Strain differences in the cytotoxic activity and TNF production of murine macrophages stimulated by lentinan.

The effect of lentinan, a glucan type immunomodulatory polysaccharide was studied on the antitumor cytotoxicity and on the TNF secretion of peritoneal macrophages in inbred H-2 congeneic mouse strains under in vivo and in vitro conditions. The cytotoxic activity and TNF secretion of murine macrophages was found to be elevated by lentinan in vitro and in vivo conditions. The effectiveness of lentinan to induce cytotoxicity and TNF secretion was highly influenced by the genotype of the host. The increased cytotoxicity of macrophages was modified by the H-2 and the background genes. The black background and the H-2a and H-2d haplotypes were associated with high responsiveness, while the albino and agouti background and the H-2b and the H-2k haplotypes with low responsiveness to lentinan treatment. The degree of TNF secretion of macrophages stimulated by lentinan was influenced by the H-2 genes only. Similarly, to the macrophage cytotoxicity the TNF secretion in the H-2a and H-2d haplotypes were found to be high, on the other hand, in the H-2b and H-2k were low.

A simple, rapid and sensitive fluorimetric assay for the measurement of cell-mediated cytotoxicity.

A fluorimetric method using 4-methylumbelliferyl heptanoate (MUH) has been developed for detecting cell-mediated cytotoxicity and cell proliferation. The assay is based on the hydrolysis of the fluorochrome (MUH) by intracellular esterases of viable cells resulting in the production of highly fluorescent 4-methylumbelliferone that can be measured in a microplate fluorimeter. Because of a similarity to the principle of the widely used colorimetric MTT assay, a comparison was made between the two assays when measuring cell proliferation and LAK cell cytotoxicity to different target cell types. The results have shown that the MUH assay represents a method for evaluating both cell-mediated cytotoxicity and cell proliferation which is completely comparable to the MTT method. The rapidity of the new cytotoxicity assay, 5 h in contrast to 9 h for the MTT assay, its applicability to both adherently and nonadherently growing target cells and its high accuracy due to the avoidance of centrifugation steps make this method a serious contender for replacing conventional radioactive techniques.

Characterization of monoclonal antibodies to adenovirus type 35 hexon.

Twenty three monoclonal antibody-rich ascitic fluids (MIAFs) to human adenovirus (AV) type 35 hexon were studied by indirect ELISA using various tracer systems, passive haemagglutination (HA) as well as gel diffusion techniques. Eleven different human heterologous hexon types in addition to the homologous one, and two animal adenovirus (AV) hexons were used to determine the reactivity patterns (RPs) of the monoclonal antibodies (MoAbs). Based on the cross-reactivity with the different hexon types, the MoAbs exhibited genus, subgenus and type specificities; furthermore, a variety of intersubgenus and intertype specificities could be found. Fifteen of the MoAbs reacted in ELISA, but not in passive HA, suggesting that certain epitopes on the hexons bound to red blood cells were not available for the MoAbs in question. Four MoAbs were able to form a precipitin line with the hexon antigen in gel diffusion. Two of the four (MoAbs 35H10 and 35H51) formed with the homologous AV35 hexon a single confluent precipitin line only. In spite of the origin of these MoAbs from different hybrid cells (clones) their specificity was probably identical when recognizing the type-specific epitope of the AV35 hexon. The other two MoAbs (35H15 and 35H26) with a broad RPs were able to precipitate not only the homologous but also different heterologous hexon types.

The role of genetic factors in the immunomodulating effect of thymosin.

The possible role of hypothetical genetic factors involved in the immunomodulating effect of thymosin fraction 7 (T7) was investigated. The model system was the in vitro immunization of murine spleen cell cultures with sheep red blood cells (SRBC), and the generation of antigen specific B cells in T7 treated cultures was compared to that of control values. It was found that T7 treatment enhanced the plaque forming cell (PFC) response of BALB/c spleen cells, while it proved to be suppressive in CBA cultures. Moreover, the T7 treatment of athymic BALB/c nude spleen cells resulted in a marked PFC response to SRBC, while a similar treatment of CBA nude cultures was ineffective in the same assay. The role of possible genetic factors was further confirmed using H-2 congenic and recombinant mouse strains on the C3H and B10 background. T7 elevated the PFC values in all B10 strains tested, and was suppressive in the case of C3H strains. It seems that the outcome of T7 treatment of murine target cells is determined by the genetic background and is independent of the H-2 haplotype.