牛源坏死梭杆菌43K外膜蛋白的黏附特性研究

旨在明确牛源坏死梭杆菌43K OMP的黏附特性。将43K OMP基因克隆连接至pET-32a载体,转化入大肠杆菌BL21 DE3中,通过IPTG诱导进行原核表达,应用黏附试验、天然蛋白竞争试验、抗体抑制试验和蛋白酶水解试验,明确牛源坏死梭杆菌43K OMP的黏附性,同时将纯化的重组蛋白和提取的天然43K OMP与牛子宫内膜细胞和牛乳腺上皮细胞共孵育,观察43K OMP对细胞的黏附作用。结果显示：43K OMP基因克隆到pET-32a载体中,随后在大肠杆菌BL21 DE3以包涵体形式成功表达;携带重组质粒(H2019)的大肠杆菌经IPTG诱导后,与空载体对照相比,与宿主细胞的结合显著增强(P<0.05);天然43K OMP与细胞共孵育后,黏附细胞的细菌数量显著降低(P<0.05);H2019与43K OMP多抗或单抗预孵育后,显著降低黏附宿主细胞的细菌数量(P<0.05);经不同浓度蛋白酶K处理H2019后,黏附细胞的细菌数量显著降低(P<0.05),且与蛋白酶K浓度呈现剂量依赖关系。同时,43K OMP天然蛋白和重组蛋白能黏附于牛子宫内膜细胞和乳腺上皮细胞表面。...     

牛坏死杆菌的43 ku外膜蛋白在其黏附细胞中的作用初步分析

旨在明确牛坏死杆菌(Fusobacterium necrophorum)的43 ku外膜蛋白(43K OMP)在其黏附细胞中的作用，本研究将重组43K OMP蛋白和天然43K OMP蛋白与鼠乳腺上皮细胞共孵育，通过免疫荧光方法观察牛坏死杆菌43K OMP对鼠乳腺上皮细胞的黏附作用；同时利用天然蛋白竞争试验和抗体抑制试验明确43K OMP是否介导牛坏死杆菌与细胞的黏附，进一步利用牛坏死杆菌43K OMP基因缺失菌，评价43K OMP基因缺失对牛坏死杆菌黏附力的影响，阐明43K OMP在牛坏死杆菌黏附细胞中的作用。免疫荧光试验结果显示：天然43K OMP和重组43K OMP均能黏附于鼠乳腺上皮细胞表面，天然43K OMP与鼠乳腺上皮细胞或鼠肝细胞预孵育后，牛坏死杆菌黏附数量明显下降(P＜0.05)；牛坏死杆菌与43K OMP多抗或单抗预孵育后，黏附于鼠乳腺上皮细胞或鼠肝细胞的牛坏死杆菌数量显著降低(P＜0.05)；与牛坏死杆菌A25菌株相比，基因缺失菌A25Δ43K OMP黏附宿主细胞能力极显著下降(P＜0.01)，黏附率分别降低了94.4%和90.4%。因此，43K OMP在牛坏死杆菌黏附细胞中发挥关键性作用，深入研究其黏附机理将为揭示牛坏死杆菌致病机制提供理论基础。     

Killing of Brucella antigen-sensitized macrophages by T lymphocytes in bovine brucellosis

The present study was an investigation into the role of T lymphocytes in the killing of antigen-sensitized macrophages (MΦ) in bovine brucellosis. Following confirmation of bovine T lymphocyte cell lines derived from Brucella abortus Strain 19 vaccinated steers as antigen-specific in proliferation studies using various antigens, we adapted an apoptosis assay for evaluation of cytotoxicity by these bovine T cells against autologous monocyte-derived macrophages (MDMΦ) as target cells. Various B. abortus antigen preparations were tested including whole γ-irradiated B. abortus bacteria (γBA), a soluble cytosolic protein fraction and a membrane-associated protein fraction. Both polyclonal and cloned T lymphocyte cell lines exhibited cytotoxicity against MDMΦ targets in an antigen-specific fashion. Polyclonal and cloned T lymphocyte cell lines demonstrated cytotoxic responses to varying degrees against B. abortus antigens regardless of whether the antigen used was whole nonviable bacteria, a soluble protein extract or a membrane-associated fraction of extracted bacteria. To further develop correlation of these responses to an in vivo host defense mechanism, cytotoxicity was evaluated using target cells that had been infected with live B. abortus S19 or B. abortus Strain 2308. Cytotoxic responses were also demonstrated consistently against infected targets with either strain of B. abortus although in most cases, cytotoxicity was higher against target cells sensitized with γBA compared to those infected with live bacteria. Cloned T lymphocyte cell lines were all CD4⁺, CD8⁻ cells indicating that the observed cytotoxic responses were most likely due to an inflammatory T_h1 response and may represent an important host defense mechanism induced by vaccination with live attenuated strains of B. abortus in cattle.     

Inhibition of chicken leukocyte migration in vitro: A direct agarose plate assay

Leukocyte migration inhibition (LMI) is a widely used in vitro correlate of delayed type hypersensitivity (DTH) in mammals. This report describes the development of a direct agarose LMI assay for studying DTH in avian species. Optimum demonstration of LMI was found with leukocytes isolated on a Ficoll-diatrizoate gradient solution. The agarose culture plates were maintained at pH 7.2–7.4 in a water-vapor saturated, 39°C incubator with 2% CO₂ tension.

Antigen specific LMI was demonstrated in chickens with DTH to purified protein derivative of Mycobacterium (PPD) and ferritin. A good comparison between LMI and DTH, as measured by the delayed wattle reaction (DWR), was demonstrated. The effect of bacterial lipopolysaccharide (LPS) on LMI was examined and LPS in microgram quantities was found to inhibit in vitro migration of chicken leukocytes. Contamination of antigen preparations with LPS is a probable explanation for occasional nonspecific inhibition of leukocyte migration since endotoxin is an almost ubiquitous contaminant of antigen preparations.     

Discrimination between sheep antibodies to Brucella melitensis and to Brucella ovis

When preparations containing smooth Brucella abortus lipopolysaccharide (LPS) were used as antigens in an ELISA, strong positive reactions were obtained with sera from sheep infected with Brucella melitensis or with Brucella ovis. Oxidation of the LPS with sodium metaperiodate greatly reduced the extent of the cross-reactions with antisera to B. ovis, with little effect on the reactions with antisera to smooth B. melitensis. Periodate oxidation of hot saline extract (HSX) antigen of B. ovis markedly reduced its reactivity in ELISA with anti-B. ovis sera and eliminated cross-reactivity with anti-B. melitensis sera. The reactivity of HSX was maintained after treatment with proteinase K.

A simple ELISA system, in which replicate samples from a single serum dilution were tested in parallel against both B. ovis HSX antigen and periodate-oxidised smooth phase B. abortus LPS, was evaluated. It was found to discriminate well between antibodies induced by vaccination or virulent infection with B. melitensis strains and those induced by infection with B. ovis.     

坏死梭杆菌白细胞毒素研究进展

坏死梭杆菌是动物和人的各种坏死化脓感染的条件性致病菌.坏死梭杆菌的白细胞毒素是一种高度不稳定性分泌蛋白,被认为是主要的毒力因子.坏死梭杆菌白细胞毒素基因的开放阅读框(lktAORF)包括9 726 bp,编码3 241个氨基酸,总分子质量为336 ku的蛋白,且与其他细菌的细胞毒素没有任何相似的序列.覆盖在整个坏死梭杆菌lktA ORF上的5个短的重叠的多肽分别是BSBSE,SX,GAS,SH和FINAL,将它们在大肠埃希菌中表达,所有的多肽都有免疫原性,但GAS引起最小的抗体反应,BSBSE和SH对坏死梭杆菌攻击诱导产生了很强的保护力,比坏死梭杆菌的培养上清内全长活性lkt或无活性上清的保护性要好得多.     

HO-1在巨噬细胞中的抗炎和抗氧化作用

【目的】旨在揭示血红素加氧酶1(HO-1)在巨噬细胞中的抗炎和抗氧化作用。【方法】利用不同浓度脂多糖(LPS,0、3、5、10、15、20、25μg/mL)、HO-1(0、0.02、0.04、0.06、0.08、0.10μg/mL)及锌原卟啉(zinc protoporphyrin, ZPP;0、5、10、15、20、30 ng/mL)分别处理巨噬细胞(RAW264.7),12 h后通过CCK8法检测RAW264.7细胞活力,计算LPS、HO-1和ZPP处理RAW264.7细胞的最佳浓度。将RAW264.7细胞随机分为对照组(CT)、LPS组(LPS)、LPS+HO-1组(LH)、HO-1组(HO-1)、LPS+HO-1+ZPP组(LHZ),每组3个重复。CT组细胞用含10%胎中血清的DMEM培养基培养;LPS组细胞用LPS处理;LH组细胞用LPS和HO-1共处理;HO-1组细胞用HO-1处理;LHZ组细胞用LPS、HO-1和ZPP共处理,各组细胞的处理时间均为12 h,收集细胞和上清。采用ELISA法检测上清液中白细胞介素-6(IL-6)、IL-8、肿瘤坏死因子α(TNF-α)、活性...     

GPX4失活通过JNK通路缓解LPS诱导巨噬细胞炎症反应

【目的】 研究硒蛋白谷胱甘肽过氧化物酶4（glutathione peroxidases 4,GPX4）失活如何参与调控脂多糖（lipopolysaccharide,LPS）诱导的RAW264.7巨噬细胞炎症反应及其潜在的分子机制。【方法】 体外培养RAW264.7巨噬细胞,以DMSO为对照,使用0.1~5.0 μmol/L GPX4抑制剂FIN56处理,通过CCK-8法检测细胞活力和Western blotting检测GPX4蛋白表达水平,确定抑制剂最适浓度。将RAW264.7巨噬细胞分为4组：对照组,添加DMSO培养24 h;FIN56（GPX4抑制剂）组,添加0.5 μmol/L FIN56培养24 h;DMSO-LPS组,DMSO培养24 h后使用LPS （100 ng/mL）刺激3 h;FIN56-LPS组,FIN56培养24 h后使用LPS刺激3 h。各组细胞经培养后,利用荧光探针2',7'-二氯二氢荧光素二乙酸酯（2',7'-dichlorodi-hydrofluorescein diacetate,H₂DCFDA）检测细胞内活性氧（reactive oxygen species,ROS）水平,使用试剂盒法检测细胞内丙二醛（malondialdehyde,MDA）水平,分别使用ELISA和荧光定量PCR检测促炎细胞因子白细胞介素1β（interleukin-1β,IL-1β）、白细胞介素6（interleukin-6,IL-6）、肿瘤坏死因子α（tumor necrosis factor-α,TNF-α）的分泌水平和基因表达量,使用Western blotting法检测Toll样受体4（toll like receptor 4,TLR4）信号通路相关蛋白表达水平。【结果】 与DMSO处理相比,0.5 μmol/L FIN56处理巨噬细胞24 h对细胞活性无显著影响（P>0.05）,且显著降低GPX4蛋白表达水平（P<0.05）,故选用0.5 μmol/L FIN56用于后续实验。与对照组相比,FIN56和LPS均显著增加了ROS积累（P<0.05）,而与DMSO-LPS组相比,FIN56-LPS组ROS水平显著升高（P<0.05）。与对照组相比,LPS可显著增加小鼠巨噬细胞IL-1β、IL-6和TNF-α的mRNA表达量和IL-6含量,以及c-Jun氨基末端蛋白激酶（c-Jun N-terminal protein kinase,JNK）和c-Jun磷酸化水平（P<0.05）,而FIN56可显著下调LPS诱导的IL-6的mRNA表达量和含量及JNK和c-Jun磷酸化水平（P<0.05）,但对p38蛋白（p38 MAPK,p38）、细胞外调节蛋白激酶（phosphorylate extracellular regulated protein kinases1/2,Erk1/2）蛋白表达水平无显著影响（P>0.05）。【结论】 GPX4失活可有效阻断JNK和c-Jun磷酸化,从而特异性抑制LPS诱导的巨噬细胞的炎症反应。该结果可为以GPX4为靶点研发抗炎治疗策略提供理论依据。     

坏死杆菌白细胞毒素作为腐蹄病亚单位疫苗候选抗原的研究前景

腐蹄病（foot rot）是侵害反刍动物趾间皮肤及深层软组织为主的,严重影响奶牛生产性能和产奶质量的一种常见疾病。由于传统的灭活菌苗具有免疫效果差、副反应严重及大量生产困难等缺点,使腐蹄病基因工程疫苗的研究成为热点。笔者对坏死杆菌白细胞毒素作为腐蹄病亚单位疫苗候选抗原研究的最新进展进行综述,希望为腐蹄病亚单位疫苗的研究提供参考。     

The effect of immunity to core lipopolysaccharides (LPS) on the production of thromboxane and prostacyclin by equine peritoneal macrophages

An experiment was designed to determine whether a change in the ability of macrophages to respond to lipopolysaccharides (LPS) of gram-negative bacteria was involved in the development of cross-reactive immunity to endotoxemia. The endotoxin-induced production of thromboxane A2(TxA2) and prostacyclin (PGI2) by peritoneal macrophages from horses which were hyperimmunized against the common core region of LPS were compared to those in unimmunized horses. Bacterins used for induction of core LPS immunity were prepared from the J-5 mutant of Escherichia coli 0111:B4, and the R 595 mutant of Salmonella minnesota. Serum antibody titers to core LPS were determined by an indirect enzyme-linked immunosorbent assay. Immunized horses had a marked increase in titer to core LPS (p less than 0.05), while there was no change in titer in unimmunized control horses. The only significant difference in the in vitro LPS-induced production of TxA2 and PGI2 by peritoneal macrophages between immunized and control horses was a greater production of TxA2 by macrophages from immunized horses in response to 10 ng/ml LPS (p less than 0.05). Results of this experiment do not support the concept that cross-reactive immunity to LPS is attended by reduced production of TxA2 and PGI2 by equine peritoneal macrophages.     

坏死梭杆菌FN(A)p2001株小鼠感染模型的建立

将鹿源坏死梭杆菌FN(A)p2001分离株以3×108、3×107、3×106、3×105、3×104、3×103个/只等不同菌量,分别接种于6组小鼠,每组10只,逐日观察小鼠感染情况.结果表明,3×106、3×107、3×108个/只菌量感染组小鼠,在接种后3 d～8 d先后死亡,5 d后死亡小鼠的病理变化明显,并从死亡小鼠内脏及脓汁中均检到长丝状及小杆状等多形态典型的坏死梭杆菌;3×104个/只和3×105个/只菌量感染仅表现体重减轻,3×103个/只菌量感染无明显临床表现.由此表明,坏死梭杆菌FN(A)P2001分离株具有很强的感染毒力;对小鼠的最小致死量为106个/只菌量;腹腔接种是适宜的感染途径.从而建立了坏死梭杆菌分离株小鼠感染模型.     

鹿源坏死梭杆菌毒力菌株FN（AB）94实验动物感染模型的建立

将坏死梭杆菌FN（AB）94分离株以10^6,10^7,10^8,10^9 个/mL等不同菌量，于耳后根颈部皮下分别接种于4组的健康实验家兔，每组3只，逐日观察感染家兔的发病情况，结果，10^8,10^9个／mL，菌量感染组家兔，在接种后9－68 d内先后死亡，68d死亡家兔的病理变化明显，并从死亡家兔内脏及脓法叶，应用触片及分离培养均检到长丝状及小杆状等形态典型的坏死梭杆菌，10^7个/mL感染家兔仅表现体重减轻，10^6个／mL感染家兔无明显临床表现，由此表明，坏死梭杆菌FN（AB）94分离株具有很强的感染毒力，对家兔的最小致死量为10^8个／mL，耳后板颈部皮下接种是适宜的感染途径，从而建立了坏死梭杆菌分离株实验动物感染模型。     

Effect of Candida tropicalis,Bacillus subtilis and Lactobacillus Combination on Quality and Aerobic Stability of Sugarcane Tops Silage

The assay was aimed to investigate the effect of different microbial inoculant combinations of Candida tropicalis (CT),Bacillus subtilis (BS),Lactobacillus plantarum (LAP) and Lactobacillus buchneri (LAB) on the quality and aerobic stability of sugarcane tops silage.There were 10 groups:Sugarcane tops silages with no additives served as control group (C);GroupsⅠ1,Ⅰ2 and Ⅰ3 were treated with CT+BS at 10,20 and 30 mL/kg,respectively;Groups Ⅱ1,Ⅱ2 and Ⅱ3 were treated with CT+BS+LAP at 10,20 and 30 mL/kg,respectively;Groups Ⅲ1,Ⅲ2 and Ⅲ3 were treated with CT+BS+LAB at 10,20 and 30 mL/kg,respectively.Each treatment was treated with 0.5% fresh forge urea.The results showed that:①Compared to control group,CT+BS treatment groups had significantly higher levels of pH and acetic acid (P< 0.05),dry matter recovery (DMR) and lactic acid content were significantly decreased (P< 0.05),and the aerobic stability was lower.②Compared to control group,CT+BS+LAP treatment groups had significantly higher levels of pH and lactic acid (P< 0.05),and the aerobic stability was lower.③In CT+BS+LAB treatment groups,the levels of acetic acid were significantly higher and DMR was significantly lower than control group (P< 0.05),and the CP content and aerobic stability were highest in all groups.So the quality of sugarcane tops silage was lower in CT+BS treatment groups and CT+BS+LAB treatment could improve the quality and aerobic stability of sugarcane tops silage.     

热带假丝酵母、枯草芽孢杆菌与乳酸菌组合对甘蔗尾青贮品质和有氧稳定性的影响

试验旨在研究热带假丝酵母(CT)、枯草芽孢杆菌(BS)、植物乳杆菌(LAP)、布氏乳杆菌(LAB)不同微生物添加剂组合对甘蔗尾青贮品质和有氧稳定性影响。本试验设置了10个组。对照组(C)不添加微生物添加剂;Ⅰ1、Ⅰ2、Ⅰ3组分别添加10、20和30 mL/kg的CT+BS菌液;Ⅱ1、Ⅱ2、Ⅱ3组分别添加10、20和30 mL/kg CT+BS+LAP菌液;Ⅲ1、Ⅲ2、Ⅲ3组分别添加10、20和30 mL/kg CT+BS+LAB菌液。添加0.5%尿素作为氮源。结果显示:①与对照组相比,CT+BS处理组pH、乙酸含量显著升高(P< 0.05),乳酸含量、干物质回收率(DMR)显著降低(P< 0.05),有氧稳定性有所降低;②与对照组相比,CT+BS+LAP处理组pH、乳酸含量显著升高(P< 0.05),且可降低青贮有氧稳定性;③与对照组相比,CT+BS+LAB处理组乙酸显著上升,DMR显著降低(P< 0.05),但粗蛋白质(CP)含量、有氧稳定性是所有处理组中最高的。因此,CT+BS处理青贮甘蔗尾品质较差,而CT+BS+LAB能改善甘蔗尾青贮品质,提高有氧稳定性。     

Differential immunolocalization between lingual antimicrobial peptide and lactoferrin in mammary gland of dairy cows

Lingual antimicrobial peptide (LAP), one of the β-defensins in bovines, and lactoferrin (LF) are synthesized in mammary epithelium and have bactericidal and bacteriostatic functions. However, it is not known whether they have similar expression patterns. Therefore, the present study was undertaken to compare (1) immunolocalization of LAP and LF in the mammary gland and (2) changes in concentration of these two components in milk after lipopolysaccharide (LPS) challenge. Bovine mammary tissues without LPS challenge were collected and their sections were immunostained with antibodies to LAP or LF. Milk from our previous study was collected every hour up to 12h and twice daily from d 1 to 7 after LPS challenge (the day of infusion was considered as d 0). These milk samples were measured for LAP but not LF in our previous report. Therefore, concentration of LF was measured by enzyme immunoassay in the present study. Epithelial cells of some alveoli showed immunopositive reaction for LF, but negative for LAP. Conversely, some alveoli were LAP positive in their epithelial cells but LF negative. Many alveoli had immunoreactions for neither LAP nor LF. The concentration of LAP in milk was elevated significantly at 3h after LPS infusion compared with pre-infusion values and remained at a high level until 12h. However, LF concentration in milk remained low at d 0 and increased at d 2. These results suggest that LAP and LF were mostly differentially localized in the alveolar epithelium in mammary glands. The different spatial expressions between them may be associated with their different temporal expression mechanisms.     

坏死梭杆菌外膜蛋白提取及免疫原性分析

从坏死梭杆菌(Fusobacterium necrophorum,FN)中提取外膜蛋白(outer membrane protein,OMP)并分析免疫原性。采用无菌心脑浸液(BHI)肉汤培养基培养坏死梭杆菌,用20mmol/L HEPEs-LiCl缓冲液提取外膜蛋白,经SDS-PAGE、Western blot和接种小鼠病理学检测分析表明,具有唯一条带,分子质量为44.5ku,具有良好的免疫活性并有一定毒性,研究结果为坏死梭杆菌亚单位疫苗研制奠定了基础。     

灭活大肠杆菌与脂多糖诱导猪肠黏膜上皮细胞表达RegⅢγ的机理研究

试验旨在探索革兰氏阴性菌大肠杆菌（Escherichia coli,E.coli）及其表面分子脂多糖（LPS）诱导胰腺再生蛋白Ⅲγ（RegⅢγ）表达调控的机制。首先,用不同浓度灭活E.coli（10⁹、10⁸、10⁷、10⁶、10⁵、10⁴ CFU/mL）和LPS （0.01、0.1、1、5、10、20、40、80 μg/mL）诱导猪肠黏膜上皮细胞（IPEC-JⅡ）,用MTT法测D_{490 nm}值,检测E.coli和LPS对IPEC-JⅡ细胞活力的影响;其次,用不同浓度灭活E.coli（10⁷、10⁶、10⁵ CFU/mL）和LPS （0.01、0.1、1、5 μg/mL）处理IPEC-JⅡ细胞24 h,用实时荧光定量PCR和Western blotting检测RegⅢγ mRNA和蛋白的表达;最后,用1 μg/mL LPS处理IPEC-JⅡ细胞24 h,用实时荧光定量PCR和Western blotting检测p65、p38、JNK、ERK mRNA和蛋白表达及磷酸化水平。结果显示,除0.01 μg/mL LPS不抑制IPEC-JⅡ细胞活力外,其他浓度的灭活E.coli和LPS均可抑制IPEC-JⅡ细胞活力,且10⁹、10⁸ CFU/mL E.coli和10、20、40、80 μg/mL LPS组细胞活力极显著下降（P<0.01）;与对照组相比,10⁷、10⁶和10⁵ CFU/mL E.coli均能诱导RegⅢγ表达增加,且10⁵ CFU/mL E.coli组RegⅢγ mRNA表达量极显著高于对照组（P<0.01）,蛋白表达量显著高于对照组（P<0.05）;0.01、0.1、1和10 μg/mL LPS均能诱导RegⅢγ表达增加,且0.1和1 μg/mL LPS组RegⅢγ mRNA表达量极显著高于对照组（P<0.01）,RegⅢγ蛋白表达虽有增加趋势,但差异不显著（P>0.05）;与对照组相比,1 μg/mL LPS组p65、p38 mRNA表达量极显著增加（P<0.01）,JNK、ERK mRNA表达量显著增加（P<0.05）;同时,p38、JNK蛋白表达量和磷酸化水平均极显著增加（P<0.01）,p65蛋白磷酸化水平显著增加（P<0.05）,ERK蛋白和磷酸化水平均增加,但差异不显著（P>0.05）。以上结果表明,灭活E.coli和LPS均可诱导RegⅢγ表达,1 μg/mL LPS可增加p65、p38和JNK蛋白的磷酸化水平。     

Effects of Enolase of Streptococcus equi ssp. zooepidemicus on Phagocytic Functions of Alveolar Macrophages in Mice

To investigate the effect of enolase (Eno) of Streptococcus equi ssp. zooepidemicus (SEZ) on phagocytosis of mouse alveolar macrophages (RAW264.7). Recombinant enolase (rEno) was obtained by constructing prokaryotic expression plasmid, and the cytotoxicity of rEno protein on RAW264.7 cell proliferation was determined by trypan-blue living cell count method. After the rEno protein was incubated with RAW264.7 cells, SEZ was applied to the cells and the quantity of bacteria being phagocytosed was detected to determine the phagocytic activity of RAW264.7 cells. Further, candidate proteins that might interact with SEZ Eno in RAW264.7 cells were screened by live cell stable isotope labeling (SILAC) and protein spectrum analysis (LC-MS/MS). It was found that protein treatment (rEno,10 μg·mL^-1) had significant cytotoxic effects on RAW264.7 cells. Treatment of RAW264.7 cells with 10.0 μg·mL^-1 rEno protein for 2 and 4 hours could significantly inhibit the phagocytosis of RAW264.7 cells (P<0.01, P<0.05). In RAW264.7 cells, dynactin subunit protein 2 (Dctn), integrin alpha-M and about 17 proteins that might interact with Eno were preliminarily identified as rEno interaction proteins. The rEno recombinant expression protein was obtained in this study, and it could reduce the phagocytosis of RAW264.7 cells to SEZ. Preliminary screening of interacting proteins also laid a foundation for further revealing the mechanism of Eno in the anti-phagocytosis of SEZ.     

Inducible nitric oxide synthase expression in Leishmania-infected dog macrophages

Nitric oxide (NO) production by the inducible NO synthase (iNOS or NOS2) represents one of the main microbicidal mechanisms of murine macrophages, but its role in other animal models is poorly investigated. Therefore, the aim of this work was to evaluate NOS2 expression in dog macrophages infected with Leishmania infantum. Macrophages obtained from peripheral blood of healthy dogs were activated with recombinant human interferon (rhIFN)-γ and bacterial lipopolysaccharide (LPS) and then infected with L. infantum promastigotes, zymodeme MON1. For the immunofluorescence assay fixed macrophages were incubated with polyclonal rabbit anti-NOS2 and then with rhodamine F(ab′)₂ goat anti-rabbit IgG. For immunoblotting, cell lysates were submitted to SDS–PAGE and blots were incubated with polyclonal rabbit anti-NOS2 and then with horseradish peroxidase-conjugated goat anti-rabbit IgG. Results demonstrated that L. infantum-infected cells, after stimulation with rhIFN-γ and LPS, displayed high levels of fluorescence for the NOS2 in their cytoplasm, unlike unstimulated uninfected macrophages. In western blotting, polyclonal anti-NOS2 reacted specifically with a protein band corresponding to 130 kDa. The signal produced in Leishmania-infected cells stimulated with rhIFN-γ and LPS was higher than that produced in Leishmania-infected unstimulated cells. No band was detected in cellular lysates from uninfected unstimulated cells. These results indicate that dog macrophages can express NOS2, and suggest a role for IFN-γ and LPS in NOS2 induction also in this animal model.     

Evaluation of the ability of colistin,amoxicillin (components of Potencil®), and fluoroquinolones to attenuate bacterial endotoxin‐ and Shiga exotoxin‐mediated cytotoxicity—In vitro studies

Escherichia coli is one of the major pathogens in humans and animals causing localized and systemic infections, which often lead to acute inflammation, watery diarrhea, and hemorrhagic colitis. Bacterial lipopolysaccharide (LPS) and Shiga exotoxins (Stx) are mostly responsible for such clinical signs. Therefore, highly effective treatment of E. coli infections should include both eradication of bacteria and neutralization of their toxins. Here, for the first time, we compared the in vitro ability of common antibiotics to decrease LPS‐ and Stx‐mediated cytotoxicity: colistin, amoxicillin (used separately or combined), enrofloxacin, and its metabolite ciprofloxacin. Three experimental scenarios were realized as follows: (a) the direct effect of antibiotics on endotoxin, (b) the effect of antibiotic treatment on LPS‐mediated cytotoxicity in an experiment mimicking “natural infection,” (c) the effect of antibiotics to decrease Stx2e‐mediated cytotoxicity. Two cell lines, A549 and Vero cells, were used to perform cytotoxic assays with the methyl tetrazolium (MTT) and lactate dehydrogenase leakage (LDH) methods, respectively. Colistin and amoxicillin, especially used in combination, were able to attenuate LPS toxic effect, which was reflected by increase in A549 cell viability. In comparison with other antibiotics, the combination of colistin and amoxicillin exhibited the highest boster or additive effect in protecting cells against LPS‐ and Stx2e‐induced toxicity. In summary, in comparison with fluoroquinolones, the combination of colistin and amoxicillin at concentrations similar to those achieved in plasma of treated animals exhibited the highest ability to attenuate LPS‐ and Stx2e‐mediated cytotoxicity.