犬新孢子虫巨噬细胞转移抑制因子（McMIF）克隆表达与其免疫调节作用鉴定

对犬新孢子虫巨噬细胞转移抑制因子(NcMIF)生物学特性进行鉴定,将NcMIF在大肠杆菌中以3种不同的形式进行表达,三种蛋白分别为NcMIF (成熟的蛋白质),NcMIFm (脯氨酸突变为甘氨酸)和NcMIFhis (在N端添加多组氨酸标记),对三种蛋白的多聚体状态、互变异构酶、氧化还原酶及是否与MIF受体(CD74)结合等进行分析。实验结果显示这三种重组的NcMIFs (rNcMIF)均不具备互变异构酶和氧化还原酶活性;甘氨酸替代脯氨酸的重组NcMIF减少了二聚体和三聚物的形成;N端额外添加的HIS标签增加了三聚物的形成;rNcMIF无法与重组人MIF竞争与MIF受体(CD74)结合,这表明CD74不是NcMIF受体结合;免疫荧光染色结果证明NcMIF定位于犬新孢子虫速殖子的顶端。免疫电镜结果进一步显示NcMIF存在于微线体、棒状体、致密颗粒及细胞核中。为进一步分析NcMIF在寄生虫免疫逃逸过程中发挥的作用提供参考。     

犬新孢子虫巨噬细胞转移抑制因子生物学特性鉴定

目的 通过原核系统表达犬新孢子虫巨噬细胞转移抑制因子(NcMIF),并对该蛋白的免疫调节作用进行分析。方法 通过GenBank发表的序列,利用分子生物学软件设计了一对特异性引物,通过RT-PCR方法扩增出NcMIF全基因,经测序分析后,将NcMIF亚克隆到原核表达载体pET28a(+),然后将鉴定为阳性的重组质粒转化到E. coli BL21(DE3)中用IPTG进行诱导表达。利用HPLC对可溶性表达的重组NcMIF蛋白进行纯化,去除内毒素,最后对NcMIF的免疫调节作用进行鉴定。结果 NcMIF没有明显的抗糖皮质激素的免疫抑制作用,也没有上调巨噬细胞TNF-α和NO表达量的作用。结论 实现了NcMIF在大肠杆菌系统中的可溶性表达并对其功能进行了鉴定,为进一步探究NcMIF生物学功能及其MIF在宿主免疫调节中的作用奠定基础。     

Expression of Aquaporin Water Channels in Equine Endometrium is Differentially Regulated During the Oestrous Cycle and Early Pregnancy

The expression of 12 different aquaporin subtypes in equine endometrium was examined at the mRNA and protein level. Endometrial samples were obtained during anoestrus, oestrus, 8, and 14 days after ovulation in non‐pregnant mares, and 14 days after ovulation in pregnant mares. Quantitative PCR revealed a time‐dependent pattern for all aquaporin subtypes examined except for AQP10 and 12. AQP3, 5 and 7 showed highest mRNA abundance 8 days after ovulation, while AQP0 and 2 were most abundant at Day 14 of the cycle in non‐pregnant mares. At 14 days of pregnancy, AQP1, 4, 8, 9 and 11 displayed highest expression levels. Western blot analysis confirmed protein expression of AQP0, 2 and 5. Immunohistochemistry localized protein expression to luminal and glandular epithelial and stromal cells. AQP0 staining intensity was highest in samples obtained on Day 14 of the oestrous cycle. AQP2 immunoreactivity seemed to be stronger in samples collected 14 days after ovulation from non‐pregnant animals, in particular luminal epithelial staining. Samples collected 8 days after ovulation from cyclic animals were characterized by intense AQP5 staining of glandular epithelium, predominantly in the deeper glands. Progesterone treatment of anoestrous mares did not enhance expression of AQPs, indicating that factors other than progesterone are required for the up‐regulation of certain AQP subtypes during dioestrus. In conclusion, it seems that an equine‐specific collaboration of aquaporin subtypes contributes to changes in endometrial fluid content occurring throughout the oestrous cycle and contributes to endometrial receptivity during early pregnancy in the mare.     

Effects of Hysteroscopic and Uterine Body Insemination on the Presence of Selected Immune Cells in the Equine Endometrium

The effects of standard uterine body and hysteroscopic insemination on endometrial health were investigated. For this purpose, 33 mares were assigned to five different protocols: control (no insemination; n = 7), sham AI (sham uterine body insemination; n = 6), sham HysAI (sham hysteroscopic insemination; n = 7), standard AI (standard uterine body insemination, 300 × 10⁶ progressively motile sperms (PMS); n = 7) and HysAI (hysteroscopic insemination, 100 × 10⁶ PMS; n = 6). Sampling included uterine swabbing for microbiological examination, cytology for determination of polymorphonuclear neutrophils (PMNs) in the uterus, and endometrial biopsy collection for histology and characterization of endometrial immune cells on day 18 after ovulation (B1) as well as 8–10 hours (B2, day 20) and 72 hours after insemination (B3, day 23). Microbial contamination increased throughout the experiment in the sham insemination groups. Significant effects (P < .05) over time were detected for PMNs (cytology: sham HysAI, standard AI, and HysAI; histology: standard AI and HysAI), macrophages (immunohistochemistry: standard AI and HysAI) and T cells (immunohistochemistry: standard AI), showing an increase at B2 and a subsequent decrease toward baseline levels at B3. At B2, significant differences (P < .05) existed for PMNs (mean ± SEM) between control (1.3 ± 1.9%) and sham AI (2.2 ± 2.7%) versus standard AI (12.2 ± 4.7%) and for macrophages between control (4.1 ± 3.5%) and sham AI (2.5 ± 1.3%) versus standard AI (25.4 ± 15.8%). Thus, the cellular immune response of the endometrium depends on sperm deposition in the uterus and does not differ between hysteroscopic and standard uterine body insemination.     

绵羊痒螨巨噬细胞迁移抑制因子重组蛋白对兔外周血单个核细胞Th1/Th2型和Th17/Treg型特征因子表达的影响

为了初步探讨绵羊痒螨巨噬细胞迁移抑制因子（PoMIF）对健康新西兰兔外周血单个核细胞（PBMC）中Th1/Th2和Th17/Treg细胞平衡的变化。采用RT-PCR从绵羊痒螨总RNA中扩增得到MIF全长基因,经原核表达、纯化重组PoMIF（rPoMIF）蛋白,并分析其氧化还原酶和互变异构酶活性。筛选与健康新西兰兔PBMC孵育的最佳rPoMIF浓度,且用最佳浓度rPoMIF（0.2 μg·mL^-1）与PBMC共同孵育0、1、6、12、24、36 h后收集细胞,用荧光定量PCR检测其Th1/Th2/Th17/Treg细胞相对应的特征性转录因子T-bet/GATA-3/RORc/Foxp3和特征性细胞因子IFN-γ/IL-4/IL-17A/IL-10 mRNA表达变化。结果表明：PoMIF全长363 bp,rPoMIF大小为32 ku（含pET32a标签蛋白19 ku）,且具有互变异构酶活性;rPoMIF刺激后PBMC中Th1细胞的T-bet和IFN-γ先下降后上升,Th2细胞的GATA-3和IL-4均呈下降趋势,且T-bet/GATA-3和IFN-γ/IL-4比值在12、24和36 h均升高;Th17细胞的RORc和IL-17A在各时间点下降,而Treg细胞的Foxp3和IL-10在各时间点升高,且RORc/Foxp3和IL-17A/IL-10比值在各时间点均变低。rPoMIF可造成家兔外周血单个核细胞的Th1/Th2和Th17/Treg平衡分别向Th1和Treg偏移。     

白血病抑制因子及其受体与子宫内膜的关系

白血病抑制因子(LIF)是一种多生物活性的细胞因子,研究表明,LIF在人及动物的子宫内膜的腺上皮细胞、妊娠期蜕膜及胎盘上均有表达;胚泡是动物生殖过程中的关键,一些细胞因子起重要作用,它们能够充当子宫内膜微环境的"局部调节者",LIF对哺乳动物胚胎发育到胚泡阶段具有一定的作用,LIF在子宫内膜中特异性表达,促进胚胎生长发育和启动胚泡植入;LIF在生殖周期中影响子宫的功能及调节子宫内膜生长,这可能与LIF作用于子宫使其能发生蜕膜反应有关;LIF通过与LIF受体及gp130蛋白作用而发挥重要的生物学功能。LIF在哺乳动物早期妊娠中的作用机制具有重要意义。文章对LIF及其受体与子宫内膜的关系做了综述。     

Incidence of Transfusion Reactions and Retention of Procoagulant and Anticoagulant Factor Activities in Equine Plasma

Background: The extent of preservation of clotting factors and incidence of transfusion reactions to noncommercial equine plasma is not documented.
Hypothesis: Equine frozen plasma would retain its coagulation factor activity within the reference range and the incidence of transfusion reactions would be low.
Animals: Ten plasma donor horses. Fifty clinically ill hospitalized horses receiving plasma were reviewed to determine the incidence of reactions.
Methods: In vitro study and retrospective case review. Plasma was prepared by gravity sedimentation from whole blood refrigerated for 48 hours. The activities of factors VII through XII, antithrombin (AT), and Protein C were measured. Factor activities were compared for plasma samples obtained before blood collection (S0), after 48 hours of gravity sedimentation at 5 °C and after plasma separation (S1), and after 90 days of storage at −20 °C (S90). The medical records of 50 consecutive clinically ill horses receiving frozen plasma were reviewed to determine the incidence of transfusion reactions.
Results: The combined effect of plasma harvest, gravity sedimentation, decantation, and freezing caused significant reductions in factors IX, (43% P = .0013), X, (33% P = .0001), XI, (48% P = .0008), AT, (10% P = .02), and Protein C (26% P = .0001). Activities for all factors analyzed, except factor X, remained within the reference ranges. Transfusion reactions were recorded for 5/50 horses.
Conclusions and Clinical Relevance: Clotting factors, AT, and Protein C were well preserved. The incidence of reactions to frozen plasma was 10%.     

Minimum Inhibitory Concentration and Postantibiotic Effect of Amikacin for Equine Isolates of Methicillin-Resistant Staphylococcus aureus In Vitro

Objective— To report the minimum inhibitory concentration (MIC) of amikacin sulfate for equine clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and characterize the initial kill and duration of the postantibiotic effect (PAE) for selected strains.
Study Design— Experimental study.
Methods— Isolates of MRSA (n=35) had their amikacin MIC determined using the E-test agar diffusion method. Two isolates with MICs>256 μg/mL limit were further characterized using broth macrodilution. Six distinct isolates with amikacin MICs of 32, 48, 128 (2 isolates) and 500 (2 isolates) μg/mL had PAE determinations made over a range of amikacin concentrations from 31.25–1000 μg/mL using standard culture-based techniques.
Results— Median MIC of the 35 isolates was 32 μg/mL (range 2 to >256 μg/mL). Mean PAE of selected MRSA strains had an overall mean (all amikacin doses) of 3.43 hours (range 0.10–9.57 hours). PAE for MRSA exposed to amikacin at 1000 μg/mL was 6.18 hours (range 3.30–9.57 hours), significantly longer than that for all other concentrations ( P <.0001). There was no statistically significant effect of isolate MIC on PAE.
Conclusions— Isolates had a wide range of MIC; however, growth of all 6 selected strains were inhibited within the range of concentrations tested, including 2 strains with MICs of 500 μg/mL. PAE duration was not influenced by the MIC of amikacin but was significantly longer with treatment at 1000 μg/mL than at lower concentrations.
Clinical Relevance— Clinical isolates of MRSA are susceptible to amikacin at concentrations achieved by regional perfusion: however, the modest duration of PAE observed suggest that further laboratory and in vivo evaluation be conducted before recommending the technique for clinical use.     

Equine cervical intervertebral disc degeneration is associated with location and MRI features

Morphology of the equine cervical intervertebral disc is different from that in humans and small companion animals and published imaging data are scarcely available. The objectives of this exploratory, methods comparison study were (a) to describe MRI features of macroscopically nondegenerated and degenerated intervertebral discs (b) to test associations between spinal location and macroscopic degeneration or MRI‐detected annular protrusion and between MRI‐detected annular protrusion and macroscopic degeneration, and (c) to define MRI sequences for characterizing equine cervical intervertebral disc degeneration. Ex vivo MRI of intervertebral discs was performed in 11 horses with clinical signs related to the cervical region prior to macroscopic assessment. Mixed‐effect logistic regression modeling included spinal location, MRI‐detected annular protrusion, and presence of macroscopic degeneration with “horse” as random effect. Odds ratio and 95% confidence interval were determined. Reduced signal intensity in proton density turbo SE represented intervertebral disc degeneration. Signal voids due to presence of gas and/or hemorrhage were seen in gradient echo sequences. Presence of macroscopic intervertebral disc degeneration was significantly associated with spinal location with odds being higher in the caudal (C5 to T1) versus cranial (C2 to C5) part of the cervical vertebral column. Intervertebral discs with MRI‐detected annular protrusion grades 2‐4 did have higher odds than with grade 1 to have macroscopic degeneration. It was concluded that MRI findings corresponded well with gross macroscopic data. Magnetic resonance imaging of the equine cervical intervertebral disc seems to be a promising technique, but its potential clinical value for live horses needs to be explored further in a larger and more diverse population of horses.     

鸡转移因子脂质体对小鼠巨噬细胞吞噬功能及肠绒毛长度的影响简

为探讨鸡转移因子脂质体（chicken transfer factor liposome，CTFL）对小鼠腹腔巨噬细胞及消化吸收的影响，本试验选取了大小相近的18～22 g SPF级雌性小鼠30只，随机分成3组，每组10只，分别腹腔注射鸡转移因子（chicken transfer factor，CTF）、CTFL和生理盐水2 mL/只，隔日注射1次，连续注射5次，第11和21天检测小鼠腹腔巨噬细胞对鸡红细胞的吞噬率，同时分别取十二指肠、空肠和回肠固定到10%福尔马林液中、石蜡切片、苏木素—伊红染色，显微镜下观察，测量各段小肠的绒毛长度。结果显示，CTF和CTFL组对小鼠腹腔巨噬细胞吞噬率的影响与对照组相比均差异极显著（P＜0.01），CTF和CTFL组相比差异不显著（P＞0.05）。小鼠饲养11和21 d时，CTF和CTFL组的十二指肠绒毛平均长度均显著长于对照组（P＜0.05），且CTFL组的十二指肠绒毛平均长度比CTF组长，而空肠和回肠平均绒毛长度均无显著差异（P＞0.05）。结果表明，CTFL和CTF均能显著提高机体免疫功能，CTFL对小鼠消化吸收的促进作用比CTF强。     

Antibody Responses Against Equine Influenza Virus Induced by Concurrent and by Consecutive Use of an Inactivated Equine Influenza Virus Vaccine and a Modified Live Equine Herpesvirus Type 1 Vaccine in Thoroughbred Racehorses

An inactivated equine influenza virus (EIV) vaccine and a live equine herpesvirus type 1 (EHV-1) vaccine are usually administered concurrently to Thoroughbred racehorses in Japan. The objective of this study was to evaluate whether concurrent administration of an inactivated EIV vaccine and a live EHV-1 vaccine in Thoroughbred racehorses influences the antibody response against EIV. We compared the antibody response against EIV in horses administered both vaccines on the same day (Group A; n = 27) and the response in horses administered an inactivated EIV vaccine first and then a live EHV-1 vaccine 1–2 weeks later (Group B; n = 20). In both groups, geometric mean hemagglutination inhibition (HI) titers against A/equine/Ibaraki/1/2007 and A/equine/Yokohama/aq13/2010 increased significantly after EIV vaccination. However, the percentage of horses that showed a twofold increase or greater in HI titers against A/equine/Yokohama/aq13/2010 was significantly higher in Group B (75%) than in Group A (37%; P = .02). These results suggest that the concurrent use of an inactivated EIV vaccine and a live EHV-1 vaccine reduced the immune response against EIV to some extent, and it would be better to use these vaccines consecutively, especially for naïve horses or horses whose vaccination history is incomplete.     

鸡粒细胞-巨噬细胞集落刺激因子的原核表达及生物学活性分析

通过PCR方法扩增不含信号肽的鸡GM-CSF成熟蛋白基因,克隆至原核表达载体pET-32a（＋）,构建原核表达质粒p32GM-CSF,通过IPTG诱导重组鸡GM-CSF蛋白表达,经镍离子亲和层析纯化后,用MTT法检测表达重组蛋白的生物学活性,并制备兔抗鸡GM-CSF多克隆抗体。结果表明成功地构建了p32GM-CSF原核表达质粒,SDS-PAGE结果显示表达的重组蛋白约34 ku,主要以包涵体形式表达,纯化后得到高纯度目的蛋白。West-ern Blot结果表明,该重组蛋白能与制备的抗鸡GM-CSF抗体特异性结合。MTT试验证实,该重组蛋白具有明显增强鸡骨髓细胞增殖的生物学活性;这些研究结果表明,表达的重组chGM-CSF蛋白及其多克隆抗体拥有相应的生物学功能,这将为进一步研究鸡GM-CSF蛋白的生物学功能及其应用奠定基础。     

Comparative Effects of Phenylbutazone, Naproxen and Flunixin Meglumine on Equine Platelet Aggregation and Platelet Factor 3 Availability in vitro

Nonsteroidal anti-inflammatory drugs are commonly used in the treatment of inflammatory conditions, and have potential value in the treatment of thrombotic disease in the horse. This study compares the potency of three nonsteroidal anti-inflammatory drugs phenylbutazone, naproxen (equiproxen) and flunixin meglumine (banamine) with respect to their effects on equine platelets. Two functional responses of horse platelets were evaluated in vitro: their ability to aggregate and their ability to make available platelet factor 3 procoagulant activity.

Flunixin at a concentration of 10^-6 M significantly depressed the maximum degree of adenosine diphosphate-induced (10^-6M) aggregation while much higher concentrations of phenylbutazone and naproxen (5 X 10⁵M) were required to produce similar effects. None of the non-steriodal anti-inflammatory drugs significantly affected the duration of the lag phase or the initial velocity of adenosine diphosphate-induced aggregation within the range of drug concentrations used (10^-6-10^-3M). The lag phase and initial velocity of acid-soluble collagen-induced aggregation were significantly affected by 10^-6 M flunixin and 10^-4 M phenylbutazone or naproxen was required to produce equivalent effects. Concentrations of 5 X 10^-6 M flunixin and 5 X 10^-4 M phenylbutazone or naproxen were required to significantly depress the degree of collaen-induced aggregation of horse platelets.

Although the effects of the nonsteroidal anti-inflammatory drugs were qualitatively similar, flunixin was a much more potent inhibitor of platelet aggregation than either of the other two drugs (which were equipotent). At very high drug concentrations (5 X 10^-4 M and greater), all three drugs produced the same degree of inhibition of equine platelet aggregation.

Platelet factor 3 activity was made available by exposing horse platelets to 10^-5 M adenosine diphosphate or 1:800 acid-soluble collagen; but not by exposure to a suspension of kaolin particles. Only a small portion of the total platelet factor 3 activity was made available on stimulation with either adenosine diphosphate or collagen. Pretreatment of horse platelets with any of the nonsteroidal anti-inflammatory drugs (10^-4 M concentration) had no significant effect on adenosine diphosphate or collagen-induced platelet factor 3 availability.

     

白血病抑制因子及其受体在月经周期猕猴输卵管内的表达

应用免疫细胞化学方法对白血病抑制因子(LIF)、白血病抑制因子受体(LIFR)和gp130在月经周期猕猴输卵管内的表达进行了研究。结果表明，LIF、LIFR和gp130主要在输卵管上皮细胞内表达，而在固有层、肌层及泉膜内的表达量较少。LIF、LIFR和gp130在猕猴输卵管内的表达量随月经周期的不同而变化，在增殖中期到分泌中期的输卵管内表达量较高，而在增殖早期及分泌晚期输卵管内表达量较低。LIF及其受体在猕猴输卵管内的表达可能由卵巢分泌的类固醇激素所调控，LIF可能在猕猴排卵及早期胚胎发育过程中起重要的调节作用。