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

BackgroundPremature 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.MethodsAP was induced by 7 hourly intraperitoneal injections of cerulein (50 μg/kg) in wild-type and pancreas-specific conditional Ctsb knockout (Ctsb^Δpan), Ctsl knockout (Ctsl^Δpan), and Ctsb;Ctsl double-knockout (Ctsb^Δpan;Ctsl^Δpan) 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.ResultsDouble 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. Ctsb^Δpan;Ctsl^Δpan mice had comparable serum amylase activity and histopathological changes and displayed similar levels of proinflammatory cytokines, apoptosis, and autophagy activity compared with wild-type, Ctsb^Δpan, and Ctsl^Δpan mice.ConclusionDouble 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.     

Ovine Iliac Vein Model for Endovascular Thrombectomy of Acute Deep Venous Thrombosis

An ovine iliac vein thrombosis model was devised to test a wall-contacting rotational thrombectomy device. Thrombosis was successfully induced in 9 sheep with an average clot length of 31 mm ± 12 and >60% vessel occlusion on angiography. The thrombus was subsequently removed, maintaining normal intraoperative pulmonary arterial pressure (5.9 mm Hg ± 3.6) and complete distal reperfusion after thrombectomy. Additionally, the sheep were without signs of vascular trauma or embolic complications on gross necropsy and histopathologic analysis. The findings from this study support the use of an ovine iliac deep vein thrombosis model for testing of a lower extremity thrombectomy device.     

What is the impact of zinc deficiency for pancreatectomies in patients with pancreatic ductal adenocarcinoma?

Backgroundand purpose: Zinc is an essential element for human health and plays an important role in metabolic, immunological and other biological processes. The present study was conducted to investigate the association between zinc deficiency (ZD) and the perioperative clinical course in patients with pancreatic ductal adenocarcinoma (PDAC).MethodsOf 216 patients with PDAC who underwent elective pancreatectomy between 2013 and 2017 at our institution, 206 patients with sufficient clinical data were retrospectively reviewed. The perioperative variables were compared and the risk factors associated with infectious complications were identified.ResultsZD was preoperatively present in 36 (17.5%) of 206 patients with PDAC. In the patients of the ZD group, a higher proportion of males, higher preoperative modified Glasgow prognostic scores, a higher neutrophil-to-lymphocyte ratio, and a higher occurrence of postoperative infectious complications after pancreatectomy were observed, compared to the non-ZD group. By a univariate analysis, three risk factors were significantly associated with infectious complications after pancreatectomy: ZD (vs non-ZD: p = 0.002), serum albumin <3.5 g/dl (vs ≥ 3.5 g/dl: p = 0.005), and the procedure of pancreaticoduodenectomy (vs others: p = 0.013). By multivariate logistic regression analysis, the occurrence of infectious complications was significantly associated with ZD (OR 3.430, 95%CI 1.570 to 7.490, p = 0.002) and the procedure of pancreaticoduodenectomy (OR 2.030, 95%CI 1.090 to 3.770, p = 0.025).ConclusionsThe current study newly demonstrated that ZD could serve as a preoperative predictor of infectious complications after pancreatectomies in the patients with PDAC.     

放疗干预对宫颈癌荷瘤小鼠的抑瘤作用及对Th1/Th2免疫细胞比例的影响

目的探讨放疗干预对宫颈癌荷瘤小鼠的抑瘤作用及其对辅助性T细胞1(Th1)/Th2细胞比例的影响。方法建立宫颈癌荷瘤小鼠模型,随机分为荷瘤组和放疗组,每组各10只,另设对照组10只。放疗组进行放疗干预14天,荷瘤组和对照组不治疗。放疗后4、6、8、10、12、14天测量肿瘤体积;末次治疗后,ELISA法测定血清干扰素-γ(IFN-γ)、白介素-2(IL-2)、IL-4、IL-10含量;计算抑瘤率、胸腺指数和脾脏指数;HE染色观察肿瘤组织学变化;TUNEL染色观察肿瘤组织细胞凋亡情况;流式细胞术检测脾脏Th1/Th2细胞比例;RT-qPCR和Western blot检测脾脏T盒子转录因子(T-bet)、GATA结合蛋白3(GABA-3)mRNA和蛋白表达。结果与荷瘤组比较,放疗组小鼠4、6、8、10、12、14天肿瘤体积及瘤质量减小,血清IL-2、IFN-γ升高,IL-4、IL-10降低,胸腺指数、脾脏指数升高,Th1细胞比例、Th1/Th2增加,Th2细胞比例减少,T-bet mRNA和蛋白及T-bet/GATA-3表达升高,GATA-3 mRNA和蛋白表达降低(P<0.05)。HE染色显示,荷瘤组肿瘤细胞数量较多,核大深染,无明显坏死;放疗组肿瘤细胞数量减少,出现大量坏死组织。TUNEL染色显示,荷瘤组TUNEL阳性细胞较少,放疗组TUNEL阳性细胞明显增多。结论放疗对宫颈癌荷瘤小鼠具有明显抑瘤作用,可能是通过调节T-bet/GATA-3表达,促进Th1/Th2分化平衡,增强机体免疫功能发挥作用。     

基于转录组测序方法研究加替沙星对小鼠的肝损伤作用

目的： 探讨加替沙星（GAT）对小鼠肝脏的损伤作用及其机制。方法： 选取32只SPF级雄性昆明小鼠作为研究对象，随机分为4组：低、中、高剂量（分别为25、50、100 mg/kg） GAT组和对照组。给药体积按10 mL/kg，连续灌胃给药7 d，对照组给予对应体积的生理盐水。通过检测各组小鼠血清中的谷丙转氨酶（ALT）、谷草转氨酶（AST）、碱性磷酸酶（AKP）、肌酐（CRE）和甘油三酯（TG）浓度，初步评价加替沙星导致的小鼠肝组织损伤。进一步利用转录组测序技术检测各组小鼠肝脏的基因表达谱，筛选差异表达基因，对差异基因进行基因本体论（GO）功能分类，并采用京都基因与基因组百科全书（KEGG）数据库进行信号通路富集分析。结果： 与对照组比较，高剂量GAT组小鼠肝脏质量显著降低（P<0.01）；低、中剂量GAT组肝脏系数显著降低（P<0.01）；低、中剂量GAT组小鼠血清ALT浓度显著降低（P<0.05或0.01）；低剂量GAT组小鼠血清AST浓度显著降低（P<0.01）。与对照组比较，中剂量GAT组共筛选出27个差异表达基因（包括20个上调基因，7个下调基因）。GO功能分类提示这些基因主要富集在免疫系统、多细胞生物、多生物及生殖过程等17个生物过程中。KEGG通路分析提示差异基因主要富集于脂质代谢、萜类化合物和聚酮化合物的代谢等22条通路中。结论： GAT可导致小鼠肝功能发生变化，并通过影响小鼠肝脏内胆汁酸和胆固醇等内分泌系统和脂质代谢等的平衡，造成肝组织损伤。     

眼表铁过载小鼠干眼模型的构建和机制研究

目的：通过给小鼠腹腔注射右旋糖酐铁，建立小鼠铁过载干眼模型并初步探索其可能的机制。

方法：将40只雄性C57BL/6小鼠(取右眼为实验眼)用随机数字表法将小鼠分为4组：对照组10只，每次腹腔注射生理盐水0.2mL； 低剂量组、中剂量组、高剂量组各10只为模型组，每次分别腹腔注射浓度为12.5、25、50mg/mL的右旋糖酐铁溶液0.2mL。每3d注射1次，共注射28d。注药后第7、14、28d观察各组小鼠眼表炎症指数、角膜荧光素染色、泪膜破裂时间(BUT)及泪液分泌量(SⅠt)，评估干眼及眼表炎症程度。28d后处死小鼠，取角膜、结膜及泪腺组织，进行HE染色、普鲁士蓝染色以及组织铁检测，评估小鼠炎症反应及铁过载情况； 采用酶联免疫吸附试验(ELISA)检测炎症因子白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)、基质金属蛋白酶-9(MMP-9)的表达情况。

结果：与对照组相比，模型组小鼠出现一系列干眼症状，小鼠眼表炎症指数增高，角膜荧光素染色评分增加，BUT缩短，泪液分泌量减少(均P<0.05)； 模型组小鼠角膜、结膜及泪腺组织均受到不同程度的损伤，各组织眼表铁沉积情况较对照组加重，组织铁含量明显增加(均P<0.01)； 模型组小鼠角膜、结膜及泪腺组织中炎症因子(IL-1β、TNF-α、MMP-9)的含量均高于对照组(均P<0.01)，随着右旋糖酐铁注药时间及浓度增加，小鼠干眼及眼表炎症程度逐渐加重。

结论：腹腔注射右旋糖酐铁可成功建立小鼠铁过载干眼模型，其机制可能与铁过载加重眼表炎症反应有关。     

生慧汤对APP/PS1雄性AD小鼠海马内APP代谢产物的昼夜变化影响＊

目的研究生慧汤对APP/PS1（β-amyloid precursor protein/presenilin 1246E）小鼠海马内β淀粉样前体蛋白（Amyloid precursor protein，APP）代谢产物昼夜表达的影响。方法 将56只雄性APP/PS1双转基因痴呆模型小鼠随机分为4组，分别为：痴呆模型组、褪黑素治疗组（0.78 mg·kg^-1·d^-1）、生慧汤高剂量组（27.0 g·kg^-1·d^-1）、生慧汤低剂量组（13.5 g·kg^-1·d^-1），每组14只；对照组为14只雄性C57BL/6J小鼠，连续给药30天。采用Morris水迷宫检测小鼠学习记忆能力，采用酶联免疫吸附检测（ELISA）检测不同时间段取出的海马组织sAPPα含量。对海马β淀粉样蛋白_1-40、β淀粉样蛋白_1-42表达进行免疫印迹分析。免疫组化（IHC）检测海马CA1区Aβ_1-40蛋白的表达。结果 Morris水迷宫结果表明，与对照组相比，模型组上平台潜伏期明显延长、穿越平台次数明显减少（P<0.01）；与模型组相比，生慧汤不同剂量组上平台潜伏期明显缩短（P<0.01、P<0.05），穿越平台次数明显增加（P<0.05）。ELISA结果表明，与对照组相比，模型组的sAPPα总量、各时间段（12：00、24：00）sAPPα含量明显减少（P<0.01）；与模型组相比，生慧汤不同剂量组sAPPα总量明显增多（P<0.01、P<0.05），生慧汤高剂量组仅能增加12：00 sAPPα含量（P<0.01），生慧汤低剂量组仅能增加24：00 sAPPα含量（P<0.05）。对照组sAPPα含量具有明显昼夜差异（P<0.01）。模型组sAPPα含量的昼夜差异性消失（P>0.05）。生慧汤高剂量组sAPPα含量具有昼夜差异（P<0.01），生慧汤低剂量组sAPPα含量不具有昼夜差异（P>0.05）。Western blot结果显示，与对照组相比，模型组Aβ_1-40、Aβ_1-42蛋白表达明显增加（P<0.01）；与模型组相比，生慧汤高剂量组Aβ_1-40、Aβ_1-42蛋白表达减少（P<0.05、P<0.01）；生慧汤低剂量组仅能减少Aβ_1-40蛋白的表达（P<0.05），对Aβ_1-42蛋白表达无影响（P>0.05）。免疫组化结果表明，与对照组相比，模型组Aβ_1-40表达明显增加（P<0.01）；与模型组相比，生慧汤不同剂量组Aβ_1-40表达不同程度减少（P<0.01、P<0.05）。结论 生慧汤能够改善5月龄APP/PS1阿尔茨海默病模型小鼠的学习记忆障碍，其机制可能与恢复海马内APP代谢产物sAPPα的昼夜节律性表达、从而减少Aβ的沉积有关。     

Immunogenicity and protective efficacy of adenoviral and subunit RSV vaccines based on stabilized prefusion F protein in pre-clinical models

Respiratory Syncytial Virus (RSV) remains a leading cause of severe respiratory disease for which no licensed vaccine is available. We have previously described the derivation of an RSV Fusion protein (F) stabilized in its prefusion conformation (preF) as vaccine immunogen and demonstrated superior immunogenicity in naive mice of preF versus wild type RSV F protein, both as protein and when expressed from an Ad26 vaccine vector. Here we address the question if there are qualitative differences between the two vaccine platforms for induction of protective immunity. In naïve mice, both Ad26.RSV.preF and preF protein induced humoral responses, whereas cellular responses were only elicited by Ad26.RSV.preF. In RSV pre-exposed mice, a single dose of either vaccine induced cellular responses and strong humoral responses. Ad26-induced RSV-specific cellular immune responses were detected systemically and locally in the lungs. Both vaccines showed protective efficacy in the cotton rat model, but Ad26.RSV.preF conferred protection at lower virus neutralizing titers in comparison to RSV preF protein. Factors that may contribute to the protective capacity of Ad26.RSV.preF elicited immunity are the induced IgG2a antibodies that are able to engage Fcγ receptors mediating Antibody Dependent Cellular Cytotoxicity (ADCC), and the induction of systemic and lung resident RSV specific CD8 + T cells. These data demonstrate qualitative improvement of immune responses elicited by an adenoviral vector based vaccine encoding the RSV preF antigen compared to the subunit vaccine in small animal models which may inform RSV vaccine development.     

Intervention rates and outcomes in medically managed uncomplicated descending thoracic aortic dissections

ObjectiveTo evaluate the long-term incidence and outcome of aortic interventions for medically managed uncomplicated thoracic aortic dissections.MethodsBetween January 2012 and December 2018, 91 patients were discharged home with an uncomplicated, medically treated aortic dissection (involving the descending aorta with or without aortic arch involvement, no ascending involvement). After a median period of 4 (first quartile: 2, third quartile: 11) months, 30 patients (33%) required an aortic intervention. Patient characteristics, radiographic, treatment, and follow-up data were compared for patients with and without aortic interventions. A competing risk regression model was analyzed to identify independent predictors of aortic intervention and to predict the risk for intervention.ResultsPatients who underwent aortic interventions had significantly larger thoracic (P = .041) and abdominal (P = .015) aortic diameters, the dissection was significantly longer (P = .035), there were more communications between both lumina (P = .040), and the first communication was significantly closer to the left subclavian artery (P = .049). A descending thoracic aortic diameter exceeding 45 mm was predictive for an aortic intervention (P = .001; subdistribution hazard ratio: 3.51). The risk for aortic intervention was 27% ± 10% and 36% ± 11% after 1 and 3 years, respectively. Fourteen patients (47%) underwent thoracic endovascular aortic repair, 11 patients (37%) thoracic endovascular aortic repair and left carotid to subclavian bypass, 3 patients (10%) total arch replacement with the frozen elephant trunk technique, and 2 patients (7%) thoracoabdominal aortic replacement. We observed no in-hospital mortality.ConclusionsThe need for secondary aortic interventions in patients with initially medically managed, uncomplicated descending aortic dissections is substantial. The full spectrum of aortic treatment options (endovascular, hybrid, conventional open surgical) is required in these patients.