity compared to ELISA (90% vs. 75%). As RPPA is a high-throughput method that shows results comparable to that of ELISA, we propose that RPPA is a viable technique for rapid experimental screening and validation of candidate biomarkers in blood samples.