Serum phospholipase A2 in intensive care patients with peritonitis,multiple injury,and necrotizing pancreatitis

Summary To study the source and role of circulating phospholipase A₂ (PLA₂) catalytic activity we monitored the serum from patients with necrotizing pancreatitis (n=8), diffuse peritonitis (n=6), and multiple injuries (n=11). Immunoreactive PLA₂ serum protein concentration was analysed using a fluoroimmunoassay based on an antibody against human pancreatic PLA₂. Serum PLA₂ catalytic activity was analysed using a radiochemical method based on a substrate with tritiated palmitic acid in beta position. In necrotizing pancreatitis immunoreactive PLA₂ and PLA₂ catalytic activity both increased. Obviously, in necrotizing pancreatitis the major part of serum catalytic activity stems from the pancreas. In patients with diffuse peritonitis and multiple injuries, as a rule, immunoreactive phospholipase A₂ serum concentration appears to be within the normal range. In contrast, in these patients we demonstrated high serum catalytic PLA₂ activity comparable to that in necrotizing pancreatitis. The source of catalytic PLA₂ activity in peritonitis and multiple injuries seems not to be the pancreas. There was a correlation between pulmonary insufficiency and serum PLA₂ catalytic activity in patients with necrotizing pancreatitis, peritonitis, and multiple injuries.     

Diagnostic value of immunoreactive phospholipase A2 in acute pancreatitis

Summary In a prospective clinical trial 85 patients with acute pancreatitis were analysed for serum total amylase, pancreatic amylase, pancreatic lipase, trypsin, elastase 1, and immunoreactive phospholipase A₂ (IR-PLA₂). The diagnostic sensitivity of serum IR-PLA₂ was comparable to that of serum total amylase, pancreatic amylase, and trypsin. The specificity of IR-PLA₂ is superior to that of serum total amylase determination due to the fact that the IR-PLA₂ determination is based on an antibody against human pancreatic PLA₂.     

Characterization of phospholipase A2 activity in aspirates of human pancreatic pseudocysts after isolation by reversed-phase high performance liquid chromatography

Summary Phospholipase A (PLA) is able to attack membrane phospholipids and thereby plays a putative role in the pathogenesis of pancreatic pseudocysts. We looked for PLA₂-like activity in aspirates from human pancreatic pseudocysts. In material originating from one cyst which occurred shortly after an acute pancreatitis attack, hydrolyzing enzymatic activity measured by a sensitive bioassay system for PLA₂ activity was found without prior trypsin activation (67×10³ U/min/100 µl). A biochemical characterization of this hydrolyzing enzymatic activity was provided after resolution of the respective proteins contained in the cyst fluid by HPLC. High hydrolyzing activities were found in correspondence to one specific, early eluting peak. The purified enzyme had pH optima at 3.5 and 6. Addition of EDTA (5 mM) to the test system abolished the enzymatic activity which mirrored the requirement for calcium ions. The activity was optimal at calcium concentrations ranging from 1–2 mM. Higher calcium concentrations reduced the enzymatic activity. The enzyme showed high heat stability. SDS-gel analysis of the peak showed one single band with a molecular weight of about 20,000 Daltons. Our findings demonstrate the possibility of activated, PLA-like activity in human pancreatic pseudocyst fluid. We speculate that an inappropriate activation of this enzyme in peri- or intrapancreatic fluid collections could account for pseudocyst formation after an acute pancreatitis attack.     

Expression of group XIIA phospholipase A2 in human digestive organs

Cellular distribution of group XIIA phospholipase A₂ (GXIIA PLA₂) was studied in human digestive organs by immunohistochemistry. GXIIA PLA₂ protein was detected in epithelial cells of normal gastrointestinal tract, gallbladder and pancreatic acinar cells. The GXIIA PLA₂ protein was evenly distributed in the cytoplasm in contrast to secretory granular distribution of GIB PLA₂ and GIIA PLA₂ in pancreatic acinar cells and small intestinal Paneth cells respectively. Epithelial cells of intestinal glands in Crohn's disease and ulcerative colitis expressed abundant GXIIA PLA₂, whereas inflammatory cells were devoid of the enzyme protein. Tumour cells in colonic adenomas and carcinomas and pancreatic ductogenic carcinomas expressed GXIIA PLA₂ protein at varying intensity levels. The putative functions of GXIIA PLA₂ remain to be investigated and its role in healthy and diseased digestive organs can only be speculated on at present.     

Prognostic value of serum phospholipase A in acute pancreatitis

Summary Serum from 48 patients with acute pancreatitis (21 with interstitial-edematous and 27 with necrotizing pancreatitis) was monitored for immunoreactive (IR) phospholipase A₂ (PLA₂) protein concentration and PLA catalytic activity. In both groups within 48 h after start of acute pancreatitis an up to tenfold increase of IR-PLA₂ was demonstrable. Determination of IR-PLA₂ revealed no differences between the groups. In contrast, determination of PLA catalytic activity allowed us to differentiate between patients with interstitial-edematous and necrotizing pancreatitis.     

Immunocytochemical evidence of phospholipase A2 in pancreatic tumors — Diagnostic values

Summary Phospholipase A₂ is an enzyme which is produced in acinar cells, and persists even in regressive states of chronic pancreatitis, when the production of other enzymes diminishes. We therefore tested this enzyme as a marker of acinar descent in various pancreatic tumors. This enzyme could be seen in 50% of the acinar-cell carcinomas, in 60% of solid and papillary pancreatic tumors, and in 50% of microglandular carcinomas. Ductal cancers and isletcell cancers were negative. In contrast to other markers of acinar matrix (amylase, antitrypsin), phospholipase A₂ gave fewer false-positive or false-negative results.Supported by a grant of the J. and F. Marohn Stiftung at the University of Erlangen/Nürnberg     

The epigenetic regulators Bmi1 and Ring1B are differentially regulated in pancreatitis and pancreatic ductal adenocarcinoma

Chronic pancreatitis and pancreatic ductal adenocarcinoma (PDAC) are associated with major changes in cell differentiation. These changes may be at the basis of the increased risk for PDAC among patients with chronic pancreatitis. Polycomb proteins are epigenetic silencers expressed in adult stem cells; up‐regulation of Polycomb proteins has been reported to occur in a variety of solid tumours such as colon and breast cancer. We hypothesized that Polycomb might play a role in preneoplastic states in the pancreas and in tumour development/progression. To test these ideas, we determined the expression of PRC1 complex proteins (Bmi1 and Ring1b) during pancreatic development and in pancreatic tissue from mouse models of disease: acute and chronic pancreatic injury, duct ligation, and in K‐Ras^G12V conditional knock‐in and caerulein‐treated K‐Ras^G12V mice. The study was extended to human pancreatic tissue samples. To obtain mechanistic insights, Bmi1 expression in cells undergoing in vitro exocrine cell metaplasia and the effects of Bmi1 depletion in an acinar cancer cell line were studied. We found that Bmi1 and Ring1B are expressed in pancreatic exocrine precursor cells during early development and in ductal and islet cells—but not acinar cells—in the adult pancreas. Bmi1 expression was induced in acinar cells during acute injury, in acinar–ductal metaplastic lesions, as well as in pancreatic intraepithelial neoplasia (PanIN) and PDAC. In contrast, Ring1B expression was only significantly and persistently up‐regulated in high‐grade PanINs and in PDAC. Bmi1 knockdown in cultured acinar tumour cells led to changes in the expression of various digestive enzymes. Our results suggest that Bmi1 and Ring1B are modulated in pancreatic diseases and could contribute differently to tumour development. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

N-乙酰半胱氨酸对急性胰腺炎模型大鼠的保护作用

背景：急性胰腺炎是由胰腺腺泡细胞损伤介导的常见炎性疾病,白细胞浸润是其主要的发病特征。近来发现N-乙酰半胱氨酸能够控制白细胞游走并在一些严重的炎症疾病中发挥调节炎症反应的作用。 目的：观察N-乙酰半胱氨酸在体内对牛黄胆酸盐诱导的急性胰腺炎大鼠模型的保护作用。 方法：90只SD大鼠随机等分为正常对照组、急性胰腺炎组和N-乙酰半胱氨酸组,后2组以十二指肠大乳头逆行注射牛黄胆酸盐制备急性大鼠胰腺炎模型。N-乙酰半胱氨酸组大鼠由尾静脉给予N-乙酰半胱氨酸治疗。 结果与结论：急性胰腺炎模型诱导后大鼠血浆淀粉酶活性较正常对照组大鼠显著升高(P < 0.05)。急性胰腺炎组白细胞介素1β,6,10和肿瘤坏死因子α表达水平明显高于正常对照组(P < 0.05)。免疫荧光化学显示N-乙酰半胱氨酸主要表达在胰岛细胞上,急性胰腺炎组织N-乙酰半胱氨酸的表达从mRNA水平到蛋白水平都明显低于正常组织。N-乙酰半胱氨酸治疗显著降低了大鼠血清淀粉酶水平,髓过氧化物酶活性以及促炎性细胞因子产物生产和胰腺及肺组织损伤,但N-乙酰半胱氨酸治疗并没有明显抑制胰腺组织核因子κB的激活。提示N-乙酰半胱氨酸能改善急性胰腺炎大鼠胰腺和肺脏的组织损伤,并发挥抗炎症的作用。 中国组织工程研究杂志出版内容重点：肾移植;肝移植;移植;心脏移植;组织移植;皮肤移植;皮瓣移植;血管移植;器官移植;组织工程全文链接：     

Flavonoids as Phospholipase A2 Inhibitors: Importance of Their Structure for Selective Inhibition of Group II Phospholipase A2

The inhibitory effect of the plant flavonoid, rutin, on group I phospholipase A₂ (PLA₂-I) from porcine pancreas and Naja naja, and on group II phospholipase A₂ (PLA₂-II) from Vipera russelli and Crotalus atrox was investigated. Rutin efficiently inhibited PLA₂-II from both Vipera russelli and Crotalus atrox but was only a weak inhibitor of PLA₂-I from porcine pancreas and Naja naja. The lack of strong inhibition of pancreatic PLA₂-I was not due to contaminating proteins in the enzyme preparation, since the same weak inhibition was obtained against pancreatic PLA₂ purified to homogeneity as judged by two-dimensional gel electrophoresis. Rutin also efficiently inhibited human PLA₂-II from synovial fluid but was only a weak inhibitor of human PLA₂-I from pancreatic juice, suggesting that rutin is a selective PLA₂-II inhibitor. A number of structurally similar flavonoids were tested for their ability to inhibit PLA₂-II from Crotalus atrox and, for comparison, PLA₂-I from porcine pancreas. The results obtained indicate that the hydroxyl group in 5-position as well as the double bond and the double-bonded oxygen in the oxane ring are all important for the overall ability of flavonoids to inhibit PLA₂ activity, and that the hydroxyl groups in 3- and 4-position are required for selective inhibition of PLA₂-II.     

垂体腺苷酸环化酶激活多肽可加剧实验性急性胰腺炎

脑肠肽垂体腺苷酸环化酶激活多肽 (Pituitaryadenylatecyclaseactivatingpolypeptide ,PACAP)在急性胰腺炎 (Acutepancreatitis,AP)发病机理和治疗中的作用尚不清楚 ,为此 ,本实验观察外源性PACAP对正常大鼠胰腺、实验性AP病程的影响。结果发现 :5～ 30 μg/kg的PACAP可促使血清淀粉酶轻微增高、胰腺水肿形成 (实验组2 3 88%± 2 .5 32 %～ 2 5 .86 %± 1.974 %vs正常组 2 9.2 1%± 5 .6 5 7% )、炎性细胞浸润、腺泡细胞空泡化、部分病例可见脂肪坏死和实质坏死灶。 15 μg/kg和 30 μg/kgPACAP可加重蛙皮缩胆囊肽诱发的AP ,胰腺组织水肿更明显(实验组 13.4 5 %± 2 .0 4 5 %～ 17.6 6 %± 4 .6 5 2 %vs蛙皮缩胆囊肽组 2 1.83%± 3.0 13% ,P <0 .0 5 ) ,血清淀粉酶增高 ,出现腹水、胰腺出血、脂肪坏死和实质坏死 ,腺泡细胞明显空泡化。 5 μg/kg和 10 μg/kgPACAP对牛磺胆酸钠诱发的急性出血坏死性胰腺炎病程的影响有所不同 ,可稍减轻胰腺水肿 (实验组 19.18%± 2 .10 2 %～ 2 0 .87%±5 5 97%vs牛磺胆酸钠组 17.5 2 %± 1.5 0 5 % ;下同 )、降低血清淀粉酶 (1986 .91%± 710 .97%～ 2 94 4 .33± 1182 .4 7IU/Lvs 36 90 .87± 2 2 77.99IU/L ,P <0 .0 5 ) ,而胰腺出血和坏死加剧。除蛙皮缩胆囊肽诱发     

Fibrosis of the pancreas: the initial tissue damage and the resulting pattern

Fibrosis in the pancreas is caused by such processes as necrosis/apoptosis, inflammation or duct obstruction. The initial event that induces fibrogenesis in the pancreas is an injury that may involve the interstitial mesenchymal cells, the duct cells and/or the acinar cells. Damage to any one of these tissue compartments of the pancreas is associated with cytokine-triggered transformation of resident fibroblasts/pancreatic stellate cells into myofibroblasts and the subsequent production and deposition of extracellular matrix. Depending on the site of injury in the pancreas and the involved tissue compartment, predominantly inter(peri)lobular fibrosis (as in alcoholic chronic pancreatitis), periductal fibrosis (as in hereditary pancreatitis), periductal and interlobular fibrosis (as in autoimmune pancreatitis) or diffuse inter- and intralobular fibrosis (as in obstructive chronic pancreatitis) develops.     

Experimental immune pancreatitis in the mouse by rabbit immune sera directed against purified enzymes of the exocrine pancreas.

An experimental xenogeneic immune pancreatitis was induced in AB-mice by repeated intraperitoneal injections of rabbit immune sera directed against purified pancreatic enzymes (alpha-amylase, lipase, trypsin) for 3 hours up to 8 days. Histologically, the immune pancreatitis is characterized by three different findings: 1. Multiple acinar cell necroses on the 2nd, 3rd and 5th day of immune serum application. 2. A dedifferentiating acinar cell atrophy with development of pseudocanalicular acini on the 5th and 9th day. 3. An increasing interstitial histiolymphoplasmocytic pancreatitis on the 5th and 9th experimental day. Ultrastructurally, the acinar cell necroses proved as the final stage of a step-by-step developing acute lethal cell damage. The dedifferentiating acinar cell atrophy corresponds to a chronic sublethal cell injury with alteration of different cytoplasmic components. The interstitial pancreatitis in immune serum treatment is characterized by differently activated histiocytes and lymphocytes as well as by mature plasma cells. Because of immune histological findings (peri- and intraacinar deposition of rabbit globulin, specific fixation of guinea-pig complement, and appearance of mouse globulin in the mouse exocrine pancreas) and control experiments with rabbit and mouse normal serum as well as with physiological saline, the pathogenesis of the induced xenogeneic immune pancreatitis is regarded as a twophase process: 1. The acinar cell necroses are mainly due to a cytotoxic immune reaction (possibly in combination with an immune complex reaction) caused by specific anti-pancreatic enzyme antibodies of the applied immune sera. The dedifferentiating acinar cell atrophy may be the result of a specific action of the anti-enzyme antibodies against the corresponding pancreatic enzymes in the apical secretion granules of the pancreatic acinar cells. 2. The interstitial histiolymphoplasmocytic pancreatitis is mainly the morphologic substrate of an extravascularly (intraperitoneally) induced serum sickness reaction (immune complex reaction) due to the foreign proteins applied with the xenogeneic immune sera.     

Effects of ORP150 on appearance and function of pancreatic beta cells following acute necrotizing pancreatitis

Pancreatic beta cells produce and release insulin, which decreases the blood glucose level. Endoplasmic reticulum stress caused pancreatic beta cell dysfunction and death in acute necrotizing pancreatitis (ANP). The 150 kD oxygen-regulated protein (ORP150) took part in the process of endoplasmic reticulum stress. This study investigated the effect of ORP150 on appearance and function of pancreatic beta cells in ANP.Acute necrotizing pancreatitis relied on retrograde infusion of 5% sodium taurocholate into the bile-pancreatic duct. The severity of ANP was estimated by serum amylase, secretory phospholipase A_2, and pancreatic histopathology. The changes in appearance and function of pancreatic beta cells were detected by light and electron microscopy and the levels of serum glucose, insulin, and C-peptide. ORP150 expression was studied using western blot and immunohistochemisty assay.The expression of ORP150 mainly appeared on pancreatic beta cells and decreased gradually during the pathogenesis of ANP. The results of light and electron microscopy indicated pancreatic beta cell dysfunction and death, concomitant with elevation of serum glucose, insulin, and C-peptide in ANP.These results imply a probable role of ORP150 in the changes in appearance and function of pancreatic beta cells following acute necrotizing pancreatitis, through the pathway of endoplasmic reticulum stress.     

Serotonin promotes acinar dedifferentiation following pancreatitis‐induced regeneration in the adult pancreas

The exocrine pancreas exhibits a distinctive capacity for tissue regeneration and renewal following injury. This regenerative ability has important implications for a variety of disorders, including pancreatitis and pancreatic cancer, diseases associated with high morbidity and mortality. Thus, understanding its underlying mechanisms may help in developing therapeutic interventions. Serotonin has been recognized as a potent mitogen for a variety of cells and tissues. Here we investigated whether serotonin exerts a mitogenic effect in pancreatic acinar cells in three regenerative models, inflammatory tissue injury following pancreatitis, tissue loss following partial pancreatectomy, and thyroid hormone‐stimulated acinar proliferation. Genetic and pharmacological techniques were used to modulate serotonin levels in vivo. Acinar dedifferentiation and cell cycle progression during the regenerative phase were investigated over the course of 2 weeks. By comparing acinar proliferation in the different murine models of regeneration, we found that serotonin did not affect the clonal regeneration of mature acinar cells. Serotonin was, however, required for acinar dedifferentiation following inflammation‐mediated tissue injury. Specifically, lack of serotonin resulted in delayed up‐regulation of progenitor genes and delayed the formation of acinar‐to‐ductal metaplasia and defective acinar cell proliferation. We identified serotonin‐dependent acinar secretion as a key step in progenitor‐based regeneration, as it promoted acinar cell dedifferentiation and the recruitment of type 2 macrophages. Finally, we identified a regulatory Hes1–Ptfa axis in the uninjured adult pancreas, activated by zymogen secretion. Our findings indicated that serotonin plays a critical role in the regeneration of the adult pancreas following pancreatitis by promoting the dedifferentiation of acinar cells. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Interleukin-1β induces autophagy by affecting calcium homeostasis and trypsinogen activation in pancreatic acinar cells

The strong up-regulation of inflammatory mediators has been reported to play a key role in acute pancreatitis (AP). Elevated serum levels of interleukin-1β (IL-1β) are associated with the development of AP. However, the precise effect and mechanism of IL-1β in AP remains obscure. In this study, we investigated the potential role and mechanism of IL-1β in AP. We measured autophagy activation in response to IL-1β in AR42J cells. The disrupting effects of IL-1β on cellular Ca²⁺ were observed. To determine whether the disruption of Ca²⁺ signaling has protective effects in vivo during AP, male C57BL/6 mice were treated with cerulein to induce AP. We found that the treatment of AR42J cells with IL-1β triggered autophagy and that the autophagic flux was impaired. In addition, IL-1β induced Ca²⁺ release from the ER. Furthermore, the expression of the ER stress markers GRP78 and IRE1 also increased. 2APB, an antagonist of the InsP₃ receptor, inhibited increased expression of autophagy markers. Subsequent biochemical assays revealed that co-culture with IL-1β could induce the activation of trypsinogen to trypsin and reduce the viability of acinar cells. Pathological changes of the pancreas were also observed in vivo. We found that the pathological injuries of the pancreas were significantly alleviated in mice co-treated with 2APB. Taken together, our results indicate that IL-1β can induce trypsin activation and decrease cellular viability in pancreatic acinar cells. These effects depend on impaired autophagy via intracellular calcium changes. Ca²⁺ signaling may become a promising therapeutic target in the treatment of pancreatitis.     

Electron microscopic studies of viral pancreatitis in coxsackie B4 virus infected mice.

Using a high titer culture of Coxsackie B₄ virus, cytonecrosis consistent with picornaviral infection and cytopathic changes indicative of viral pancreatitis were produced in the pancreas of newborn mice. Virocytonecrosis was manifested within acinocytes by formation of characteristic membrane-vesicle complexes, dilatation of rough endoplasmic reticulum, margination of nuclear chromatin, pyknosis of nuclei, and acute inflammation. Viral pancreatitis was characterized by formation of cytosegresomes, fibroid bodies, and sloughed damaged acinar cytoplasm apparent in autophagic vacuoles of macrophages. Immature leucocytic cells were closely aligned to the wall of acinar ducts. Beta cell damage was also observed. Of significance was the observation of aggregates of Coxsackie B₄ virus particles in various pancreatic cells of 11 of the 23 mice studied. These aggregates appeared in the typical crystalline form with cubic and hexagonal lattice configurations and as viral particles circularly arranged into eight-sided and ten-sided “rosettes.” These findings strongly support the concept that viruses are etiologic agents in pancreatitis and diabetes mellitus.     

Mitochondrial function and malfunction in the pathophysiology of pancreatitis

As a primary energy producer, mitochondria play a fundamental role in pancreatic exocrine physiology and pathology. The most frequent aetiology of acute pancreatitis is either gallstones or heavy alcohol consumption. Repeated episodes of acute pancreatitis can result in the development of chronic pancreatitis and increase the lifetime risk of pancreatic cancer 100-fold. Pancreatic cancer is one of the most common causes of cancer mortality with only about 3?C4?% of patients surviving beyond 5?years. It has been shown that acute pancreatitis involves Ca²⁺ overload and overproduction of reactive oxygen species in pancreatic acinar cells. Both factors significantly affect mitochondria and lead to cell death. The pathogenesis of inflammation in acute and chronic pancreatitis is tightly linked to the induction of necrosis and apoptosis. There is currently no specific therapy for pancreatitis, but recent findings of an endogenous protective mechanism against Ca²⁺ overload??and particularly the potential to boost this protection??bring hope of new therapeutic approaches.     

Expression of insulin-like growth factor binding protein-4 (IGFBP-4) in acute pancreatitis induced by L-arginine in mice

The mechanisms of injury and regeneration after acute pancreatitis are still incompletely understood. Insulin-like growth factor binding proteins (IGFBPs) have been reported to play roles in various pancreatic diseases, but the involvement of insulin-like growth factor binding protein-4 (IGFBP-4) in acute pancreatitis is unknown. The aim of the study was to examine the expression of IGFBP-4 in mice with acute pancreatitis induced by two doses of l-arginine. IGFBP-4 expression was assayed by microarray test, real-time RT-PCR, Western blotting, ELISA and by an immunohistochemical assay. Microarray test of pancreatic mRNA showed that IGFBP-4 mRNA increased significantly after l-arginine treatment and the increase was confirmed by real-time RT-PCR. Western blotting and ELISA assay showed similar patterns of increase of IGFBP-4 in pancreatic tissues and serum. In the control pancreas, IGFBP-4 was mainly immunolocalized in the pancreatic islets. In the pancreatic tissues of mice with pancreatitis induced by l-arginine, the immunolocalization of IGFBP-4 was detected in both acinar cells and pancreatic islets. In conclusion, our results suggest that IGFBP-4 may play a potential role in pancreatic injury and regeneration in a murine model of acute pancreatitis induced by l-arginine.     

Necrotizing pancreatitis due to simian adenovirus type 31 in a rhesus monkey.

Simian adenovirus type 31 was isolated from pancreatic tissue of an 8-year-old male rhesus monkey that died as the result of acute necrotizing pancreatitis. Histologically, the pancreas showed wide-spread necrosis, extensive infiltration of polymorphonuclear leukocytes, and nuclear inclusions in pancreatic acinar cells. Large numbers of adenovirus particles were demonstrable in the acinar cells by electron microscopy. This is the second case of adenovirus-associated pancreatitis we have found in rhesus monkeys and the first case in which we attempted to isolate the virus. Our data indicate that adenovirus pancreatitis is a distinct entity in rhesus monkeys and may occur with greater frequency than is generally believed.