Taking organelles apart, putting them back together and creating new ones: lessons from the endoplasmic reticulum

The endoplasmic reticulum (ER) is a highly dynamic organelle. It is composed of four subcompartments including nuclear envelope (NE), rough ER (rER), smooth ER (sER) and transitional ER (tER). The subcompartments are interconnected, can fragment and dissociate and are able to reassemble again. They coordinate with cell function by way of protein regulators in the surrounding cytosol. The activity of the many associated molecular machines of the ER as well as the fluid nature of the limiting membrane of the ER contribute extensively to the dynamics of the ER. This review examines the properties of the ER that permit its isolation and purification and the physiological conditions that permit reconstitution both in vitro and in vivo in normal and in disease conditions.     

Regulation of purinergic signaling in biliary epithelial cells by exocytosis of SLC17A9-dependent ATP-enriched vesicles

ATP in bile is a potent secretogogue, stimulating biliary epithelial cell (BEC) secretion through binding apical purinergic receptors. In response to mechanosensitive stimuli, BECs release ATP into bile, although the cellular basis of ATP release is unknown. The aims of this study in human and mouse BECs were to determine whether ATP release occurs via exocytosis of ATP-enriched vesicles and to elucidate the potential role of the vesicular nucleotide transporter SLC17A9 in purinergic signaling. Dynamic, multiscale, live cell imaging (confocal and total internal reflection fluorescence microscopy and a luminescence detection system with a high sensitivity charge-coupled device camera) was utilized to detect vesicular ATP release from cell populations, single cells, and the submembrane space of a single cell. In response to increases in cell volume, BECs release ATP, which was dependent on intact microtubules and vesicular trafficking pathways. ATP release occurred as stochastic point source bursts of luminescence consistent with exocytic events. Parallel studies identified ATP-enriched vesicles ranging in size from 0.4 to 1 μm that underwent fusion and release in response to increases in cell volume in a protein kinase C-dependent manner. Present in all models, SLC17A9 contributed to ATP vesicle formation and regulated ATP release. The findings are consistent with the existence of an SLC17A9-dependent ATP-enriched vesicular pool in biliary epithelium that undergoes regulated exocytosis to initiate purinergic signaling.     

双歧三联活菌胶囊对急性胰腺炎患者肠黏膜屏障功能的保护作用

目的探讨双歧三联活菌胶囊对急性胰腺炎患者肠黏膜屏障功能的保护作用。方法选取急性胰腺炎患者66例,随机分为观察组和对照组。两组患者均予以禁食水、持续胃肠减压、解痉镇痛、抗感染、抑制胃酸分泌、生长抑素、改善局部微循环和保持水电解质酸碱平衡等常规治疗。观察组患者加用双歧杆菌三联活菌胶囊420mg水化后自胃管灌注,夹管1．5h,3次／d,连用7d。观察两组患者的治疗前后肠黏膜屏障功能的变化情况,并比较其临床疗效。结果治疗7d后,两组患者血清内毒素、TNF—α和D-乳酸水平均有明显下降（P〈0．05或P〈0．01）,且观察组下降值比对照组更明显（P〈0．05）;同时观察组患者临床总有效率为93．94％,明显高于对照组的75．76％（χ2=4．24,P〈0．05）。结论双歧杆菌三联活菌胶囊治疗急性胰腺炎效果较好,能降低血清内毒素、TNF-α和D-乳酸水平,对患者肠黏膜屏障功能具有良好的保护作用。     

A single-nucleotide polymorphism in tumor necrosis factor-α (− 308 G/A) as a biomarker in chronic pancreatitis

Objective

Chronic pancreatitis is a gradual, long-term inflammation of the pancreas that results in alteration of its normal structure and function. The study aims to investigate the role of − 308 (G/A) polymorphism of TNF-α gene in chronic pancreatitis.

Material and methods

A total of 200 subjects were included in this case–control study. A total of 100 in patients admitted in the Gastroenterology Unit of Gandhi Hospital and Osmania General Hospital, Hyderabad were included in the present study. An equal number of healthy control subjects were randomly selected for the study. The genotyping of TNF-α gene was carried out by tetra-primer ARMS PCR followed by gel electrophoresis. The TNF-α levels were assayed by enzyme-linked immunosorbent assay.

Results

A significant variation with respect to the genotypic and allelic distribution in the disease group when compared to control subjects [OR = 2.001 (1.33–3.005), p < 0.0001**] was observed. Subjects homozygous for the A allele had higher TNF-α levels compared to G allele.

Conclusion

The present study revealed a significant association of the TNF-α gene promoter polymorphism with chronic pancreatitis. Thus, TNF-α genotype can be considered as one of the biological markers in the etiology of chronic pancreatitis.     

重症急性胰腺炎患者血清IL-6水平与内毒素移位及肠黏膜紧密连接蛋白表达关系的研究(英文)

目的:大量研究表明重症急性胰腺炎(SAP)患者血清中高浓度IL-6和肠黏膜低表达的紧密连接蛋白可促进内毒素移位的发生。本文主要研究重症胰腺炎患者血清IL-6水平对内毒素移位和肠黏膜紧密连接蛋白表达的影响。方法:50例重症胰腺炎患者,其中12例在患病早期因结肠受累合并腹胀,对12例结肠受累患者应用结肠镜行结肠灌洗进行腹腔减压,同时取结肠黏膜进行活组织检查。所有病人在治疗的第3天,第7天,第10天,第14天抽取外周静脉血。40例健康志愿者作为对照组。应用ELISA方法检测血清IL-6水平,鲎试验(LAL)方法检测血清内毒素含量,应用免疫荧光和Western blotting方法检测肠黏膜紧密连接蛋白表达水平。结果:SAP患者血清IL-6和内毒素含量明显高于健康对照组,而结肠黏膜紧密连接蛋白表达低于对照组;在临床治疗过程中,早期SAP患者血清IL-6和内毒素水平高于晚期(P值均0.05)。SAP早期血清高浓度的IL-6与结肠黏膜紧密连接蛋白的低表达具有相关性,差异有统计学意义(r=0.735,P0.05)。结论:血清IL-6水平可作为早期评价重症急性胰腺炎严重程度的一项指标,IL-6水平与重症急性胰腺炎临床病程有相关性,可能导致肠道内毒素移位。     

胆总管内注射无水乙醇致胆道闭锁动物模型的建立

目的应用胆总管内注射无水乙醇建立SD大鼠胆道闭锁模型。方法将SD雄性大鼠随机分为实验组和对照组,在实验组中经静脉留置针插入胆总管注入无水乙醇,对照组注入生理盐水。观察SD大鼠的生化及病理结果。结果在实验组中SD大鼠根据病理及生化检测分为肝功能持续恶化组和肝功能修复组,肝功能持续恶化组在8周以后生化明显高于对照组及肝功能修复组。常规HE染色及SMA、Masson染色也出现明显变化。结论胆总管无水乙醇注射诱导胆道闭锁模型是一种可靠的动物模型,此动物模型会帮助人们进一步研究胆道闭锁提供更多的研究手段。     

Abdominal paracentesis drainage attenuates intestinal mucosal barrier damage through macrophage polarization in severe acute pancreatitis

Abdominal paracentesis drainage (APD), as an effective treatment of severe acute pancreatitis (SAP) in clinical settings, can ameliorate intestinal barrier damage and the overall severity of SAP. However, the mechanism underlying therapeutic effects of APD on damaged intestinal mucosal barrier during SAP is still unclear. Here, SAP was induced by injecting 5% Na-taurocholate retrograde into the biliopancreatic duct of rats to confirm the benefits of APD on enteral injury of SAP and further explore the possible mechanism. Abdominal catheter was placed after SAP was induced in APD group. As control group, the sham group received no operation except abdominal opening and closure. By comparing changes among control group, sham group, and APD group, APD treatment obviously lowered the intestinal damage and reduced the permeation of intestinal mucosal barrier, which was evidenced by intestinal H&E staining, enteral expression of tight junction proteins, intestinal apoptosis measurement and detection of serum diamine oxidase, intestinal fatty acid binding protein and D-lactic acid. Furthermore, we found that APD polarized intestinal macrophages toward M2 phenotype by the determination of immunofluorescence and western blotting, and this accounts for the benefits of APD for intestinal injury in SAP. Importantly, the protective effect against intestinal injury by APD treatment was mediated through the inhibited ASK1/JNK pathway. In summary, APD improved the intestinal mucosal barrier damage in rats with SAP through an increasing portion of M2 phenotype macrophages in intestine via inhibiting ASK1/JNK pathway.     

Retracted: microRNA-542-5p protects against acute lung injury in mice with severe acute pancreatitis by suppressing the mitogen-activated protein kinase signaling pathway through the negative regulation of P21-activated kinase 1

Severe acute pancreatitis (SAP) is a condition associated with high rates of mortality and lengthy hospital stays. In the current study, SAP mouse models were established in BALB/c wild-type and P21-activated kinase 1 (PAK1) knockdown mice with the objective of determining the expression of microRNA-542-5p (miR-542-5p) and the subsequent elucidation of the mechanism by which it influences acute lung injury (ALI) by mediating mitogen-activated protein kinase (MAPK) signaling and binding to PAK1. The targeting relationship between miR-542-5p and PAK1 was verified using the bioinformatics prediction website and by the means of a dual-luciferase reporter assay. Following the SAP model establishment, the mice were assigned into various groups with the introduction of different mimic and inhibitors in an attempt to investigate the effects involved with miR-542-5p on inflammatory reactions among mice with SAP-associated ALI. Our results indicated that PAK1 was targeted and negatively mediated by miR-542-5p. Mice with SAP-associated ALI exhibited an increased wet-to-dry weight ratio, myeloperoxidase activity, serum amylase activity, TNF-α, interleukin-1 beta (IL-1β), and intercellular adhesion molecule-1 (ICAM-1) contents, p-p38MAPK, p-ERK1/2, and p-JNK protein levels as well as PAK1 positive expression, while decreased miR-542-5p levels were observed. Functionally, overexpression of miR-542-5p improves ALI in mice with SAP via inhibition of the MAPK signaling pathway by binding to PAK1.Based on the evidence from experimental models, miR-542-5p was shown to improve ALI among mice with SAP, while suggesting that the effect may be related to the inactivation of the MAPK signaling pathway and downregulation of PAK1 gene. Thus, miR-542-5p could serve as a promising target for ALI treatment.     

The substance P content of peripheral tissues in several mammals

Levels of substance P immunoreactivity (SPI) were determined in several skin and mucosal areas, in parts of the sympathetic nervous system, the urinary, biliary and respiratory systems of cats, rabbits and guinea-pigs, and in various skin and mucosal areas of humans by radioimmunoassay. Salient findings are (1) The general distribution pattern of SPI in rabbits was similar to that in rodents. (2) The highest SPI tissue levels were found in the sympathetic nervous system, notably in guinea-pigs. (3) The guinea-pig also had the highest SPI levels in ureter, urinary bladder and bile duct. (4) The aorta, pulmonary artery and portal vein of the rabbit contained very low amounts of SPI, the concentration in the carotid sinus being several fold higher. (5) Skin SPI content was generally highest in the cat, especially in the hindpaw-pad, and lowest in abdominal and back skin. (6) SPI levels found in postmortem human skin and mucosal samples are comparable to those found in other mammals. The observations are discussed in view of the sensory innervation of the various tissues.     

Adoptive transfer of CD3+ T cells and CD4+CD44high memory T cells induces autoimmune pancreatitis in MRL/MpJ mice

The immunopathogenesis of autoimmune pancreatitis (AIP) is poorly understood. Here, we have used MRL/MpJ mice, a model of spontaneous AIP, to address the role of cellular autoimmune processes in the initiation and progression of the disease. Therefore, different T cell subpopulations were adoptively transferred from sick to still healthy (but susceptible) MRL/MpJ mice. Unpurified splenocytes and CD3⁺ T cells both efficiently induced AIP, while CD4⁺ and CD8⁺ T cells alone, as well as splenocytes from healthy mice, were insufficient to trigger the disease. Strikingly, CD4⁺CD44^high memory T cells, although transferred at lower numbers than other T cells, also induced AIP in recipient mice. Employing a modified experimental design, we also evaluated the effects of regulatory T cells (T_regs) on the progression of AIP in already diseased mice. Under the given experimental conditions, there was no significant suppressive effect of adoptively transferred T_regs on pancreatic histopathology. The results of our studies suggest a key role of T cell‐mediated processes in murine AIP. The effects of CD4⁺CD44^high memory T cells are in accordance with genetic studies of our group, which had previously implicated this cell type into the pathogenesis of AIP. In follow‐up studies, we will focus on the interplay of cellular and humoral autoimmunity in the context of AIP.