嗜肺军团菌感染多噬棘阿米巴及其增殖的实验观察

目的　初步建立国内嗜肺军团菌感染原生动物自由生活阿米巴的实验感染模型。方法将多噬棘阿米巴和嗜肺军团菌进行实验室共同培养 ,于 8h、2 4h、4 8h、72h后将预置盖玻片上生长的虫体Giemsa染色后作油镜观察 ,各阶段的培养物作透射电镜观察 ,研究嗜肺军团菌感染多噬棘阿米巴后形态学的变化。结果　共同培养 2 4h后Giemsa染色显示阿米巴内出现明显的细菌菌体 ,后期菌体布满整个胞浆 ;透射电镜下阿米巴滋养体和囊胞中都充满健康完整的军团菌菌体 ,并观察到不同阶段的二分裂现象。结论　嗜肺军团菌能在自由生活阿米巴内生长并增殖 ,初步证实了阿米巴可能是军团菌在自然界的贮存宿主。     

营养缺乏对自由生活阿米巴自噬的影响

目的研究在营养缺乏的环境中自由生活阿米巴自噬的变化和自噬结构的形态学特征。方法对照组自由生活阿米巴用涂有大肠埃希菌的琼脂培养基培养。实验组将在大肠埃希菌中培养的阿米巴转移至不含大肠埃希菌的琼脂培养基上培养12 h。扫描电镜下观察不提供细菌的培养环境中阿米巴的形态学变化,透射电镜下观察阿米巴自噬的变化及自噬前体、自噬体和自噬溶酶体的结构特点,图像分析仪测量虫体内自噬结构与细胞质的断面面积。单丹磺酰尸胺(MDC)染色法标记自由生活阿米巴虫体内的自噬体,在共聚焦激光扫描显微镜下观察和定量分析。结果对照组中自由生活阿米巴均为滋养体形式;实验组中,滋养体逐渐向包囊转变。对照组阿米巴虫体内充满细菌碎片,只发生轻微的自噬,自噬结构数目较少。与对照组比较,实验组阿米巴自噬水平显著提高,自噬结构数目增多,自噬前体、自噬体和自噬溶酶体与细胞质的断面面积比增大(P0.05或0.01);部分阿米巴虫体内残存未消化的细菌碎片。结论在缺乏营养的环境中,自由生活阿米巴由滋养体向包囊转变,虫体内自噬功能显著增强。     

致病性自由生活阿米巴感染灵长类的实验:卡氏棘阿米巴的易感性

关于自由生活的Naegleria属阿米巴是人体原发性阿米巴脑膜脑炎的病原体的报道,引起了对自由生活的棘阿米巴属(Acanthamoeba)的研究。本文作者为了阐明棘阿米巴属虫种对人的致病性,以猴为模型进行了一系列的实验。本实验对卡氏棘阿米巴(Acanthamoebaculb6rtsoni)的易感性作了观察。这种阿米巴是先后用(月示)蛋白胨酵母葡萄糖培养基和酪蛋白胰酶水解物大豆肉汤培养基保持下来的虫株。这种阿米巴放在37C盛有75毫升培养基的500毫升三角烧瓶中培     

人眼内棘阿米巴的分离、鉴定及生物学特性

已有报告自由生活的纳氏虫属(Naegleria)和棘阿米巴属(Acanthamoeba)能使人发生致死的原发性脑膜脑炎。纳氏虫属可引起急性暴发病症,棘阿米巴属则引起慢性病症。曾从上呼吸道疾病及健康人的鼻咽内分离出棘阿米巴属的虫种,并在上呼吸道疾病、视神经炎及斑性病患者的血清中查出了对这属阿米巴的补体结合抗体。这些发现说明了棘阿米巴属可引起不明显的非致死     

自由生活阿米巴感染的流行病学

棘阿米巴属和耐格里属的小型自由生活阿米巴遍布全世界。它们已从各种生境中分离出来。由这些阿米巴引起的中枢神经系统感染的病例已多达200例。大部分病例是由福氏耐格里阿米巴引起的急性原发性阿米巴脑膜脑炎。其余病例据报道是由于棘阿米巴或其它一些自由生活阿米巴引起的一种称之为肉芽肿性阿米巴脑炎的亚急性或慢性感染。此外,棘阿米巴还能引起疼痛的,威胁视力的棘阿米巴角膜炎。     

柯氏棘阿米巴毒力相关的cDNA片段上调表达[英]

自由生活的棘阿米巴可引起阿米巴肉芽肿脑炎和角膜炎,其毒力可通过长期体外培养逐渐减弱,但又可经鼻内接种或小鼠脑传代感染而恢复。本研究将体外保种多年的柯氏棘阿米巴(Acanthamoeba culbertsoni),小鼠接种进行脑传代,采用mRNA差异显示聚     

半胱氨酸蛋白酶在棘阿米巴成囊过程中作用的研究

棘阿米巴是一种机会性致病原虫,在环境中普遍存在,一定条件下可以引起人体严重的甚至危及生命的感染。棘阿米巴生活史中具有致病性的滋养体期和有感染性的包囊期两个阶段。棘阿米巴成囊有两个阶段,分别是包括自溶和蛋白质降解的早期阶段和伴随着包囊特异性基因表达的晚期阶段。半胱氨酸蛋白酶在棘阿米巴成囊过程中的早期阶段起着重要作用,其成囊过程可以被半胱氨酸蛋白酶抑制剂E64d抑制。半胱氨酸蛋白酶是具有保守催化位点的组织蛋白L样酶,在滋养体和包囊中连续表达,分布在溶酶体内并在酸性条件下表现出最高的催化活性。与丝氨酸蛋白酶不同,半胱氨酸蛋白酶在成囊过程中水解多种蛋白质。包囊特异性半胱氨酸蛋白酶通过介导线粒体的自噬从而在棘阿米巴的成囊过程中起到关键作用,本文综述了内源性半胱氨酸蛋白酶抑制剂调节成囊过程的作用。     

Cpn感染与急性脑梗死的相关性研究

肺炎衣原体（Cpn）是一种细胞内寄生的G-菌,具抗原特异性,有双相生命周期,其感染性要素体（EB）可在细胞外生存,在人类中经气雾滴传播。Cpn感染普遍,在成人中有50％Cpn抗体阳性,且男性阳性率高于女性,这与男性易患动脉粥样硬化（AS）的结论相一致。近年研究发现,Cpn感染与脑梗死有关。     

致人体呼吸道感染的似棘阿米巴形态学和分子生物学鉴定

目的鉴定某反复咳嗽患者痰液样本中形态学似棘阿米巴的寄生虫种属。方法分离患者痰液中原虫进行体外培养,显微镜观察原虫滋养体和包囊形态,并提取原虫DNA,采用阿米巴科18S rRNA通用引物Ami6F1和Ami9R、棘阿米巴属18S rRNA通用引物JDP1和JDP2、葛氏棘阿米巴(Acanthamoeba griffini)S-7ATCC 18S rRNA全长序列引物AacGF和AacGR进行PCR扩增鉴定。以样本的18S rRNA基因为分子标志,与GenBank中各棘阿米巴序列进行同源性分析,筛选相关物种序列,采用最大似然法构建系统进化树,分析亲缘关系。结果镜下可见,患者痰样中的滋养体具有棘阿米巴特征性的棘状伪足凸起,并呈无规则的变形虫状;包囊为两层膜结构,内膜具有棘阿米巴特征性的星状突起。PCR检测结果显示,采用3组引物分别扩增出830、479和1 954 bp的条带,与预期片段大小一致。经BLAST比对后,3个扩增产物序列与棘阿米巴S-7 ATCC相似度分别达99%、99%、100%。系统进化树结果显示,样本棘阿米巴与引起角膜炎的卡氏棘阿米巴(A.castellanii)、多噬棘阿米巴(A.polyphaga)、柯氏棘阿米巴(A.cullbertsoni)和条脊棘阿米巴(A.rhysodes)同源性较高,分别为91.4%、99.6%、94.5%和91.8%。结论该呼吸道感染患者痰液样本中的寄生虫鉴定为葛氏棘阿米巴。     

兔衣原体病病原体的分离及鉴定

本文报道一起暴发流行的兔繁殖障碍综合征,主要表现为子宫炎、流产、死胎、产弱仔和不孕等。经过鸡胚分离、感染McCoy细胞、特异性免疫荧光染色、姬姆萨染色观察包涵体、碘染色、磺胺抑制等试验,初步确认该病原为鹦鹉热衣原体。     

Survival of Helicobacter pylori in milk and tap water

The survival capacity of Helicobacter pylori in artificially contaminated milk and tap water was investigated in the study. Helicobacter pylori could survive for up to 10 days in milk at 4°C storage but only 4 days in tap water with a steady decrease of colony forming units. However, electron microscopy clearly showed that the non-culturable coccoid form was present in tap water which had been kept at 4°C for 7 days. It is concluded that H. pylori may survive in tap water as well as in milk, with the implication that they may, thereby, act as a vehicle of transmission.     

A monoclonal antibody recognizing the 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) trisaccharide alphaKdo(2-->4)alphaKdo(2-->4)alphaKdo of Chlamydophila psittaci 6BC lipopolysaccharide

A monoclonal antibody (mAb) S45-18 was generated against a synthetic neoglycoconjugate containing the trisaccharide alphaKdo(2-->4)alphaKdo(2-->4)alphaKdo (Kdo, 3-deoxy-D-manno-oct-2-ulopyranosonic acid) which represents a structure of the lipopolysaccharide (LPS) from Chlamydophila psittaci 6BC. The antibody was characterized by binding and inhibition assays in ELISA using: (i) the immunizing antigen and chemically synthesized derivatives thereof; (ii) chlamydial elementary bodies (EB); and (iii) LPS of Chl. psittaci 6BC and Chlamydia trachomatis L2. The specificity was determined in comparison to that of mAb S25-23 recognizing the alphaKdo(2-->8)alphaKdo(2-->4)alphaKdo trisaccharide which represents an epitope shared by all species of the family. MAb S45-18 bound to an epitope of the structure alphaKdo(2-->4)alphaKdo(2-->4)alphaKdo, with lower reactivity with the (2-->8)-(2-->4)-linked analog. Using chlamydial EB or LPS, mAb S45-18 bound preferentially to LPS and EB of Chl. psittaci. Therefore, Chl. psittaci LPS contains, in addition to the known genus-specific epitope, a species-specific epitope.     

Weibel-Palade bodies in endothelial cells of normal thoracic ducts and deep cervical lymphatics in rabbits

The endothelial cells of normal thoracic ducts and deep cervical lymphatics were examined by electron microscopy using conventional staining methods and acid-phosphatase and ruthenium red (RR) reactions. The endothelial cells contained rod-shaped, circular and elliptical bodies of moderate density. The shape and structure of all these bodies were the same as those of the Weibel-Palade bodies (WPB) in the endothelial cells of blood vessels. They were usually found near the Golgi complex in groups, and the long axis of the rods paralleled the Golgi saccules. In addition, a peculiar vacuolated rod with a bulge was found adjacent to the WPB. Single coated vacuoles were occasionally located next to the WPB. Acid-phosphatase activity and RR positive material were not seen in the WPB and the vacuoles. Our observations suggest that the WPB have a close relationship, morphological as well as functional, to the Golgi complex in lymphatic endothelial cells.     

Cytochemistry of myocardial structures related to degenerative processes in spontaneously hypertensive and normotensive rats

Using electron microscopy and cytochemical techniques we investigated structures which are associated with long-term hypertension and ageing in the myocardial cell of the rat. Lysosomes, demonstrated by acid phosphatase and aryl sulfatase activities, were found mainly in the perinuclear region in young rats. With age these organelles appeared with increasing frequency in other regions of the cell. Spontaneously hypertensive rats (SHR) showed an earlier apparent migration of lysosomes than did normotensive rats (WKY). Our observations indicate that lysosomes were closely associated with autophagic vacuoles, membrane swirls , translucent mitochondria, myelin figures and other structures linked with degenerative events. In the oldest SHR lysosomes, autolysosomes (autophagic vacuoles with lysosomal activity), and degenerative structures were observed in various regions of the myocardial cell. Peroxisomes, as demonstrated by catalase activity, did not seem to be affected by hypertension or age. A number of dense osmophilic structures did not react for any of the enzymes studied; these included myelin figures, mitochondrial inclusions and diffuse dense bodies. Our observations implicate both ageing and hypertension in the enhancement of lysosomes and their end products.     

Isolation and characterization of rabbit lung lamellar bodies

A method has been devised for the isolation of a highly purified preparation of lamellar bodies from rabbit lung. The purity of the preparation was confirmed by electron microscopy, marker enzymes, phospholipid composition, and isopycnic centrifugation on continuous density sucrose gradients. Contamination of the lamellar bodies by such subcellular components as mitochondria, nuclei, lysosomes and plasma membranes could be excluded; however, reduced nicotinamide adenine dinucleotide phosphate (NADPH) cytochrome c reductase, an enzyme specific for the endoplasmic reticulum components was a persistent contaminant in the preparation of the isolated lamellar bodies. When the lamellar bodies were subject to isopycnic centrifugation, all of the NADPH cytochrome c reductase activity was associated with the lamellar bodies in the low density peak; no reductase activity could be detected in the region of the density gradient demonstrated to localize microsomes. Use of 3H-radiolabeled microsomes confirmed that all of the NADPH cytochrome c reductase activity present in the lamellar body preparations could be accounted for by microsomal contamination. When lamellar bodies or liposomal membranes synthesized from the total phospholipid fraction of lamellar bodies were analyzed by the electron paramagnetic resonance probe, 5-dioxyl-methylstearate, they exhibited a high degree of fluidity at physiological temperature. This was in contrast to the low fluidity of liposomal membranes composed of pure dipalmitoylphosphatidylcholine, the major component (50%) of rabbit lamellar body phospholipids. Furthermore, the major temperature-dependent phase transition in lamellar body membranes occurred at a different temperature (30.5 degrees C) from that of dipalmitoyl-phosphatidylcholine (41.0 degrees C). It is clear, therefore, that the membrane fluidity of lamellar bodies must be highly influenced by the minor lipid component.     

IFN-γ抗鹦鹉热衣原体急性感染保护作用研究

目的探讨IFN-γ对鹦鹉热衣原体(Chlamydia psittaci,Cps)的抗感染作用,为进一步阐明机体抗衣原体免疫机制提供参考数据。方法不同浓度的重组人IFN-γ(5ng/mL、25ng/mL和50ng/mL)作用于感染Cps 6BC的HeLa细胞,48h后计数包涵体数量,并观察包涵体形态的改变。2×10~6 IFUs Cps 6BC滴鼻感染C57BL/6J小鼠,于感染前、后24h腹腔内注射10μg重组鼠IFN-γ,观察小鼠体重、活动状态、生存率等一般指标,分别于感染后5d和10d处死小鼠,取肝、肺组织进行HE染色检测其病理变化;并取感染后5d的肺组织,匀浆,计数其中Cps包涵体数量。结果感染48h后,Cps 6BC在5ng/mL、25ng/mL、50ng/mL重组人IFN-γ处理的HeLa细胞中包涵体数量低于对照组(包涵体数分别为(23.8±5.1)×10~6,(10±3.58)×10~6,(8.0±2.22)×10~6,(43.3±11.05)×10~6,衣原体包涵体形状不规则,体积变小。小鼠实验显示,腹腔注射IFN-γ可明显提高Cps 6BC感染小鼠生存率,并减轻急性临床表现及脏器病变。结论 IFN-γ可发挥早期抗Cps感染的保护作用。     

Morphology and immunotyping of AC-43 strain of Chlamydia pneumoniae isolated from a Japanese child]

A strain of Chlamydia pneumoniae (C. pneumoniae) which had been isolated from a Japanese child (AC-43) was examined morphologically and serologically using micro-immunofluorescent (micro-IF) test. Inclusions of AC-43 were stained by an indirect immunofluorescent method using C. pneumoniae specific monoclonal antibody. They were dense round inclusions which had been reported as a characteristic figure for C. pneumoniae. An elementary body (EB) of AC-43 was pear-shaped by electron micrograph, which was the same as previous reports for C. pneumoniae. We produced monoclonal antibodies using purified EB of AC-43 as antigen. Culture fluids of these clones reacted with C. pneumoniae antigens, but did not react with C. trachomatis or C. psittaci antigens by micro-IF tests. There was no difference in morphological and serological findings among standard strains and Japanese isolate of C. pneumoniae.     

Red cell vacuoles: their size and distribution under normal conditions and after splenectomy

The frequency of occurrence of vacuoles in red blood cells was studied by transmission electron microscopy. Small vacuoles were found in about 13% of the cell sections, and they had a mean diameter of 130 +/- 72 nm (mean +/- SD). It can be estimated that there were about 20 small vacuoles per erythrocyte. The frequency of vacuoles was similar in density-separated cell fractions. In splenectomized patients, the small vacuoles were 4 times more frequent; there was again no difference in vacuole density between top and bottom fractions of density-separated red blood cells. The bottom fraction of red blood cells from splenectomized patients, however, had a high incidence of large vacuoles (greater than 300 nm in diameter) and clustering of small vacuoles. These large vacuoles were probably the result of aggregation and fusion of small vacuoles, and their size allowed detection by light microscopy. Hence, the well-known "pocked" or "pitted" red blood cells of splenectomized individuals were more frequent in the bottom fraction. We conclude that small vacuoles occur normally in erythrocytes, that they tend to cluster and fuse during cell aging, and that the spleen is capable of removing these structures when they reach a certain size.     

Development of serodiagnostic kit "HITAZYME Chlamydia Ab" for Chlamydia trachomatis infections using extracted antigen]

To develop EIA kit with low cross-reactivity for the quantitative detection of anti-chlamydial antibodies, we examined the preparation of trachomatis antigens and its specificity to mouse antisera and human sera. The chlamydial elementary body (EB) purified from C. trachomatis L2/434/Bu strain was treated by Sarkosyl, dithiothreitol and SDS by turns to obtain the soluble EB outer membrane (COMC). SDS-PAGE showed that the major components of the COMC were 96K, 60K and 39.5 KDa peptides. The reactivity of the COMC immobilized to 96 wells microtiter plate to mouse anti-serum to C. trachomatis was higher than the other two mouse anti-sera to C. psittaci and pneumoniae. In human sera, the cut off values were calculated from an average optical density plus its two-fold standard deviation obtained by the testing of 100 samples of healthy human sera. We evaluated the specificity of the kit to 17 anti-C. pneumoniae, 9 C. trachomatis and 4 C. psittaci antibodies positive patients' sera judged by the MFA method respectively. The results showed that the concordance ratio of IgG and IgA were 88%, 100% in anti-C. pneumoniae, 89%, 78% in anti-C. trachomatis and 50%, 50% in anti-C. psittaci respectively. From the results obtained in this study, we concluded that the HITAZYME method which had a very low cross-reactivity to C. pneumoniae is clinically useful in the serodiagnosis of C. trachomatis infections, even if it has a little common antigenicity with C. psittaci antigen.     

The influence of specific antibodies and complement on the motility and the viability of Entamoeba histolytica trophozoites

The distribution of peroxidase-labeled normal or specific rabbit immunoglobulin (Ig) in Entamoeba histolytica trophozoites was studied by transmission electron microscopy. Small amounts of normal Ig became attached to the cell surface but did not redistribute. Internalized specific Ig was firmly bound to the inner membranes of phagocytosis vacuoles, while aggregates of normal Ig were dispersed over the vacuolar lumen. The distribution of fluorescein isothiocyanate-labeled Ig in living amebae was correlated with the cell motility at different times. Immobilization coincided with the cellular metabolic processing of internalized material. Remobilization occurred when the Ig was degraded and antibodies again bound to the reappearing surface antigens. E. histolytica activated complement by the classical pathway. Fresh guinea pig serum alone did not produce lysis which, however, did occur when it was added together with normal rabbit Ig. Normal rabbit Ig may constitute a complement-fixing substrate and activate complement by the classical pathway. Immobilization of the amebae by cytochalasin B (10 micrograms/ml) increased the susceptibility to cytolytic effects of specific antibodies or complement, or both. Pharmacological inhibition of cell motility might augment the immunological defence of the host against amebic infection.