Expression of inhibitory receptors for MHC class I molecules on T cells

Inhibitory receptors (IRs) specific for MHC class I molecules and originally described on natural killer (NK) cells are also expressed on a fraction of peripheral T cells. The presence of these receptors on T cells is poorly understood. In this review, the different antigen specificities described to date for IR+ T cells and the expression pattern of these receptors on T cells are analyzed. This analysis indicates that the population of T cells defined by IR expression is heterogeneous and that different IRs (or families of IRs) may play different roles in T-cell biology.     

Microcarrier cell culture: Vero cells on cytodex® 1

Summary All the necessary steps required for the cultivation of Vero cells on microcarriers are described. These procedures are used routinely in our laboratories for the growth of Vero cells on Cytodex 1. Consistent, high density, final yields of more than 10⁶ cells/ml of culture medium are obtained. The protocols described here can be modified for the growth of a variety of anchorage-dependent cell types. Culture parameters important for the successful exploitation of microcarrier cell culture technology are discussed.     

The fine structure of chordoma with particular reference to the physaliphorous cell

A chordoma was examined by electron microscopy. It was composed of stellate and physaliphorous cells. The fine structure of these cells is described. Transitional type cells are also described and it is suggested that the physaliphorous cells develop from the stellate cells through a process of vacuolation. Two types of vacuoles are described, `smooth-walled' and `villous' endowed with numerous structureless microvilli. Some observations on stained sections are discussed.     

Antigens of the surface of mouse ascites tumour cells: I. Studies with rabbit anti-mouse cell sera

Studies with rabbit antisera have revealed the presence of three distinct agglutinogens on the surface of mouse ascites tumour cells and `L' cells. The T-antigen is the dominant agglutinogen on the mouse nucleated cells and is absent from mouse and sheep erythrocytes; the F-antigen is common to the mouse nucleated cells and sheep erythrocytes but absent from mouse erythrocytes; the C-antigen is common to the surface of mouse nucleated cells and erythrocytes but absent from sheep erythrocytes. The C-antigen is not the major agglutinogen of the mouse erythrocyte. Methods are described for the independent assay of the T, C and F antigens and their occurrence on a number of mouse tumour cells described.     

Further Miniaturization and Automation of in vitro Lymphocyte Cultures

A microtechnique (10 mul culture volume) for in vitro lymphocyte cultures is described, which, compared with current miniaturized techniques, permits a four to 20-fold reduction of both medium volume and cell numbers. Furthermore, two alternative procedures for harvesting and washing radioisotope labelled cells are described. The first, making use of a conventional harvesting device, collects cells on glass fibre filters in a two to three-fold shorter time than that needed for the current semi-automatic collectors. The second takes advantage of the possibility of washing and fixing the cells in their culture wells and allows processing of more cultures with fewer manual operations. Various technical aspects of the micro-culture system are described and discussed with special reference to automation problems.     

Morphology of Pneumocystis carinii and activation of the plasmalemmal vesicular system in alveolar epithelial cells of the host. An ultrastructural study

Lungs from rats with dexamethasone-induced Pneumocystis carinii pneumonia were examined. The ultrastructure of Pneumocystis carinii and their zone of attachment on type I alveolar epithelial cells are described. An activation of the plasmalemmal vesicular system of type I alveolar epithelial cells was observed and is described here for the first time. The significance of this observation is discussed.     

Procedures to test for the serum-free cultivation of adherent cell lines on microcarriers

Summary Procedures are described for determining whether it is possible to cultivate adherent cell lines as suspension cultures on microcarriers in serum-free medium. The method is described for Gelibead, Cytodex 1, and Cytodex 3 microcarriers. It is performed in two parts. First, cells are cultivated on microcarriers in unstirred cultures in hydrophobic petri dishes in order to test whether the cells will grow on microcarriers in serum-free medium at all. If the cells attach and grow, suspension cultures are set up with microcarriers in spinner flasks in order to test the potency of the serum-free medium under stirred conditions. This method indicates whether the cultivation of an individual adherent cell line in a particular serum-free medium is practical on microcarriers. If the cells grow without problems on the microcarriers, such a cultivation has a good chance for succes in scale-up in controlled fermenters.     

Intracellular vesicular stomatitis virus nucleocapsids and virions visualized by surface spreading

Spreading of cells on a solution surface could visualize vesicular stomatitis virus nucleocapsids and virions in infected cells easily and clearly without the need for any purification. Characteristic structures observed by the spreading of the infected cells are described and discussed.     

Functional-morphological model of organization of the epithelium of normal, hyperplastic and neoplastic human endometrium]

A model of functional-morphological organization of epithelium of human normal, hyperplastic, and tumour endometrium is described. The model is based on a hypothesis that a population of stem cells capable of indefinite unlimited self-maintenance exists in normal glandular epithelium of the endometrium. The population of normal stem cells of endometrium epithelium is the source of emergence of differentiated types of epithelial cells among which, according to the model, three cell types can be distinguished: cells sensitive to estradiol alone, cells sensitive to both estradiol and progesteron, and cells sensitive to progesteron alone. The conditions necessary for transition of one cell type to another as well as histochemical characteristics of each of the cell types are described. The model has been used for the analysis of morphological changes developing in hyperplastic processes and cancer of the endometrium as well as for the substantiation of the current schedules for hormone therapy of these diseases.     

Interactions of macrophages and erythrocytes

An in vitro technique is described which demonstrates a difference in the interaction of fresh and stored, effete, red cells with autologous macrophages. The stored erythrocytes adhere to macrophages whereas fresh erythrocytes do not.

The effects on this difference of the medium in which the red cells are stored, and of the storage temperature, are described. Results are presented which indicate that serum opsonic factors are not necessary for the adherence of the stored red cells to macrophages.

     

Antigen-specific proliferation of murine lymph node cells in vitro

Summary A method is described for determining the antigen-specific proliferative response of murine cells to foreign antigens in vitro. Methods are given for immunizing mice and for preparing lymphoid cells. Culture of lymphoid cells with antigen and quantitation of the proliferative response by determination of the uptake of tritiated thymidine is described. Applications of this methodology are indicated.     

Direct protein transfer to terminally differentiated muscle cells

Recently, a new approach for direct protein transfer to mammalian cells based on the herpes simplex virus type 1 protein VP22 has been described. This protein has the remarkable property of intercellular trafficking, which is independent of direct cell contacts and is also retained when fused to heterologous proteins. However, the spreading has only been described for proliferating cells and has also been controversially discussed. In this study we describe the generation of a GFP-VP22 fusion protein which is able to spread in COS-7 cells after transient transfection. Moreover, we show in coculture experiments with transfected COS-7 cells and C2C12 myotubes that this fusion protein is also able to spread into terminally differentiated skeletal muscle cells. These results suggest that VP22 might be a novel therapeutic tool for direct protein transfer not only in proliferating but also in terminally differentiated cells.     

'Autoreactivity' of some hybridomas may be caused by a three-cell interaction

We have produced hybridomas by the fusion of BALB/c (H-2d) bone marrow cells, bone marrow cells from BALB/c-nu/nu mice, BALB/c fetal liver cells, and BALB/c fetal thymocytes with the AKR (H-2k) thymoma BW5147. The hybridomas were selected for the expression of the Thy-1.2 antigen of the normal cell donor and for their ability to produce IL-2 upon co-culture with irradiated normal spleen cells. The hybridomas produce IL-2 when co-cultured with H-2k, H-2u, H-2j, or H-2v cells of some strains but not in mixtures with H-2p, H-2s, H-2f, H-2b, H-2q, or H-2d cells. An investigation into the nature of these differences revealed a novel type of interaction between hybridomas, mature T lymphocytes and class II-positive spleen or lymph node cells. The experiments described in this communication suggest that irradiated L3T4+, Ly-1 High T cells recognize syngeneic class II-positive spleen or lymph node cells, but only in some strains. The ability to recognize the syngeneic cells depends both upon the H-2 complex and on the non-H-2 genes. The recognition leads, in the absence of proliferation, to the secretion of an as yet unidentified and largely hypothetical factor which then acts on the hybridoma cells. Upon stimulation with the T lymphocyte-derived factor, the hybridoma cells begin to secrete IL-2, which can then be measured by the proliferation of the IL-2-dependent CTLL line. The IL-2 production by the hybridoma cells is independent of their proliferation. The described interaction apparently does not involve the T-cell receptor of the hybridoma cells. The interaction resembles the autoreactivity of thymocyte hybridomas described by other investigators, and therefore it is possible that this 'autoreactivity' may in fact be generated by a similar mechanism to the one described here.     

An immunoperoxidase technique for the identification of gastrin-producing cells

A technique for the identification of gastrin-producing cells (G cells) is described. This is applicable to formalin-fixed, paraffin-embedded material. It is based on an immunohistochemical method using peroxidase-labelled antibodies.     

Methodology of using vaccinia virus to express foreign genes in tissue culture

Summary A basic technique is described for inserting any foreign gene into poxvirus by in vitro recombination. Also described is a method for identifying and plaque-purifying recombinant poxvirus containing the foreign gene using nitrocellulose filters and DNA hybridization. Immunologic techniques are presented for analyzing expression of the foreign gene, either on the surface membrane or inside infected cells.     

Binding of type-III group-B streptococci to buccal epithelial cells

A binding assay was used to study the attachment of type-III group-B streptococci (GBS) to buccal epithelial cells. Results indicate that an adhesin, with the characteristics of a protein, is the molecule at the streptococcal cell surface responsible for attachment to the buccal cells. The bacterial adhesin probably recognises a sugar on the surface of the mucosal cell, because periodate oxidation of the buccal cells caused a significant reduction in subsequent adherence of GBS. A sonicate to type-III GBS blocked the binding of the organism to buccal cells. The effects of physical and chemical modifications of the sonicate on its ability to prevent bacterial attachment are described; these corroborate the evidence gained from heat and periodate treatments of the buccal cells and GBS. Results suggest a lectin type of attachment mechanism for type-III BGS which can be blocked by N-acetyl-D-glucosamine, rather than attachment by means of a lipoteichoic acid as described for group-A streptococci.     

An antigenic study of human plasma cells in normal tissue and in myeloma: identification of a novel plasma cell associated antigen.

A mouse monoclonal antibody named BU11 which detects an antigen strongly expressed on human plasma cells is described. The antibody stains plasma cells in tonsil sections, fresh and cultured plasmacytoid cells from the bone marrow of patients with multiple myeloma and cells of the plasmacytoid cell line RPMI 8226 used as the immunogen. In vitro studies of pokeweed mitogen (PWM) stimulated peripheral blood B cells and Epstein-Barr virus (EBV) stimulated tonsil B cells show that the antigen is present mainly on cells coexpressing the OKT10 antigen and containing cytoplasmic immunoglobulin (cIg). The BU11 antigen is expressed weakly on some normal B cells and is not present on T cells, monocytes or granulocytes. The antigen is of molecular weight 58kD under reducing conditions and is biochemically distinct from previously described plasma cell antigens.     

Measuring the blood-flow velocity in microvessels with a pulse counter

A photometric trigger method of measuring the velocity of red blood cells, by means of which the linear velocity and the cell flow in microvessels can be measured for a long period of time, is described. The use of a pulse counter to time the intervals at which the red cells pass enables the signals of the measuring trigger to be transformed directly into a digital code. The results of trials of the apparatus on microvessels of the frog mesentery are described.     

Production and isolation of anti-idiotypic B cells

Summary Techniques are described for the production of anti-idiotypic B cells by immunization with idiotypic antibodies complexed to rheumatoid factor. Screening for anti-idiotypic antibody production and immunoaffinity isolation of the B cells, actively producing the anti-idiotype antibody, by idiotypic antibody-coated magnetic beads is described. This methodology can be used to isolate anti-idiotypic B cells for immune regulation studies or to provide fusion partners for the production of monoclonal anti-idiotypic antibodies.     

Intravenous tufted angioma

A case of a rare vascular tumor, intravenous tufted angioma, is described. A 51-year-old Japanese man presented with a 12x8 mm solitary reddish nodule on the right foot, which had been found at birth. Histologically, the tumor was confined to a malformed vein and was characterized by nodular aggregates of plump cells. The aggregates showed a compact proliferation of round cells, including capillary-forming cells. Venous angiomatous areas were also observed. No multinucleated giant cells were seen. Immunohistochemically, the capillary-forming cells in the aggregates and the endothelial cells in the angiomatous areas were positive for endothelial markers (factor VIII-related antigen, CD31, CD34). Pericyte-like cells expressing alpha-smooth muscle actin and muscle actin, and macrophage-like cells, which stained for factor XIIIa, were intermingled in the cellular aggregates. Flow cytometric analysis showed diploidy. The tumor may be a hamartomatous lesion modified by secondary reactive changes, and it may represent a distinctive clinicopathological entity that is closely related histogenetically and perhaps pathologically to tufted angioma and the recently described "giant cell angioblastoma".