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B Hevin

Publications and source records attributed to B Hevin.

11 recordsLinked to original sources

Quantitation of messenger RNA by competitive RT-PCR: a simplified read out assay.

A competitive RT-PCR method that permits reliable quantification of minute amounts of reverse-transcribed mouse lymph node mRNA is described. Using this technique, an absolute number of cDNA copies ranging from 10(3) to 10(5) can be determined, with a precision superior to 25%. The standard templates described in the present study permit the quantitation of beta-actin, IFN gamma, IL2, IL3, IL4, IL10, IL12 (p40 subunit), TGF beta 1, inducible nitric oxide synthase, ELAM-1, VCAM-1, and ICAM-1 mouse mRNA. The expression of a particular transcript is normalized to an arbitrary number of actin transcripts. The standard templates and wild-type cDNA have nearly identical sequences, but they can be distinguished by unique restriction sites. Known amounts of these standard templates, are co-amplified with serial dilutions of the cDNA derived from the mRNA of interest. Oligonucleotide primer pairs possessing 3' octamers found infrequently in the mouse genome (< or = 0.26 x 10(-6)) are used to amplify sequences, chosen to contain no GC stretches longer than 8 (PCRare software) (Griffais et al., 1991). Samples of each PCR product are digested separately with restriction endonucleases unique either for the wild-type or the standard amplicon. The quantitation of the test product and the standard product is easily carried out following their electrophoresis in an ethidium bromide-stained agarose gel.

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The biology of macrophages.

The main properties of the mononuclear phagocytic system (MPS) are summarized, focusing on their relevance within the framework of the steady-state and the inducible functions of the mammalian immune system, more specifically the immune system of the laboratory mouse, a reference vertebrate which remains the best studied. A peculiar attention is given to the rationale underlying the generation of so-called specific tools and reagents whose use is promoted to characterize this lineage, whatever the level under study, i.e. tissular, cellular, or subcellular levels. As one lineage among other lineages of the hemopoietic system, the MPS is characterizable by constitutive and inducible phenotypic and functional markers whose combination is unique for a given tissular micro-environment. Considering our present understanding of the innate and adaptive immune system functions, some of the properties of the MPS are discussed in relation with properties of another recently recognized hemopoietic lineage, namely the dendritic leukocyte system.

Animals↗

Maintenance of granuloma macrophages in serum-free medium.

Granulomas were induced by injecting polyacrylamide beads into subcutaneous pouches created by divulsion of the dorsal skin of mice. More than 10(7) phagocytic cells (60% macrophages, 40% polymorphonuclear cells) could be recovered from this granuloma. The separation of the phagocytic cells can be achieved either following sedimentation in Percoll or following the incubation of cells on plastic petri dishes. Phagocytosis of zymosan was observed in macrophages maintained in vitro for 1 month in Eagle's serum-free medium.

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Immunostimulatory mouse granuloma protein.

Earlier studies have shown that from subcutaneous talc-induced granuloma in mice, a fraction could be extracted that fully protected mice against Listeria monocytogenes. Using standard biochemical procedures--i.e., ammonium sulfate fractionation, preparative electrophoresis, gel filtration chromatography, isoelectric focusing, and preparative polyacrylamide gel electrophoresis--we have now purified an active factor to homogeneity. A single band was obtained in NaDodSO4/polyacrylamide gel with an apparent Mr of 55,000. It migrated with alpha 1-globulins and the isoelectric point was 5 +/- 0.1. The biological activity was destroyed with Pronase but not with trypsin and a monospecific polyclonal rabbit antiserum was obtained. The intravenous injection of 5 micrograms of this "mouse granuloma protein" fully protects mice against a lethal inoculum of L. monocytogenes. Moreover, after their incubation with 10 nM mouse granuloma protein, mouse peritoneal cells became cytostatic against Lewis carcinoma cells.

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[Increased resistance in mice to Listeria monocytogenes after treatment with a fraction from an inflammatory granuloma].

The multiplication of Listeria monocytogenes in the spleen was decreased between the 3rd and the 11th day following the inflammatory reaction induced in Mice by subcutaneous implantation of talc embedded in a calcium phosphate gel into the dorsal area. A similar activity was observed after injection of SO4 (NH4)2 fractionated extracts from granuloma. The major activities were found in the 33% saturation precipitate and in the 80% saturation supernatant.

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Antiinflammatory effects of murine malignant cells.

Development of teratocarcinoma does not impair immunization of mice against Listeria monocytogenes. Endotoxin injection a short time before tumor cell inoculation allows the growth of teratocarcinoma in non syngenic mice despite immune stimulation. In contrast with this absence of impaired systematic immunity, teratocarcinoma cells were found to repulse macrophages in vitro. This effect on macrophages was also found with three other malignant cells and with trophoblast cells. In vivo, teratocarcinoma cells were found to impair local inflammation. These cells and other malignant cells are able to produce a compound(s) of molecular weight between 10(3) and 10(4), which prevents inflammatory reaction. These results suggest that mouse teratocarcinomas and other tumors by-pass the host immunological system of surveillance by at least two mechanisms: a direct toxic effect on macrophages and the release of an inhibitor of inflammation. The possible relations between these properties of malignant cells and physiological functions of trophoblast are discussed.

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Immunostimulation with bacterial phospholipid extracts.

Injection of bacterial phospholipid extracts (EBP) into mice increased their resistance towards a Listeria monocytogenes infection. The blood clearance of virulent Salmonella typhimurium was enhanced and the degree of clearance correlated with the dose of extract injected. The multiplication of Listeria monocytogenes in spleen and liver of mice was inhibited and this inhibition was also correlated with the amount of extract injected. The absence of apparent toxicity in mice, of splenoand hepatomegaly, and of lymphoid hyperplasia, distinguish this immunostimulant from other known bacterial stimulants of host resistance to infection.

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