Biomedical subjects

J Giaimis

Publications and source records attributed to J Giaimis.

7 recordsLinked to original sources

PubMed1994

Flow cytometry distinction between adherent and phagocytized yeast particles.

Our laboratory recently developed a light microscopy staining technique that provides a mean to distinguish between yeast that are simply bound to the surface of macrophages and yeast that have actually been phagocytized by macrophages (7). We adapted this technique by using fluorescent probes in order to test phagocytic activity by flow cytometry. Thus we are able to distinguish unambiguously extracellular from intracellular yeast during phagocytosis with the fast rate of flow cytometry (approximately 200 cells/s). The fluorescence quenching induced by a 1% tannic acid solution (w/v) can be applied to any FITC-labeled, heat-killed yeast cell or bacteria. The yeast cells already engulfed in the macrophage remain with their native fluorescence (internal and external pH equilibrated by 50 microM monensin 30 min/4 degrees C) protected from the action of tannic acid, a nonmembrane permeable molecule. The results presented here validate this new technique. An application is presented showing the inhibition of endocytosis by cytochalasin-B.

Animals↗

PubMed1994

A new method for studying the binding and ingestion of zymosan particles by macrophages.

One of the major problems encountered during quantitative studies of phagocytosis is the discrimination without ambiguity between intracellular and extracellular particles. This difficulty is especially acute when zymosan particles are used because of their poor affinity for dyes. We show in this paper that zymosan particles may be stained by a mixture of a basic dye and tannic acid in water (or in an isotonic non saline solution). Crystal violet is one of the most suitable dyes and by combining this staining step with May-Grünwald Giemsa staining, it was possible to observe two populations of macrophage-associated particles. One comprises blue violet particles (BVP), and the second consists of particles with a purple-stained core (PPC). Treating macrophages in culture with various concentrations of cytochalasin B decreases the number of PPC and increases the number of BVP, in a dose dependant manner. Moreover, treatment with alpha-mannan or laminarin decreases the number of cell-associated particles, especially that of PPC. From these observations we concludes that BVP are extracellularly located and PPC are ingested.

Animals↗

PubMed1994

Functional beta-glucan receptor expression by a microglial cell line.

In the central nervous system, the functions of microglia appear crucial after brain damage, when phagocytes eliminate cell debris, acting as the scavengers of the brain. Diseases where an active role for microglia has been proposed recently include Alzheimer's disease, the acquired immune deficiency syndrome (AIDS) and multiple sclerosis. Only recently has it been possible to obtain a microglial cell line retaining morphological and functional aspects of these cells and their secretory products. Sugar receptors are expressed by a variety of phagocytes in primary cultures, but in contrast, are absent on the majority of the described macrophage-like cell lines. We here establish, by 4 degrees C binding experiments, that this murine cell line, called BV-2, expresses a high level (9.86 +/- 0.91 x 10(5); n = 3) of beta-glucan receptors. At 37 degrees C, BV-2 cells show high phagocytic power that can only be inhibited by the free polysugar beta-laminarin (a poly-glucose) and not by mannan (a poly-mannose) as described for macrophages. The beta-glucan receptor expressed by the microglial cell line BV-2 is fully functional in phagocytosis of unopsonized heat-killed yeast particles.

Animals↗

PubMed1993

Both mannose and beta-glucan receptors are involved in phagocytosis of unopsonized, heat-killed Saccharomyces cerevisiae by murine macrophages.

We studied the involvement of lectin-like receptors in phagocytosis of unopsonized heat-killed yeast (Saccharomyces cerevisiae) by murine macrophage-like cell lines and murine peritoneal resident macrophages. For this purpose we used a technique that allowed us to discriminate ingested and adsorbed heat-killed yeast. The internalization can be partly inhibited by soluble polyosides such as laminarin (beta-glucan) or alpha-mannan. However, when they were used together (0.4 mg/ml alpha-mannan and 0.4 mg/ml laminarin), almost complete inhibition of phagocytosis was obtained. These observations suggest that phagocytosis of unopsonized heat-killed yeast by murine macrophage-like cell lines as well as murine peritoneal resident macrophages is mediated by both mannose and beta-glucan receptors. The respective activity of these two types of receptors is a function of in vitro cell differentiation. To achieve maximal phagocytosis of unopsonized heat-killed yeast, coexpression of both mannose and beta-glucan receptors is required.

Animals↗

PubMed1992

A new and simple method for studying the binding and ingestion steps in the phagocytosis of yeasts.

Autoclaved yeasts are stained light pink by May-Grünwald Giemsa (MGG). If treated with tannic acid solution just before MGG staining, they display a deep violet color. It seemed possible that these properties could be used to discriminate between extra- and intracellular yeasts in a phagocytosis test, extracellular yeasts being violet and intracellular yeasts being pink. To validate this protocol, quantitative studies of phagocytosis by MALU cells (a murine macrophage cell line) were performed in the presence or absence of drugs known to interfere with phagocytosis. After treatment of cells with cytochalasin B, the mean number of pink yeasts per cell decreased in a dose-dependent manner, the mean number of violet yeasts increased in a dose-dependent manner, whereas the total number of cell-associated yeasts remained almost unchanged whatever the dose used. After treatment with alpha-mannans or chloroquine, the mean numbers of both violet and pink yeasts decreased in a dose-dependent manner. These results confirmed that (i) violet yeasts are extracellular, (ii) autoclaved yeasts recognize lectin receptors, and (iii) unstained (pink) yeasts are intracellular. We show that this simple method can be used for quantitative light microscopic analysis of both the attachment and internalization steps in the phagocytosis of yeasts.

Animals↗

PubMed1992

Phenotypic characterization of three long-term-cultured murine resident macrophage lines.

The antigenic phenotypes of three long-term cultured murine resident macrophage lines selected in vitro from cell suspensions of different tissues--namely MAY 1 (from the peritoneal cavity), MASP (from the spleen) and MALU (from lung tissue)--were determined using a panel of monoclonal antibodies. The results indicate that all three cell lines belong to the mononuclear phagocyte system and express characteristics indicating a rather high differentiation state. However, there was a significant difference in antigen expression between the two macrophage lines obtained from solid tissues (MASP from spleen and MALU from lung), which were very similar in their antigenic pattern, and the MAY 1 line obtained from the peritoneal cavity, which seemed to be less well differentiated. The antigenic profile of the "mesothelial" cell population associated with the MASP line indicates that this cell population is difficult to characterize and to include in a particular lineage.

Animals↗

PubMed1988

Immunotherapy of cancer: experimental approach with activated macrophages proliferating in culture.

Murine macrophages isolated from the peritoneal cavity or from the lung were continuously grown and expanded in vitro on a confluent layer of "mesothelial or endothelial" feeding cells. These cell lines could be obtained from C57B16 or BalbC mice and were nontumorogenic in nude mice. The macrophages were characterized by their capacity to phagocytose yeasts and by the presence of nonspecific esterases, of Fc receptors, and of specific antigens (MAC1 ...). In vitro, these macrophages were fully activated and were tumoricidal against different tumor cell lines. In vivo, adoptive transfer of the expanded macrophages to mice bearing EMT6 sarcoma or 3LL metastasizing carcinoma inhibited the growth of the primary tumors and the development of metastases. Local injection in the vicinity of the primary tumor and i.v. transplantation were effective. The adoptive transfer of expanded macrophages could lead to a new kind of immunotherapy of neoplastic diseases combining selective amplification with effective activation of macrophages as key effector cells.

Animals↗