Identification of a C-type lectin with antiviral and antibacterial activity from pacific white shrimp Litopenaeus vannamei

C-type lectins (CTLs) play crucial roles in innate immune responses in invertebrates by recognizing and eliminating microinvaders. In this study, a CTL from pacific white shrimp Litopenaeus vannamei (LvCTL3) was identified. LvCTL3 contains a single C-type lectin-like domain (CTLD), which shows similarities to those of other shrimp CTLs and has a mutated ‘EPD’ motif in Ca²⁺-binding site 2. LvCTL3 mRNA can be detected in all tested tissues and expression of LvCTL3 in gills was up-regulated after Lipopolysaccharides, poly (I:C), Vibrio parahaemolyticus and white spot syndrome virus (WSSV) challenges, suggesting activation responses of LvCTL3 to bacterial, virus and immune stimulant challenges. The 5′flanking regulatory region of LvCTL3 was cloned and we identified a NF-κB binding motif in the LvCTL3 promoter region. Dual-luciferase reporter assays indicated that over-expression of L. vannamei dorsal can dramatically up regulate the promoter activity of LvCTL3, suggesting that LvCTL3 expression could be regulated through NF-κB signaling pathway. As far as we know, this is the first report on signaling pathway involve in shrimp CTLs expression. The recombinant LvCTL3 protein was expressed in Escherichia coli and purified by Ni-affinity chromatography. The purified LvCTL3 can agglutinate Gram-negative microbe Vibrio alginolyticus and V. parahaemolyticus and Gram-positive bacteria Bacillus subtilis in the presence of calcium ions, but cannot agglutinate Gram-positive bacteria Streptococcus agalactiae. The agglutination activity of LvCTL3 was abolished when Ca²⁺ was chelated with EDTA, suggesting the function of LvCTL3 is Ca²⁺-dependent. In vivo challenge experiments showed that the recombinant LvCTL3 protein can significantly reduce the mortalities of V. parahemolyticus and WSSV infection, indicating LvCTL3 might play significant roles in shrimp innate immunity defense against bacterial and viral infection.     

Expression and prognostic role of Spy1 as a novel cell cycle protein in hepatocellular carcinoma

ObjectivesSpy1 is a novel cell cycle regulatory gene, which can control cell proliferation and survival through the atypical activation of cyclin-dependent kinases. Recent studies suggested that deregulation of Spy1 expression plays a key role in oncogenesis. To investigate the potential roles of Spy1 in hepatocellular carcinoma (HCC), expression of Spy1 was examined in human HCC samples.MethodsImmunohistochemistry and Western blot analysis was performed for Spy1 in 61 hepatocellular carcinoma samples. The data were correlated with clinicopathological features. The univariate and multivariate survival analyses were also performed to determine their prognostic significance.ResultsSpy1 was overexpressed in hepatocellular carcinoma as compared with the adjacent normal tissue. High expression of Spy1 was associated with histological grade and the level of alpha fetal protein (AFP) (P = 0.009 and 0.003, respectively), and Spy1 was positively correlated with proliferation marker Ki-67 (P < 0.001). Univariate analysis showed that Spy1 expression was associated with poor prognosis (P = 0.03). Multivariate analysis indicated that Spy1 and Ki-67 protein expression was an independent prognostic marker for HCC (P = 0.001 and 0.012, respectively). While in vitro, following release from serum starvation of HuH7 HCC cell, the expression of Spy1 was upregulated.ConclusionsOur results suggested that Spy1 overexpression is involved in the pathogenesis of hepatocellular carcinoma, it may be a favorable independent poor prognostic parameter for hepatocellular carcinoma.     

Thrombospondin I activates the macrophage Toll-like receptor 4 pathway

Previously, we demonstrated that macrophages from thrombospondin 1 （TSP1）-deficient mice have a reduced inflammatory phenotype, suggesting that TSP1 plays a role in macrophage activation. In this study, we determined how TSP1 regulates macrophage function. We found that recombinant or purified piatelet human TSP1 treatment stimulated tumor-necrosis factor （TNF）-α expression in bone marrow-derived macrophages in a time- and dose-dependent manner. Toll-like receptor 4 （TLR4） expression （at the mRNA and protein levels） and nuclear factor-kappaB （NF-KB） activity were also stimulated by TSP1 treatment. The TSPl-mediated increase in TNF-a production was abolished in TLR4-deficient macrophages, suggesting that TSP1 activates macrophages through a TLR4-dependent pathway. TSP1 also stimulated TLR4 activation in macrophages in vivo. Furthermore, TSPl-mediated macrophage activation was attenuated by using a peptide or an antibody to block the association between TSP1 and CD36. Taken together, these data suggest that the stimulation of the macrophage TLR4 pathway by TSP1 is partially mediated by the interaction of TSP1 with its receptor, CD36.     

人源抗破伤风毒素单链二硫键稳定抗体的重组设计与表达

目的 构建人源抗破伤风毒素重链C端单链二硫键稳定抗体原核表达载体,并进行原核表达和生物学特性鉴定.方法 采用PCR定点突变的方法,获得二硫键稳定的抗破伤风毒素重链C端(TeNT-Hc)单链抗体基因(27G-scdsFv).连接pET22b(+)载体,转化大肠杆菌BL21 (DE3)工程菌,IPTG诱导表达,SDS-PAGE、Western blot法鉴定表达产物.ELISA检测27G-scdsFv体外抗原特异结合活性和抗体相对稳定性;非竞争酶免法检测抗体亲和力.采用免疫荧光法检测27G-scdsFv体外中和活性.结果 测序结果显示获得正确的27G-scdsFv基因.原核表达scdsFv以包涵体形式存在,表达量约占菌体总蛋白的50％.复性后的27G-scdsFv保持了与TeNT-Hc的特异结合活性,亲和力较其scFv形式略有提升,KD =0.93×10-7 mol/L,1L培养物可获得5 mg scdsFv蛋白.27G-scdsFv的稳定性较scFv形式明显增强.27G-scdsFv在体外可以明显抑制TeNT-Hc与神经元细胞的结合.结论 成功构建人源抗破伤风毒素重链C端单链二硫键稳定抗体原核表达载体,并获得有活性的目的蛋白,为27G-scdsFv的进一步生物学功能研究奠定基础.     

Web-based self-management support training for health professionals: A pilot study

Objective

To evaluate a web-based self-management training for health professionals. Patients spend 99% of their time outside the healthcare system. Thus self-management support from health professionals is central to optimal care. Our objective was to teach health professionals the skills to provide this support.

Methods

Primary care residents and practicing providers enrolled in six groups. Each group received four web-based interactive training sessions derived from self-efficacy theory. Retrospective-pre/post assessed changes in self-management beliefs and confidence. Wilcoxon signed-rank tests with Bonferroni correction compared responses. Focus groups solicited qualitative feedback.

Results

Fifty-seven residents and providers across the United States enrolled. Residents demonstrated positive changes on all belief questions (P 0.001–0.012). Practicing providers had a non-significant positive change on one and significant changes on the remainder (P 0.001–0.018). Both types of participants demonstrated significant increases on confidence questions regarding their ability to support self-management (P < 0.01 for all). Participants described learned techniques as being useful, reducing burnout, and increasing acceptance of patient involvement in care planning.

Conclusion

The web-based self-management support training for health professionals was feasible and changed beliefs and confidence.

Practice implications

The program may maximize patient self-management by increasing provider self-efficacy and skill for self-management support.     

Apoptosis-dependent acute lung injury and repair after intratracheal instillation of noradrenaline in rats

Earlier work in this laboratory showed that noradrenaline (NA) induces apoptosis in primary cultures of alveolar epithelial cells (AECs). Apoptosis of alveolar epithelial cells may promote the collapse of lung barrier function. On this basis we hypothesized that exogenous NA, administered by intratracheal (I.T.) instillation, might induce AEC apoptosis in vivo followed by acute lung injury. Delivery of NA (10 microM) I.T. into male Wistar rats increased labelling of both fragmented DNA, measured by in situ end labelling (ISEL), and the active form of caspase 3 (anti-Casp3) 6 and 20 h after administration (P < 0.05), but instillation of the vehicle alone (PBS) had no effect. Both ISEL and anti-Casp3 labelling were attenuated by concurrent I.T. delivery of the broad-spectrum caspase inhibitor ZVADfmk. After 6 h, most ISEL- and Casp3-positive cells were located in the surfaces of alveolar walls, but after 20 h more were found in alveolar spaces (P < 0.05). Instillation of NA also increased the bronchoalveolar lavage (BAL) content of fluorescent albumin (BODIPY-alb), which had previously been injected intravenously; the increase was reversed by concurrent ZVADfmk administration. These data suggest that NA-induced apoptosis of AECs in vivo is sufficient to invoke transient collapse of AEC barrier function that is rapidly repaired.     

Essential roles for angiotensin receptor AT1a in bleomycin-induced apoptosis and lung fibrosis in mice

Apoptosis of alveolar epithelial cells (AECs) has been implicated as a key event in the pathogenesis of lung fibrosis. Recent studies demonstrated a role for the synthesis and binding of angiotensin II to receptor AT1 in the induction of AEC apoptosis by bleomycin (BLEO) and other proapoptotic stimuli. On this basis we hypothesized that BLEO-induced apoptosis and lung fibrosis in mice would be inhibited by the AT1 antagonist losartan (LOS) or by targeted deletion of the AT1 gene. Lung fibrosis was induced by intratracheal administration of BLEO (1 U/kg) to wild-type C57BL/6J mice. Co-administration of LOS abrogated BLEO-induced increases in total lung caspase 3 activity detected 6 hours after in vivo administration and reduced by 57% BLEO-induced caspase 3 activity in blood-depleted lung explants exposed to BLEO ex vivo (both P < 0.05). Co-administration of LOS in vivo reduced DNA fragmentation and immunoreactive caspase 3 (active form) in AECs, measured at 14 days after intratracheal BLEO, by 66% and 74%, respectively (both P < 0.05). LOS also inhibited the accumulation of lung hydroxyproline by 45%. The same three measures of apoptosis and lung fibrosis were reduced by 89%, 85%, and 75%, respectively (all P < 0.01), in mice with a targeted disruption of the AT1a receptor gene (C57BL/6J-Agtr1a(tm1Unc)). These data indicate an essential role for angiotensin receptor AT1a in the pathogenesis of BLEO-induced lung fibrosis in mice and suggest that AT1 receptor signaling is required for BLEO-induced apoptosis of AECs in mice as it is in rat and human AECs.     

长航对女性黄褐斑的影响研究

目的分析长航对女性黄褐斑的影响。方法选取参加长航任务的17例女性黄褐斑患者(黄褐斑组),并设对照组15例健康女性(健康组),检测长航前、后症状及血清促黑素细胞激素、过氧化脂质、超氧化物歧化酶水平的变化。结果长航3个月后黄褐斑患者皮损面积和颜色较长航前均不同程度扩大和加深(P<0.05),伴随症状失眠、便秘、月经不调均不同程度加重(P<0.05)。黄褐斑组和健康组长航3个月后血清促黑素细胞激素水平均较前升高(P<0.05),黄褐斑组血清促黑素细胞激素长航前、后较健康组均高(P<0.05);黄褐斑组长航3个月后过氧化脂质水平升高、超氧化物歧化酶水平降低(P<0.05),而健康组长航前、后差异无统计学意义(P>0.05)。结论长航能使黄褐斑皮损扩大、颜色加深,伴随症状加重,并使黄褐斑患者的血清促黑素细胞激素和过氧化脂质水平升高、超氧化物歧化酶水平降低。     

活体肝移植术后肝淤血的多层螺旋CT评价及原因分析

目的 探讨CT双期增强扫描对活体右半肝移植(LDLT)受体术后肝淤血的诊断价值,并对比供体术前CT图像分析肝淤血的原因.资料与方法回顾性分析48例不包含肝中静脉LDLT术后2～4周的CT增强扫描图像,观察原肝中静脉引流区域肝实质密度、肝中静脉属支显影情况,并对照不同的手术方式--肝中静脉属支重建或结扎,比较两种处理方式肝淤血发生率有无差异,分析肝淤血的原因.结果 16例(33.3%)显示Ⅴ/Ⅷ段肝实质密度异常.门静脉期淤血区域肝中静脉属支未见显影12例(75.0%),显影4例(25.0%).原肝中静脉Ⅴ/Ⅷ段属支重建和结扎两种手术方式肝淤血发生率差异无统计学意义(P＞0.05).16例淤血区域肝体积为(44.75±1.6)～(126.76±1.8)cm3中位体积(84.62±1.7)cm3,移植肝总体积为(1064.81±6.9)～(1547.37±8.3)cm3中位体积(1183.14±7.5)cm3,两者之比为(5.2±2.3)%(3.1%～12.4%),1个月后复查,肝静脉属支均显影,13例淤血区基本消失,3例淤血区范围缩小.结论多层螺旋CT双期增强扫描有助于LDLT术后受体肝淤血的诊断,测量肝淤血的范围可评价淤血的程度,门静脉期淤血区肝中静脉属支显影情况与患者预后相关.