Changes in T-lymphocyte subsets during childhood Epstein-Barr virus infectious mononucleosis

Lymphocyte subsets were measured using monoclonal antibodies in 11 children with Epstein-Barr virus-induced infectious mononucleosis and compared with those of 10 normal children. In acute infectious mononucleosis the percentage of T8⁺ lymphocytes was greater while the percentage of T4⁺ lymphocytes and the T4⁺ to T8⁺ ratio were less than those measured in normal children. The percentage and absolute number of T lymphocytes, as enumerated by E rosetting, did not differ from the values for normal children. The children with acute infectious mononucleosis had a somewhat lower T8⁺ response than that observed in four adult infectious mononucleosis patients. With clinical recovery, the T lymphocyte-subset values returned toward normal. T8⁺ lymphocytes, a phenotype subset with predominantly suppressor activity, presumably reduce normal cellular immune functions transiently and may limit the continued proliferation of Epstein-Barr virus-infected B lymphocytes.     

人外周血淋巴细胞酶活性光镜细胞化学定位

目的　探讨人外周血淋巴细胞酶结构定位与活性。方法　应用光镜酶细胞化学方法对人外周血淋巴细胞ATP酶与G 6 P酶进行酶的定位与活性分析。结果　ATP酶阳性反应颗粒清晰 ,见于淋巴细胞膜内侧 ,G 6 P酶阳性反应颗粒见于淋巴细胞胞质内。结论　二种酶存在于人淋巴细胞内。     

Chronic Tick-Borne Encephalitis Virus Antigenemia: Possible Pathogenesis Pathways

Study of the proliferative potential and immunophenotype of lymphocytes and cytokine-producing capacity of mononuclear cells in patients with chronic tick-borne encephalitis virus antigenemia showed changes in T cell lymphoproliferative response, relative content of T cells and T helper inductors, immunoregulatory index, and imbalance in the production of immunoregulatory Th1 and Th2 cytokines.__________Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 139, No. 4, pp. 446–450, April, 2005     

Electron Microscopic Characteristics of Peripheral Blood Lymphocytes in Children with Infectious Mononucleosis

Infectious mononucleosis is associated with pronounced changes in surface architectonics of peripheral blood lymphocytes persisting during convalescence and remote period after the disease. The degree of these changes depends on the disease agent and age-specific characteristics of the body. The most pronounced and sustained disorders in the morphostructural organization of lymphocytes are caused by Epstein-Barr virus (in comparison with agents of other etiology); these disorders are more pronounced in children aged 7-14 years than in those aged 3-6 years.     

Surface Architectonics of Peripheral Blood Erythrocytes in Patients with Mental Diseases

Scanning electron microscopy demonstrated altered surface topography of peripheral erythrocytes in patients with nonpsychotic mental diseases, nonmetabolic mental retardation, and paranoid schizophrenia. Maximum decrease in the number of biconcave diskocytes and accumulation of transitional, prehemolytic, and degenerative forms of erythrocytes were found in schizophrenia.     

EB病毒和ConA诱导抑制性T细胞对EB病毒感染B细胞作用探讨

本文探讨了用灭活的EB病毒(EBV)和ConA诱导产生的抑制性T细胞(Ts),对EBV感染自身B细胞的影响,结果表明,EBV抗原诱导产生的抑制性T细胞(Ts)能使EBV感染B细胞中的EBNA阳性细胞数,~3H-TdR掺入量和IgA、IgG及IgM分泌量减少;而ConA诱导产生的Ts则使EBNA阳性细胞数和~3H-TdR掺入量增加,但三种Ig含量无明显变化(P>0.05)。结果提示前者对EBV感染B细胞的激活,增殖和分化均有明显抑制作用,而后者的作用则相反,具有明显促进EBV感染B细胞的作用。     

Structure and organization of odontoblasts

Differentiation of odontoblasts involves cell-to-cell recognition, contact stabilization involving the formation of attachment specializations, cytoplasmic polarization, development of the protein synthetic and secretory apparatus, and the active transport of mineral ions. The secretory odontoblast is characterized by an extensive rough-surfaced endoplasmic reticulum, a highly developed Golgi complex, and the presence of specific secretion granules. Type I collagen, a major constituent of dentin matrix, appears to be secreted by the odontoblast into predentin at the proximal portion of the odontoblast process, the major cytoplasmic process extending from the odontoblast cell body into the dentin. The odontoblast process contains a rich network of microtubules and microfilaments. The proximal portion of the process is also a site of fluid-phase endocytosis. Adjacent odontoblasts are held together by numerous macula adherens junctions and a well-developed distal junctional complex adjacent to the predentin. Junctional strands of the occludens type have been observed to be a component of this junctional complex. Tracer studies employing horse-radish peroxidase indicate that this junctional complex does not form a tight barrier to the diffusion of tissue fluid from the interodontoblast spaces into the predentin. Many well-developed gap junctions are formed between adjacent odontoblasts and between odontoblasts and the fibroblasts that make up the subodontoblastic layer. Ca-ATPase activity is demonstrated in the Golgi complex and mitochondrial cristae and along the distal plasma membranes of odontoblasts. ALPase activity is also intense along the entire odontoblast cell surface. The osmium tetroxide-pyroantimonate technique for calcium localization demonstrates prominent reaction precipitates in mitochondria of odontoblasts. Energy-dispersive x-ray microanalysis of anhydrously fixed and processed odontoblasts detected Ca and P peaks throughout the cytoplasm. A sulfur peak is noted in the distal cytoplasm of odontoblasts and in matrix vesicles. Together, these results demonstrate the complexity and variety of cell functions involved in dentinogenesis. © 1996 Wiley-Liss, Inc.     

Computational Prediction and Identification of Epstein-Barr Virus Latent Membrane Protein 2A Antigen-Specific CD8^＋ T-Cell Epitopes

Epstein-Barr virus （EBV） associated nasopharyngeal carcinoma （NPC） is a high incidence tumor in Southeast Asia. Among EBV encoded proteins, latent membrane protein 2A （LMP2A） is an important antigen for T cell therapy of EBV. In this study, we predicted six HLA-A2 restricted CTL candidate epitopes of LMP2A by SYFPEITHI, NetMHC and MHCPred methods combined with the polynomial method. Subsequently, biological functions of these peptides were tested by experiments in vitro. In ELISPOT assay, the positive response of the LMP2A specific CTL stimulated by three （LMP2A264.272, LMP2A426-434 and LMP2A3s6.364） of six peptides respectively showed that the numbers of spots forming cells （SFC） ranged from 55.7 to 80.6 SFC/5 x 104 CO8^＋ T cells and the responding index （RI） ranged from 5.4 to 7. These three epitope-specific CTLs could effectively kill specific HLA-A2- expressing target cells. As a result, LMP2A264.272 （QLSPLLGAV）, LMP2A426.434 （CLGGLLTMV） and LMP2A356.364 （FLYALALLL） were identified as LMP2A-specific CD8^＋ T-cell epitopes. It would be useful to clarify immune response toward EBV and to develop a vaccine against EBV-correlative NPC.     

Pattern of Epstein-Barr virus (EBV)-specific proteins synthesized during primary lytic infection of mouse lymphocytes by EBV

Normal mouse lymphocytes were implanted with EBV receptors and exposed to the virus of P3HR-1 strain. 5% of the cells expressed early (EA) and viral capsid (VCA) antigens as assayed by immunofluorescence 24 h after the infection. Only 0.1% of cells expressed nuclear-like antigen (EBNA) 48 h post-infection. When labelled metabolically with [35S]methionine, extracted, immunoprecipitated with EBV-positive sera, and analyzed by SDS-gel electrophoresis and autoradiography, about 20 EBV-determined proteins ranging from 19 to 165 kd were detected. Their pattern and relative quantitative expression differed from those in P3HR-1 virus superinfected Raji cells. Polypeptides of approximate molecular size 78, 72, 65, 48 and 26.5 kd were predominant in EBV-infected mouse lymphocytes. In contrast, 130, 98, 59, 50.5 and 36 kd proteins were predominant in the induced Raji cells. Our results demonstrate that rodent lymphocytes can be used for the direct biochemical analysis of EBV-translational products during primary lytic infection in normal cells.     

Immunogold-Silver Staining Method for Lymphocyte Cell Surface Antigens with Light,Transmission Electron,and Scanning Electron Microscopy

Abstract

The immunogold-silver staining (IGSS) method combined with light, transmission electron, and scanning electron microscopy (LM, TEM and SEM, respectively) was used for detecting lymphocyte surface antigens. Two different sizes of colloidal gold particles (5 nrn and 15 nm) were applied as markers and IntenSEII kit as a physical developer for gold particles.

The silver enhanced gold particles were clearly observed on cell surfaces as black dots in LM and TEM and as white dots in SEM equipped with a mixed signal of secondary electron and back-scattered electron (SE/BE) signals. Monoclonal antibody (MAb)-positive cells possessing the complexes on their well preserved surfaces were easily identified among other lymphoid cells at low magnifications of LM or SEM equipped with SE/BE signals. Thus, the IGSS method has a great advantage for a qualitative screening such as the percentage of lymphocyte subsets in a cell suspension. However, the IGSS method was inadequate for semiquantitative study with antigen density on cell surfaces because gold particles enhanced with the physical developer were considerably enlarged, and a silver-gold complex was not considered to show one antigen site on cell surface. (The J Histotechnol 16:217, 1993)     

Absence of Epstein-Barr virus-specific, HLA class II-restricted CD4+ cytotoxic T lymphocytes in infectious mononucleosis.

Cytotoxic T lymphocytes (CTL) with the CD4+ phenotype that recognize major histocompatibility complex (MHC) class II antigens are detectable very frequently in cultures of human alloreactive or virus-specific T cells. The significance of these CD4+ CTL for an immune reaction in vivo is not clear. Since Epstein-Barr virus (EBV) transformed B cells express HLA-class I and class II antigens equally well both CD8+ and CD4+ CTL should be stimulated during an acute EBV infection. We analysed the MHC specificity and the phenotype of EBV-specific CTL from patients with infectious mononucleosis (IM). When tested directly without any previous culture, T cells from patients in the acute phase of IM showed specific MHC-restricted cytotoxicity against the autologous B cell line. Addition of a HLA class I specific monoclonal antibody (MoAb) but not of a HLA class II specific MoAb resulted in a complete blocking of the lytic activity. Cell sorting revealed that the entire cytotoxic activity was present in the CD8+ fraction whereas no specific CTL were detectable in the CD4+ fraction. The absence of cytotoxicity in CD4+ cells was not due to a lack of activation of these cells since both CD8+ and CD4+ cells were activated in situ, showing spontaneous growth in interleukin-2 (IL-2) and expressing the activation marker TP103. Frequency estimation revealed that 1/300-1/600 CD8+ but only 1/2000-1/4000 CD4+ T cells gave rise to a specific CTL colony after 10 days. If CD4+ colonies were tested repeatedly for cytotoxicity we found that CD4+ CTL acquired their cytotoxicity during in vitro culture. In addition, we isolated EBV-specific CD4+ T cell clones able to lyse their stimulator cells in the presence but not in the absence of lectin, even after a long period of culture. Taken together our results show that cytotoxicity mediated by CD4+ T cells does not play a role in an anti-viral immune response.     

尿毒症患者透析前后外周血B细胞和T细胞及其亚群的研究

应用单克隆抗体技术检测了３５名正常人和３１例尿毒症患者外周血Ｂ细胞和Ｔ细胞亚群。结果显示：尿毒症患者外周血Ｂ细胞数显著地高于正常人（Ｐ〈０．００１）,ＣＤ３、ＣＤ４低于正常人（Ｐ〈０．０１）,ＣＤ４／ＣＤ８又非常显著地低于正常人（Ｐ〈０．００１）。因此认为,尿毒症患者为一种自身免疫调节异常的疾病。     

老年鼠外周血淋巴细胞凋亡及基因调控的研究

本文采用TUNEL法及流式细胞仪观察不同鼠龄外周血淋巴细胞凋亡及凋亡发生率 ,并用免疫组织化学检测Bcl 2、Bcl xl及Bax基因表达变化。结果发现老年鼠组外周血淋巴细胞群中存有典型的凋亡细胞 ,凋亡细胞发生率明显高于青年组。老年鼠PBL的Bcl 2 ,Bcl xl表达下调 ,而Bax的表达量明显增加。提示在衰老过程中存在着免疫细胞凋亡 ,其发生与相关调控基因的表达一致。     

Peripheral Blood Smears of Myelodysplasia Patients: Scanning Electron Microscope Findings

The utility of scanning electron microscopy in the evaluation of ordinary glass peripheral blood smears of patients with myelodysplasia and those uncertain for myelodysplasia is emphasized. Attention is directed to changes in segmented granulocytes. Comparison of ultrastructural findings in abnormal blood smears with control cases is made. Important findings include reduced cytoplasmic granule number, increased cell size, large cytoplasmic vacuoles, condensation of the peripheral cytoplasm, prominence of large cytoplasmic granules, irregular cytoplasmic perimeter, abnormal nuclear morphology, abnormal cell shape, and a necklace-like arrangement of cytoplasmic granules. Of these findings, reduced cytoplasmic granule number was the most specific finding, while condensation of peripheral cytoplasm was the most sensitive. Combination of these two morphologic findings may provide a strong predictor of myelodysplasia. The study included a limited test of unknown cases evaluated by one author, including two uncertain for myelodysplasia. Pitfalls in evaluating temporary pancytopenia not associated with myelodysplasia are noted.     

Ultrastructural Morphometric Study of Follicular Center Lymphocytes: I. Nuclear Characteristics and the Lukes-Collins' Concept

A combined ultrastructural and morpnometric image analysis study was carried out on the nuclear profiles of follicular center and mantle zone lymphocytes of six cases of reactive hyperplasia in human lymph node biopsies. For accuracy of morphological observations and sampling at low magnifications, sections were mounted on formvar-covered slot grids. Measurements of nuclear profile features of small (untransformed) lymphocytes in mantle zones served as the standard for a supposed unimodal population in each case. Analysis of nuclear profile area values indicated that during lymphocyte transformation in follicular centers nuclei had a gradual and progressive increase in size and that the sampled nuclear profiles in both the mantle zone and follicular center were unimodal. Lymphocyte nuclear shape (contour index) was a more complex, and likely biologically independent, feature than nuclear area in both the mantle zone and follicular center. Nuclear profile contour indexes of mantle zone lymphocytes were more irregular than suspected and in some cases had mean values greater than those of follicular center lymphocytes. Furthermore, the frequency distribution of nuclear contour index was not normally distributed in either the follicular center or mantle zone due to the presence of a small proportion of highly irregularly shaped nuclear profiles in both sites. The results indicated that some premises of existing concepts of follicular center cells and the process of lymphocyte transformation in follicular centers were incorrect and should not be directly extrapolated to the nuclear profile characteristics in non-Hodgkin's lymphoma.     

Comparative Analysis of Accumulation of Chlorine e6 and Hematoporphyrin Derivatives in Subpopulations of Peripheral Blood Lymphocytes

We studied accumulation of porphyrin photosensitizers chlorine e6, hematoporphyrin, and their derivatives by different lymphocyte subpopulations. The intensity of staining of B lymphocytes and natural killer cells with photosensitizers was higher compared to T lymphocytes. T cell subpopulation differed by their ability to bind photosensitizers. Relative accumulation of dimethyl esters of chlorine e6 and hematoporphyrin in cells surpassed that of nonesterified porphyrins.     

宫颈癌患者外周血T淋巴细胞及NK细胞的检测及其临床意义

为探讨宫颈癌患者外周血T淋巴细胞亚群和NK细胞活性检测的临床意义,采用流式细胞仪测定宫颈癌患者外周血T淋巴细胞亚群和NK细胞活性,以正常人作对照分析。结果表明,宫颈癌患者外周血CD3＋、CD3＋CD4＋、NK细胞数量及CD3＋CD4＋/CD3＋CD8＋比值较正常对照组均明显下降（P〈0.05）,而CD3＋CD8＋细胞水平显著升高。外周血T淋巴细胞亚群和NK细胞数量的改变与宫颈癌临床病理分期有关,分期越晚,CD3＋细胞、CD3＋CD4＋细胞、CD3＋CD4＋/CD3＋CD8＋细胞比值及NK细胞数量越低,CD3＋CD8＋细胞水平越高;Ⅰ、Ⅱ期宫颈癌患者与Ⅲ、Ⅳ期患者之间有显著差异（P〈0.05）。实验结果提示,宫颈癌患者细胞免疫功能低下,且临床病理分期越晚,其免疫功能越低,检测T淋巴细胞亚群、NK细胞可用于宫颈癌患者的免疫监测。     

Transduction of Primary Lymphocytes with Epstein-Barr Virus (EBV) Latent Membrane Protein-Specific T-Cell Receptor Induces Lysis of Virus-Infected Cells: A Novel Strategy for the Treatment of Hodgkin's Disease and Nasopharyngeal Carcinoma

Adoptive immunotherapy with in vitro expanded cytotoxic T lymphocytes specific for Epstein-Barr virus (EBV) can successfully treat post-transplant lymphoproliferative disease (PTLD). However, extension of a similar strategy to Hodgkin's disease (HD) and nasopharyngeal carcinoma (NPC) is limited by the poor immunogenicity of the limited set of EBV latency antigens expressed in these malignancies, making T-cell expansion difficult. Retroviral transduction of LMP-specific T-cell receptors (TCR) into activated T lymphocytes may provide a universal, MHC-restricted, means to generate effector cells without the need for tissue culture based methods of CTL expansion. We report the transfer of two LMP2-specific TCRs from human T-cell clones (HLA-A2 and HLA-A23,24 restricted) that confer the ability to lyse EBV-immortalized B-lymphoblastoid cell lines (B-LCL). B-LCL are the best model for native expression of LMP2. We also demonstrate the rapid transfer of the TCR by nucleofection of primary T cells using a simple plasmid-based vector. The ability to detect nucleofected TCRVβ chain by antibody, fully assembled TCR by tetramer, and peptide-MHC-specific lytic activity indicates that nucleofection can serve as a tool for rapid screening of TCR specificity.     

Application of Pop-off Method to Bone Marrow and Peripheral Blood Specimens for Purposes of Electron Microscopy

In this paper, we describe a pop-off method applicable to the hematological field. Bone marrow or peripheral blood specimens from patients were placed on a clean glass slide and fixed immediately in 2% glutaraldehyde solution for 10 minutes. For the DAB reaction, the slide was immersed in the DAB reagent for 30 minutes, and post fixed with 1 % OsO₄ solution for 1 hour. Specimens on the slide were washed with buffer solution, dehydrated and polymerized directly on the slide. A gelatin capsule filled with Epon mixed monomer was then reversed over the specimen. After polymerization was completed, the specimen was popped off from the slide to the capsule and trimmed carefully to prepare for ultrathin sectioning. This method allows the entire sequence of tissue preparation to be carried out on the slide, from fixation to embedding, and even, especially in scanty specimens, including the DAB reaction. Electron microscopic findings in specimens prepared by this technique show excellent preservation and the absence of specific artifacts.     

Application of Pop-off Method to Bone Marrow and Peripheral Blood Specimens for Purposes of Electron Microscopy

In this paper, we describe a pop-off method applicable to the hematological field. Bone marrow or peripheral blood specimens from patients were placed on a clean glass slide and fixed immediately in 2% glutaraldehyde solution for 10 minutes. For the DAB reaction, the slide was immersed in the DAB reagent for 30 minutes, and post fixed with 1 % OsO₄ solution for 1 hour. Specimens on the slide were washed with buffer solution, dehydrated and polymerized directly on the slide. A gelatin capsule filled with Epon mixed monomer was then reversed over the specimen. After polymerization was completed, the specimen was popped off from the slide to the capsule and trimmed carefully to prepare for ultrathin sectioning. This method allows the entire sequence of tissue preparation to be carried out on the slide, from fixation to embedding, and even, especially in scanty specimens, including the DAB reaction. Electron microscopic findings in specimens prepared by this technique show excellent preservation and the absence of specific artifacts.