人角膜及角膜缘上皮细胞分化标记的检测

目的检测分化标记在人角膜及角膜缘上皮细胞的表达,以了解角膜及角膜缘细胞分化状态,旨在发现新的角膜上皮干细胞的阴性标记。方法获取人角膜及角膜缘组织,对冰冻切片及整个角膜组织行免疫荧光染色检测分化标记钙粘连素E、角蛋白3(CK3)、角蛋白12(CK12)、缝隙连接蛋白43、巢蛋白(nestin)和包壳蛋白(involucrin)的表达,经荧光显微镜及激光扫描共焦电镜观察,并行半定量RT-PCR以检测其相关分化标记基因的表达。结果分化标记CK3、CK12、缝隙连接蛋白43、巢蛋白和包壳蛋白在角膜和角膜缘上皮的表层细胞表达,角膜缘基底细胞不表达。激光扫描共焦电镜观察及RT-PCR结果显示角膜缘基底上皮细胞不表达细胞CK3、连接蛋白43和巢蛋白,而角膜上皮细胞则明显表达。结论角膜及角膜缘表层上皮较为成熟分化,而角膜缘基底细胞具有未分化细胞的特征,很可能是干细胞的部位。     

Immune responses of sheep to surface antigens of infective larvae of Ostertagia circumcincta

Immune responses to surface antigens of infective larvae of Ostertagia circumcincta were studied in 5-month old Finn-Dorset male lambs. The sheep were vaccinated subcutaneously with 2 doses of 25 micrograms/kg body weight of infective larvae surface or somatic extracts and Freund's or beryllium hydroxide adjuvants. It was found that only in lambs vaccinated with L3 surface extracts and beryllium hydroxide as an adjuvant did worm burdens differ significantly (P less than 0.01) from those of challenge controls (71.7% protection). Sheep vaccinated with the same antigenic preparation but administered with Freund's adjuvant or with beryllium adjuvant and somatic extracts demonstrated rather poor protection (32.4 and 30.5% respectively). All the vaccinated sheep showed a high IgG response to L3 surface antigens while significant levels of serum and bile IgA reacting with the surface extracts were only detected in those lambs vaccinated with L3 surface extracts and beryllium hydroxide. In immunofluorescence tests serum IgA of this group reacted with the whole surface of exsheathed larvae or with the site of opening of excretory pore while IgG antibodies reacted strongly with the anterior and posterior parts of the infective larvae. Both IgA and IgG responses to surface antigens were stage specific.     

trkC-like Immunoreactivity in the Primate Descending Serotoninergic System

In the search for substances with a potential role in plastic responses of spinal motoneurons we have studied the distribution of trkC-like immunoreactivity in the spinal cord of adult monkeys (Macaca fascicularis). The presence of trkC, which is a signal-transducing receptor for neurotrophin-3, was detected by the use of indirect immunofluorescence with a rabbit polyclonal antibody raised against a synthetic peptide corresponding to the carboxy-terminal domain of the mouse trkC-encoded protein, thus detecting only full-length signal-transducing receptors. trkC -immunoreactive fibres/varicosities could be found at all spinal cord levels and the densest innervation was found in the autonomic intermediolateral and Onuf's nuclei, but somatic motoneuron pools also received a significant contribution of trkC-immunoreactive fibres. Terminals immunoreactive for trkC were also seen in the dorsal horn. Double-labelling experiments revealed a high degree of coexistence between trkC- and 5-hydroxytryptamine (serotonin)-like immunoreactivity in all areas except in the dorsal horn. The results of the present study suggest that neurotrophic signalling with an influence on serotoninergic as well as non-serotoninergic inputs to the adult monkey spinal cord is at hand.     

In situ immunologic characterization of follicular lymphomas

Surface markers were studied in a series of follicular lymphomas with immunofluorescence on frozen sections (39 cases) and on cell suspensions (21 cases), and with immunoperoxidase on frozen sections using a panel of 15 monoclonal antibodies (17 cases). With immunofluorescence on frozen sections, 22/39 cases showed monotypic sIg (IgMK: 14 cases, IgML: 7 cases, M: 1 case). In the remaining 17 cases the neoplastic follicles were negative. Nevertheless, even if sIg is not detected, the absence of an extracellular immunoglobulin network is indicative of the neoplastic, and not of the reactive nature of lymphoid follicles. The results obtained with immunofluorescence on frozen sections and on cell suspensions were identical in about half of the cases. In 9/21 cases monotypic sIg were detected by only one of these two methods. All the 17 cases studied with immunoperoxidase on frozen sections using monoclonal antibodies demonstrated monotypic sIg. On low magnification 6/17 sIg+ exhibited a nodular staining pattern while 7/17 cases this staining was diffuse. In 4/17 cases the staining pattern for heavy and light chains was different. A thin mantle zone, with sIgM plus sIgD cells, was observed in only 4 cases. Anti-HLA-DR and Leu-10 were positive in all cases. T cells positive for OKT3 were mainly distributed in the interfollicular areas; OKT4 + cells outnumbered OKT8 + cells. Within the neoplastic follicles, T cells stained mainly for OKT4 and OKT8 + cells were scarce. Leu-7 + cells predominated within the neoplastic nodules in 5 cases. With the anti-dendritic reticulum cell monoclonal antibody, all 17 cases showed a network, usually more loosely arranged than in reactive follicles. In 4 cases, of follicular and diffuse lymphoma, this network was extremely dissociated and in some areas these cells were scanty or lacking. We concluded that immunoperoxidase on frozen sections, using monoclonal antibodies, appears to be the most reliable method for the immunological phenotyping of follicular lymphomas.     

抗支原体单克隆抗体试剂盒的研制和应用

从20株抗支原体MAb中筛选出1株能够对各种支原体起反应的MAb(3D1)组成试剂盒,用间接免疫荧光法(IFA) 检查了35株传代细胞和6份动物标本,支原体检出率为83%,为了验证此方法的准确性,将其中部分细胞同时做常规培养检查,符合率为90%。结果证明,MAb试剂盒具有特异性高,敏感性强,速度快等优点。     

Expression Analysis of Recombinant Herpes Simplex Virus Type 1 DNase

Expression of recombinant herpes simplex virus type 1 (HSV-1) deoxyribonuclease (DNase) was analyzed in BHK-21 cells, a standard cell line for virus propagation, by using mammalian cell expression systems based on vaccinia virus and on Semliki Forest virus (SFV)¹. Although the establishing of recombinant vaccinia virus failed due to the apparent toxicity of the herpesviral enzyme, soluble and functional HSV-1 DNase was efficiently expressed in BHK-21 cells by the vaccinia virus/T7 RNA polymerase hybrid system as well as by recombinant Semliki Forest virus. Using rabbit antiserum ExoC, directed against the C-terminal residues 503–626, or mouse monoclonal antibody (MAb) Q1, raised against the type 2 enzyme, a major 85-kDa protein with the identical size of the enzyme from HSV-1-infected cells was identified to be induced in both expression systems. With recombinant SFV functional HSV-1 DNase coincided with the overproduction of a single major 85-kDa protein re aching an optimum between 16 h and 36 h after infection. At later times of infection the enzymatic activity vanished. Thus, recombinant SFV may be an appropriate expression vector for biochemical studies of the enzyme when (i) packaged recombinant virus particles are used for infection and (ii) infection does not exceed 24 h. Due to the limitations of transient expression systems, the vaccinia/T7 RNA polymerase hybrid system is suited for expression analysis on a small scale, and for studying intracellular interactions of the enzyme as demonstrated by immunofluorescence microscopy studies. Using vector pTM1, recombinant HSV-1 DNase was efficiently overproduced in BHK-21 cells at 6 h after transfection and was shown to colocalize with the cellular chromatin at sites apparently distinct from the bulk of the herpesviral replication sites the way it is observed for the enzyme of lytically infected cells. The deleting of the 123 C-terminal amino acid residues did not alter this nuclear loca lization of HSV-1 DNase, suggesting that the latter sequences and other herpesviral factors are not required for the chromatin association. This revised version was published online in July 2006 with corrections to the Cover Date.     

抗病毒糖浆治疗小儿急性呼吸道病毒感染疗效观察

对３３５名首次门诊和住院的急性呼吸道感染患儿，采用直接免疫荧光法进行鼻咽部脱落细胞内流感病毒、副流感病毒、腺病毒、呼吸道合胞病毒检测，有１５７例检出病毒．将其中 １１７例患儿随机分为实验组５７例，口服抗病毒糖浆加青霉素；对照组６０例，仅用青霉素治疗．结果实验组用药 ３天后 ４４例（７７．２２％）症状消失，病毒转阴．说明抗病毒糖浆对小儿急性呼吸道病毒感染确实有显著疗效．     

Nucleolar immunofluorescence in bone marrow specimens of human hematological malignancies

Summary Leukemic cells and myeloma cells were studied in bone marrow of untreated patients with acute and chronic myeloid leukemia, chronic lymphocytic leukemia, and multiple myeloma to provide more information on the nucleolar immunofluorescence produced by antibodies to nucleoli of HeLa cells. The nucleolar immunofluorescence was mainly observed in myeloblasts of myeloid leukemias an in immature as well as mature cells of the plasmacytic or lymphocytic cell lines of multiple myeloma or chronic lymphocytic leukemia. With respect to the nucleolar immunofluorescence, both positive and negative populations of cells were noted in the specimens of all patients studied.These studies were supported in part by Cancer Research Grant CA-10893, awarded by the National Cancer Institute, Department of Health, Education, and Welfare; the Bristol-Myers Fund; the DeBakey Medical Foundation; the Pauline Sterne Wolff Memorial Foundation; and the William S. Farish Fund     

Detection of Chlamydophila pneumoniae in the bone marrow of two patients with unexplained chronic anaemia

Anaemia of chronic disease (ACD) is a common finding involving iron deficiency and signs of inflammation. Here, we report on two patients with ACD where a persistent infection with Chlamydophila (Chlamydia) pneumoniae (CP) was detected in bone marrow (BM) biopsies. Infection was suspected by routine cytology and confirmed by immunofluorescence, electron microscopy, polymerase chain reaction (PCR) including different primer sets and laboratories and sequencing of the PCR product. This is a first report of chlamydial presence in the BM of anaemic patients. The cases are presented because persistent chlamydial infection may contribute more frequently to chronic refractory anaemia than previously suspected.