Microarray analysis of endothelial differentially expressed genes in liver of cirrhotic rats

BACKGROUND & AIMS: There is a long-standing interest in the identification of endothelial-specific pathways for therapeutic targeting in cirrhosis. Therefore, the aim of this study was to evaluate differences in gene expression patterns between liver endothelial cells (LECs) from control and cirrhotic rats by using microarrays. METHODS: LECs were obtained by isopycnic centrifugation. LECs gene expression was then analyzed on high-density oligonucleotide microarrays. RESULTS: Analysis of gene expression revealed that most of the differentially expressed mRNA in cirrhosis are associated with extracellular matrix remodeling, inflammation, antioxidant/stress response, and cell signaling. CONCLUSIONS: The collective expression changes observed within some functional groups of genes indicate that LECs in cirrhotic livers may contribute to lymphangiogenesis, enhancement of fibrogenesis and inflammatory processes, changes in cell-cell interaction with up-regulation of adherens junction proteins, and alterations in the intrahepatic vascular tone because of the down-regulation of genes involved in vasodilatation.     

Chronic ethanol consumption impairs receptor-mediated endocytosis of MAA-modified albumin by liver endothelial cells

Alcoholic liver disease has been associated with abnormalities in receptor-mediated endocytosis (RME) which results in abnormal degradation of metabolically altered proteins. Model systems using formaldehyde-modified albumin (f-Alb) have shown an impairment in RME following chronic alcohol consumption utilizing both in situ perfused rat livers and isolated rat liver endothelial cells (LECs). The discovery that alcohol metabolite derived aldehydes can modify proteins prompted a study to determine if malondialdehyde-acetaldehyde-modified albumin (MAA-Alb) would be degraded similar to that reported for f-Alb, and whether ethanol-fed rats would demonstrate an impaired RME with respect to this ligand which occurs as a consequence of chronic ethanol consumption. MAA-Alb was degraded slightly more than f-Alb in both in situ perfused livers and at the single cell level. This degradation was completely inhibited with 100x unlabeled f-Alb, which suggests the use of a similar receptor. Following alcohol consumption there was a 50-60% decrease in MAA-Alb degradation in whole livers and isolated LECs. Utilizing isolated LECs it was determined that impairment in internalization was the most likely mechanism for the decrease in the amount of MAA-Alb that was degraded. These data show that chronic alcohol consumption by rats does in fact impair RME of alcohol metabolite-derived adducted proteins, and this impairment is due to a defect in the post-internalization step rather than the binding or degradation of the modified protein.     

Maintenance of pathogenic Th2 cells in allergic disorders

Immunological memory is an important protective mechanism that enables host organisms to respond rapidly and vigorously to pathogens that have been previously encountered. In addition to the protective function, memory CD4⁺ T helper (Th) cells play a central role in the pathogenesis of chronic inflammatory disorders, including asthma. Recently, several investigators have identified phenotypically and functionally distinct memory Th2 cell subsets that produce IL-5. These memory Th2 cell subsets play an important role in the pathology of allergic inflammation and function as memory-type “pathogenic Th2 (Tpath2) cells” both in mice and humans. We review the role of lung Tpath2 cells in the development of allergic inflammation and, in the context of recent findings, propose a mechanism by which Tpath2 cells not only survive but also continue to function at the sites where antigens were encountered. A greater understanding of the functional molecules or signaling pathways that regulate the inflammatory niche for Tpath2 cells may aid in the design of more effective treatments for chronic inflammatory disorders.     

Combination of cyst fluid CEA and CA 125 is an accurate diagnostic tool for differentiating mucinous cystic neoplasms from intraductal papillary mucinous neoplasms

Background/objectivesDespite advances in imaging techniques, diagnosis and management of pancreatic cystic lesions still remains challenging. The objective of this study was to determine the utility of cyst fluid analysis (CEA, CA 19-9, CA 125, amylase, and cytology) in categorizing pancreatic cystic lesions, and in differentiating malignant from benign cystic lesions.MethodsA retrospective analysis of 68 patients with histologically and clinically confirmed cystic lesions was performed. Cyst fluid was obtained by surgical resection (n = 45) or endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) (n = 23). Cyst fluid tumor markers and amylase were measured and compared between the cyst types.ResultsReceiver operating characteristic (ROC) curve analysis of the tumor markers demonstrated that cyst fluid CEA provided the greatest area under ROC curve (AUC) (0.884) for differentiating mucinous versus non-mucinous cystic lesions. When a CEA cutoff value was set at 67.3 ng/ml, the sensitivity, specificity and accuracy for diagnosing mucinous cysts were 89.2%, 77.8%, and 84.4%, respectively. The combination of cyst fluid CEA content >67.3 ng/ml and cyst fluid CA 125 content >10.0 U/ml segregated 77.8% (14/18) of mucinous cystic neoplasms (MCNs) from other cyst subtypes. On the other hand, no fluid marker was useful for differentiating malignant versus benign cystic lesions. Although cytology (accuracy 83.3%) more accurately diagnosed malignant cysts than CEA (accuracy 65.6%), it lacked sensitivity (35.3%).ConclusionsOur results demonstrate that cyst fluid CEA can be a helpful marker in differentiating mucinous from non-mucinous, but not malignant from benign cystic lesions. A combined CEA and CA 125 approach may help segregate MCNs from IPMNs.     

Expression of TGF-β2 mRNA and PCNA, FN Protein in Lens Epithelial Cells in Age-related Nuclear and Cortex Cataract

By using RT-PCR and immunohistochemistry, the expressions of transforming growth factor β2 (TGF-β2) mRNA, proliferating cell nuclear antigen (PCNA) and fibronection (FN) protein in lens epithelial cells (LECs) of age-related nuclear and cortex cataract were detected and compared. The results of RT-PCR revealed that the expression of TGF-β2 mRNA was higher in cortex cataract than in nuclear cataract. Immunohistochemistry demonstrated that the expression of PCNA protein was lower and the expression of FN protein was higher in cortex cataract than in nuclear cataract. It was suggested that TGF-β2, PCNA and FN might take important parts in the process of age-related cataract. Cortex cataract was related to the transdifferentiation of LECs, and nuclear cataract to the proliferation of LECs.     

Expression of TGF-β2 mRNA and PCNA, FN Protein in Lens Epithelial Cells in Age-related Nuclear and Cortex Cataract

Summary： By using RT PCR and immunohistochemistry, the expressions of transforming growth factor 132 （TGF-β2） mRNA, proliferating cell nuclear antigen （PCNA） and fibronection （FN） protein in lens epithelial cells （LECs） of age-related nuclear and cortex cataract were detected and compared. The results of RT-PCR revealed that the expression of TGF-β2 mRNA was higher in cortex cataract than in nuclear cataract. Immunohistochemistry demonstrated that the expression of PCNA protein was lower and the expression of FN protein was higher in cortex cataract than in nuclear cataract. It was suggested that TGF-β2 , PCNA and FN might take important parts in the process of age-related cataract. Cortex cataract was related to the transdifferentiation of LECs, and nuclear cataract to the proliferation of LECs.     

Peripheral accumulation of newly produced T and B lymphocytes in natalizumab-treated multiple sclerosis patients

The anti-α4 monoclonal antibody natalizumab inhibits lymphocyte extravasation into the central nervous system and increases peripheral T and B lymphocytes in multiple sclerosis patients. To investigate whether the lymphocyte accumulation was due to a higher lymphocyte production, an altered homeostasis, or a differential transmigration of lymphocyte subsets through endothelia, T-cell receptor excision circles and kappa-deleting recombination excision circles were quantified before and after treatment, T-cell receptor repertoire was analyzed by spectratyping, and T- and B-lymphocyte subset migration was studied using transwell coated with vascular and lymphatic endothelial cells. We found that the number of newly produced T and B lymphocytes is increased because of a high release and of a low propensity of naïve subsets to migrate across endothelial cells. In some patients this resulted in an enlargement of T-cell heterogeneity. Because new lymphocyte production ensures the integrity of immune surveillance, its quantification could be used to monitor natalizumab therapy safety.     

Ccnd1反义寡核苷酸脂质体抑制大鼠晶状体上皮细胞增殖的研究

目的:探索Ccnd1反义寡核苷酸(Ccnd1-ASON)脂质体对培养的大鼠晶状体上皮细胞(LEC)Ccnd1基因表达和细胞增殖的抑制作用。方法:分别用不同浓度的Ccnd1-ASON脂质体(反义组)、Ccnd1正义寡核苷酸(Ccnd1-SON)脂质体(正义组)、空白脂质体(空白组)转染培养的SD大鼠LEC。转染后24h、72h、120h,实时荧光定量PCR(qRT-PCR)检测LEC中Ccnd1表达变化;转染后24h、48h、72h、96h和120h,MTT法连续测定细胞增殖的状况。应用方差分析进行统计学处理。结果:转染后24h、72h、120h,反义组与正义组和空白组相比较Ccnd1表达量均下降,正义组与空白组比较无明显变化。转染后24h内3组细胞增殖未见明显差别;转染后48～120h,反义组的细胞增殖明显慢于正义组和空白组(P<0.05),正义组与空白组之间无明显差异(P>0.05)。结论:Ccnd1-ASON脂质体能有效降低大鼠LEC Ccnd1表达,抑制LEC增殖。     

PCNA反义寡脱氧核苷酸对牛晶状体上皮细胞体外增殖活性的影响

目的　使用增殖细胞核抗原反义寡脱氧核苷酸(PCNAASODN)作用于牛晶状体上皮细胞,观察其对细胞增殖活性的影响,以探讨PCNAASODN技术防治后发性白内障形成的新途径。方法　应用3H-TdR掺入法检测细胞增殖活性,用流式细胞仪检测晶状体上皮细胞PCNA蛋白的表达。结果　PCNAASODN30～50 μmol/L可使细胞增殖显著受抑制,其CPM值分别从对照组的6 699.67下降为5986.33,5 525.33和5 154.67,PCNA蛋白在晶状体上皮细胞的表达阳性率下调至12.32%,与对照组比较有显著性差异(P<0.01)PCNASODN组则作用不明显,与对照组比较无显著性差异(P>0.05),而PCNAASODN组与PCNASODN组比较差异显著(P<0.01)。结论　PCNAASODN可以显著抑制牛晶状体上皮细胞体外增殖活性,其抑制作用可能通过阻断PCNAmRNA的翻译过程而影响PCNA蛋白表达     

兔眼人工晶体植入术后TIMPs在晶体上皮细胞的表达及其意义

目的　观察白内障囊外摘除及人工晶体植入 (ECC IOL)术后基质金属蛋白酶抑制因子 (TIMPs)在晶体上皮细胞的表达 ,探讨TIMPs以ECCO IOL术后后囊膜混浊的影响。方法　 2 5只健康成年家兔 ,均一只眼行晶体囊外摘除及人工晶体植入术 ,另一只未手术眼作为对照组。每 5兔为一组 ,分别于术后 1、3、7、1 4、30d取出晶状体后囊膜 ,用RT -PCR检测各标本中的TIMPsmRNA的表达 ,对扩增产物用凝交成像系统进行定量分析 ,以TIMP/GAPDH的比值表示TIMPsmRNA的相对表达水平 ,并用羟脯氨酸试剂盒检测晶体囊膜羟脯氨酸量的变化。结果　在常晶体上皮细胞组织均有TIMP - 1、- 2、- 3和 - 4mNA的表达 ;术后第 1天 ,TIMP - 1、- 2、- 3和- 4mRNA均明显升高 ,术后第 7天 ,TIMP - 1、- 2和 - 3RNA的表达量达到最大 ,此后表达式量逐渐下降 ,术后第30天的表达量仍高于对照组 ,术后TIMP - 4mRNA则表现为下降 ;结论　白内障囊外摘除及人工晶体植入术后TIMPsmRNA在晶体上皮细胞的表达均明显升高 ,提示TIMPs可能是抑制白内障囊外摘除及人工晶体植入术后细胞外基质降解的主要因素 ,TIMPs可能是后囊膜混浊的形成和纤维化的重要原因之一。