Short-term homing assay reveals a critical role for lymphocyte function-associated antigen-1 in the hepatic recruitment of lymphocytes in graft-versus-host disease

BACKGROUND/AIMS: The liver is a major target organ of graft versus host disease (GvHD) with massive infiltration of alloreactive lymphocytes resulting in hepatitis and hepatocyte injury. Although adhesive mechanisms have been implicated in the biology of GvHD hepatitis, the identity of homing receptors involved in the initial recruitment of cells from the blood is not known. METHODS: We have developed a short-term homing assay in a model of murine GvHD. Splenocytes from donors at an active stage of GvHD were injected intravenously into adoptive recipients also undergoing GvHD. The recruitment of cells to the liver was assessed 6h after cell transfer. RESULTS: Activated donor CD8 and CD4 lymphocytes expressed lymphocyte function antigen-1 (LFA-1), alpha4-integrins, and P-selectin binding ligands, and localized more efficiently than na?ve T cells. Immunoneutralization of LFA-1 reduced the recruitment of CD8 and CD4 lymphocytes to the liver by more than 60%. Anti-LFA-1 antibody also markedly reduced infiltration of lymphocytes in periportal areas and protected against hepatocellular damage. CONCLUSIONS: We demonstrate a critical role of LFA-1 in the recruitment of activated lymphocytes to the liver and in immune-cell mediated hepatitis. LFA-1 may be an effective therapeutic target for protecting the liver following bone marrow transplantation.     

胱氨酸、半胱氨酸及乙酰半胱氨酸的傅里叶变换拉曼光谱研究

目的为研究蛋白质等高分子化合物的分子结构奠定基础。方法拉曼光谱可作为研究分子晶体中分子振动和晶格振动的重要手段。本文采用傅里叶变换拉曼光谱仪对胱氨酸、半胱氨酸及乙酰半胱氨酸进行光谱检测,对这3种氨基酸的光谱差异进行分析,并对其分子振动光谱与结构特征的相关性进行了分析和探讨。结果与结论获得了3种氨基酸的傅立叶拉曼特征谱信息,为进一步研究它们的SERS提供了基础。     

Behavioral profile of mice with impaired cognition in the elevated plus-maze due to a deficiency in neural cell adhesion molecule

The elevated plus-maze (EPM) test is one of the most used tests for screening levels of anxiety in rodents. In the present study, we studied how impaired cognition due to a deficiency in the neural cell adhesion molecule (NCAM) could affect the behavior of mice in the EPM task. NCAM-knockout mice demonstrated impaired learning in both object-recognition and fear-conditioning tasks. Analysis of the behavior of mice in the EPM task using a minute-by-minute method revealed a profound influence of genotype. Wild-type mice demonstrated quick learning of the aversive properties of the open arms during the first few minutes of a single EPM task, whereas NCAM−/− mice were unable to learn the aversive properties of the open arms of EPM. Wild-type mice also demonstrated habituation to the EPM task in a test/retest paradigm whereas NCAM-knockout mice failed to habituate during the second EPM presentation. Our data show that the anxiolytic-like behavior of NCAM-knockout mice is not just related to levels of innate anxiety but also to their inability to recognize potential danger associated with the open arms of the EPM task.     

Induction of inflammatory cell accumulation by TM-N49 and promutoxin, two novel phospholipase A2

Local inflammation is a prominent characteristic of snakebite wound. Snake venom phospholipase A₂s (PLA₂s) are one of the main components which contribute to accumulation of inflammatory cells. We have isolated TM-N49 and promutoxin from Protobothrops mucrosquamatus venom and investigated their ability in induction of cell accumulation by using an in vivo mouse model. The results showed that both TM-N49 and promutoxin are potent stimuli for induction of neutrophil, lymphocyte, macrophage and eosinophil accumulation in the mouse peritoneum. The TM-N49- and promutoxin-induced inflammatory cell accumulation was inhibited by pretreatment of animals with cyproheptadine, terfenadine and Ginkgolide B, indicating that histamine and PAF is likely to contribute to the cells accumulation. Pre-injection of antibodies against adhesion molecules ICAM-1, CD18, CD11a and L-selectin showed that ICAM-1 is a key adhesion molecule of TM-N49- and promutoxin-induced lymphocyte, macrophage and eosinophil accumulation; CD18 and CD11a plays an important role in the migration of neutrophils, eosinophils and macrophages; and L-selectin is involved in the neutrophil and eosinophil accumulation. In conclusion, induction of inflammatory cell accumulation by TM-N49 and promutoxin confirms that group II PLA₂s is pivotal stimulus for cell infiltration, through which they participate in the formation of snakebite inflammation.     

可溶性血管细胞粘附分子-1与糖尿病肾病相关分析

目的探讨分析2型糖尿病患者血清可溶性血管细胞粘附分子-1（sVCAM-1）与尿蛋白排泄率（UAC）之间的关系。方法用酶联免疫法对75例2型糖尿病患者和40例对照组血清sVCAM-1水平进行测定。结果 2型糖尿病患者血清sVCAM-1[（678±31）ng/ml]水平明显高于对照组[（536±15）ng/ml],P〈0.001,且sVCAM-1与UAC有独立的相关性。结论 2型糖尿病患者内皮和血管损伤导致sVCAM-1表达增加并与患者的肾功能减退密切相关。     

西红花酸对晚期糖基化终产物诱导牛血管内皮细胞E-选择素表达的抑制作用

目的：研究西红花酸（crocetin）对晚期糖基化终产物（advanced glycation end products,AGEs）诱导牛主动脉血管内皮细胞（bovine Endothelial cells,BECs）E-选择素（E-selectin）表达的抑制作用。方法：分离纯化新生牛全血的中性粒细胞与单核细胞,用虎红染色法测定西红花酸对牛单核细胞/中性粒细胞与BECs黏附的影响,Cell-based ELISA法和sABC免疫细胞化学法测定E-选择素蛋白表达变化。结果：AGEs（100μg/mL）能促进中性粒细胞和单核细胞对BECs的黏附（P〈0.01 vs control）,用西红花酸（1,0.1,0.01μmol/L）预孵内皮细胞12h后,再用AGEs作用24h,中性粒细胞和单核细胞对BECs的黏附较AGEs模型组降低,且呈剂量依赖性关系（P〈0.05或0.01）。Cell-based ELISA测定结果显示,AGEs作用4h内,BECs中的E-selectin表达水平上升,之后E-selectin表达下降,8h时,恢复接近正常水平。而西红花酸（1,0.1μmol/L）能使E-selectin表达下降。sABC免疫化学结果也证实西红花酸对AGEs诱导BECs中E-selectin表达有抑制作用。结论：西红花酸可通过抑制黏附分子E-selcetin蛋白表达,从而抑制中性粒细胞和单核细胞对内皮细胞的黏附作用,这可能是西红花酸减轻糖尿病血管病变的作用机制之一。     

剖宫产170例腹壁缝合临床探讨

目的:探讨剖宫产腹壁缝合的临床意义.方法:将产妇分两组,改良组70例缝合腹膜,皮肤皮内缝合.对照组100例,不缝腹膜,皮肤外缝三大针.结果:改良组术后腹壁伤口渗出液少,住院时间短,两组比较有显著差异.结论:改良组术后恢复快,粘连少,腹壁伤口渗出液少,腹部伤口平整、疤痕轻、住院时间短、医疗费用低值得推广.     

Cell adhesion molecule L1 modulates nerve-growth-factor-induced CGRP-IR fiber sprouting

Overexpression of nerve growth factor (NGF) using adenoviruses (Adts) after spinal cord injury induces extensive regeneration and sprouting of calcitonin-gene-related peptide immunoreactive (CGRP-IR) fibers, whereas overexpression of cell adhesion molecules (CAMs) has no effect on the normal distribution of these fibers. Interestingly, co-expression of cell adhesion molecule L1 and NGF significantly decreases (p<0.0001) CGRP-IR fiber sprouting within the spinal cord, when compared to NGF alone. Co-expression of cell adhesion molecules NCAM or N-cadherin had no effect on NGF-induced CGRP-IR fiber sprouting. These data demonstrate that reduced sprouting is specific to L1 co-expression and not other cell adhesion molecules. In vitro studies carried out to address potential mechanisms show that neurite outgrowth over astrocytes overexpressing L1 in the presence of NGF is comparable to controls, indicating that other factors present in vivo might be involved in the L1-mediated reduction in sprouting. One potential factor is semaphorin 3A (sema3A), which mediates growth cone collapse of CGRP-positive axons. Recent studies have shown that L1 is important in sema3A receptor signaling for cortical neurons. In our study, co-expression of sema3A indeed reduces neurite outgrowth from DRG neurons by about 40% on L1-expressing astrocytes. Based on these results, we hypothesize that overexpression of L1 potentiates sema3A signaling resulting in reduced sprouting.     

Blockade of GpIIb/IIIa inhibits the release of vascular endothelial growth factor (VEGF) from tumor cell-activated platelets and experimental metastasis

Evidence that platelets play a role in tumor metastasis includes the observation of circulating tumor cell-platelet aggregates and the anti-metastatic effect of thrombocytopenia and anti-platelet drugs. Platelets have recently been shown to contain vascular endothelial growth factor (VEGF) which is released during clotting. We therefore studied the effects of (1) tumor cell-platelet adherence and tumor cell TF activity on platelet VEGF release; and (2) the effects of GpIIb/IIIa blockade on tumor cell-induced platelet VEGF release, tumor cellinduced thrombocytopenia and experimental metastasis. Adherent A375 human melanoma cells (TF+) and KG1 myeloid leukemia (TF-) cells were cultured in RPMI containing 10% fetal bovine serum. Platelet-rich plasma was obtained from normal citrated whole blood and the presence of VEGF (34 and 44 kDa isoforms) confirmed by immunoblotting. Platelet-rich plasma with or without anti-GpIIb/IIIa (AbciximabTM) was added to A375 monolayers and supernatant VEGF measured by ELISA. Tumor cell-induced platelet activation and release were determined by CD62P expression and serotonin release respectively. In vitro, tumor cell-platelet adherence was evaluated by flow cytometry. In vivo, thrombocytopenia and lung seeding were assessed 30 min and 18 days, respectively, after i.v. injection of Lewis Lung carcinoma (LL2) cells into control or murine 7E3 F(ab') 2 (6 mg/ kg) athymic rats. Maximal in vitro platelet activation (72% serotonin release) occurred 30 min after adding platelets to tumor cells. At this time, 87% of the A375 cells had adhered to platelets. AbciximabTM significantly (P<0.05) reduced platelet adherence to tumor cells as evidenced by flow cytometry. Incubation of A375 cells with platelets induced VEGF release in a time-dependent manner. This release was significantly inhibited by AbciximabTM (81% at 30 min; P<0.05). In the presence of fibrinogen and FII, VEGF release induced by A375 (TF+) cells was significantly higher than that induced by KG1 (TF-) cells (105.5+/-24 vs. 42+/-7 pg/ml; P<0.001). Omitting fibrinogen or FII from the reaction mixture markedly decreased VEGF release. In vivo, GpIIb/IIIa blockade with murine 7E3 F(ab') 2 reduced LL2 tumor cell-induced thrombocytopenia by 90% (P<0.001) and lung seeding by 82% (P<0.05). We conclude that TF-bearing tumor cells can activate platelets largely via thrombin generation, and that such activation is associated with release of VEGF. This may enhance metastasis, possibly by increasing extravasation at points of adhesion to vascular endothelium.     

Effects of Inhibition of PAF,ICAM-1 and PECAM-1 on Gut Barrier Failure Caused by Intestinal Ischemia and Reperfusion

Background: The role of cell adhesion molecules and transmigration of PMNs through the endothelial barrier is probably essential in intestinal ischemia and reperfusion (I/R)-induced gut barrier dysfunction. Although cytokines are released in I/R, it is unclear whether cytokines directly increase permeability or if this phenomenon requires both expression of cell adhesion molecules and PMN adhesion-activation. Endothelial barrier dysfunction plays an important role in the pathogenesis of multiple organ dysfunction syndrome, inducing gut barrier failure, but the mechanisms are not fully understood. The purpose of this study was to evaluate the potential therapeutic value of inhibition of platelet activating factor (PAF), intercellular adhesion molecule-1 (ICAM-1), and platelet endothelial cell adhesion molecule-1 (PECAM1) in gut barrier dysfunction induced by intestinal I/R. Methods: