Activity In Serum Of Mice Research Articles

An improved sensitive and simple microassay of mouse complement

A simple and fast hemolytic microassay was developed for the determination of classical pathway complement activity in mouse serum. The assay is based on hemolysis of sheep red blood cells (SRBC) that are sensitized with polyclonal mouse antibodies. The degree of hemolysis was measured in the reaction supernatants by photometric reading in an ELISA plate scanner at 405 nm wavelength. It was found that some batches of unpurified mouse anti-SRBC antibodies gave insufficient hemolysis. Analysis of two antibody preparations indicated that this might be caused by anti-complementary factors in the ascites fluid, and by an excess of non-complement fixing IgG1 antibodies. For optimal and standardized results, removal of anticomplementary factors and enrichment for complement fixing IgG2 antibodies was required and was achieved using protein A purified anti-SRBC IgG. In our assay it is possible to determine CH50 titers in triplicate in 80 μl samples of individual mouse sera with high sensitivity. Using this rapid one-step method large numbers of tests could be performed in 1 day.

Enhancement of biotinidase activity in mouse serum by inhibitors of methylation.

DL-ethionine increases the activity of liver biotinidase, an enzyme which hydrolyzes biotinylesters and biotinylpeptides. Chronic DL-ethionine feeding increases transiently the activity of biotinidase in mouse and rat liver, after which it remains elevated in the serum. In the present work we show that both isomers of DL-ethionine are equally good enhancers of the liver biotinidase, while, 3-ethylthiopropionate, the toxic metabolite of DL-ethionine, has no effect on the biotinidase activity of either liver or serum. We have also employed two different combinations of inhibitors of the hydrolytic pathway of SAH, a transmethylation product and potent inhibitor of methylation. It was found that these inhibitors (EHNA and Ara-A, 2-deoxycoformycin and adenosine) increase the activity of serum biotinidase as was the case with ethionine. Because SAH does not ethylate biomolecules, these changes in biotinidase activity, which can not be prevented by adenine, biotin or lecithin are most probably related to the inhibition of methylation.

Parasitic modulation of host immune mechanisms in schistosomiasis.

Antigen antibody complexes are suspected to play a role in the pathogenesis of some of the lesions that result from schistosomiasis. To examine the effect of immune complexes on the immune system of mice experimentally infected with Schistosoma mansoni, we measured antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated serum hemolysis in normal and infected animals. ADCC activity in infected mice was depressed compared to control mice. However, preincubation of spleen cells for 24 h in medium followed by washing restored ADCC activity. This suggested that a soluble factor(s), presumably immune complexes, was bound to the Fc receptors with resultant block in ADCC activity and this was removed in vitro during the 24-hour preincubation. Furthermore, the complement activity of mouse serum was markedly depressed in mice infected for 3 or 6 weeks. Again, the presence of immune complexes could explain this depression since immune complexes bind complement. We attempted to confirm and extend these findings with an immunoperoxidase-staining technique using antibody to S. mansoni antigen. Most of the granuloma formations identified in portal tracts and intestinal mucosa were composed of macrophages and epithelial cells surrounding a central nidus of schistosome egg. In addition, schistosome antigen was seen diffusely bound to some of the lymphoid elements in the lamina propria and many of these cells appeared plasmacytoid. Furthermore, large amounts of schistosome antigen were sequestered in the medullary cords of the mesenteric lymph nodes and in the Billroth cords of the spleen. This suggests that the antigen is conveyed to the lymph nodes and the spleen through the systemic circulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Study of the optimal reaction conditions for assay of the mouse alternative complement pathway

The optimal reaction conditions for hemolytic assay of alternative complement pathway activity in mouse serum were investigated. A microtiter system was used, in which a number of 7.5×10 6 rabbit erythrocytes per test well appeared to be optimal. Rabbit erythrocytes were superior as target cells over erythtocytes from a number of other animal species. The optimal conditions were as follows: an incubation temperature of 39°C, an ionic strength of about 200 mM, and a magnesium concentration of 2.5 mM. Incubation during 60 min was not sufficient for an end-point titration. Addition of 1 mg of zymosan A per test well, however, enhanced and accelerated the hemolytic activity of mouse serum via the alternative pathway resulting in a maximum value after 45 min. This, most probably, proceeded by a mechanism involving the formation of a zymosan-C5-convertase and bystander lysis of the target cells. In contrast to the normal alternative pathway assay the zymosan-potentiated test did, most probably, not involve natural antibodies. Cobra venom factor was more efficient in enhancing the sensitivity of the assay for the mouse alternative complement pathway than zymosan. This makes this factor very useful for testing C-pour body fluids.

Antigen-specific helper activity in serum of mice primed with sheep red cells II. Partial purification of the factors involved

The nature of antigen-specific, T-cell dependent helper activity (SSHA), present in serum of mice 9 h after intraperitoneal immunization with 2 × 10 8 sheep erythrocytes, was studied in more detail. SSHA could be enriched from serum by adsorption to formalinized sheep erythrocytes, elution of the cells with a chaotropic substance, and subsequent dialysis. This resulted in an enrichment of about 900 × with a yield of 64%. Further fractionation of the material by ultrafiltration resulted in two subfractions with helper activity indicating SSHA to be composed of two factors with mol. wt's of 300,000 and about 60,000. The eventual purification for the two factors was about 1650 and 2000 × respectively, and the total yield 46%. The possible relationship between the factors and factors described by others after induction in vitro is discussed. The purification procedure appears useful for the enrichment of SRBC-specific helper factors from serum.

Anti-T-cell idiotype activity in serum of mice injected with syngeneic hapten-specific T-cell lines.

T cells able to mediate specific delayed type hypersensitivity (DH) in mice in response to the haptens azobenzenearsonate (ABA), oxazolone (OX), and picryl chloride have been grown in continuous cultures. They were tested for their ability to react with the hapten in vitro and to induce anti-idiotype antibody in syngeneic mice. Incubation in vitro with OX-glycine blocked the ability of an anti-OX cell line to produce DH in vivo but had no effect on an anti-ABA cell line. However, this was inhibited by ABA conjugated to bovine serum albumin (ABA-alb) but not by irrelevant haptens such as OX-glycine or trinitrophenyl-alb. Antisera prepared by immunizing syngeneic mice with the anti-OX line blocked the inhibitory effect of OX-glycine on that line but did not reverse the effect of ABA-alb on the anti-ABA line. Neither normal mouse serum nor inappropriate anti-line antisera could influence the effect of OX-glycine on the anti-OX line. When given alone to naive mice, a specific anti-line antiserum induced DH specific for the antigen to which the line was responsive. Thus, for example, antiserum directed to an anti-OX line produced OX-specific DH. The results suggest that anti-T-cell idiotype antibodies can be induced by immunizing mice with syngeneic antigen-specific T-cell lines and that these antibodies can block the specific in vitro interaction of antigen with the corresponding antigen-specific T cells and induce specific DH in vivo.

Estimation of classical pathway of mouse complement activity by use of sensitized rabbit erythrocytes

A simple photometric assay was devised for determining classical complement pathway activity in mouse serum using sensitized rabbit erythrocytes as target cells. These cells appeared more sensitive to lysis by mouse complement than sensitized mouse and sheep erythrocytes, most probably by their ability to escape the C3b inactivator system. Advantages of the assay over other techniques are the high sensitivity and the avoidance of the use of radioisotopes. With this test it is possible to get more insight in the complement system of an animal species that has been most widely in use in immunological research.

Determination of alternative pathway of complement activity in mouse serum using rabbit erythrocytes

Rabbit, mouse and sheep erythrocytes expressing different concentrations of membrane sialic acid were used to study possible modes of activation of the alternative complement (C) pathway in mouse, human and guinea pig serum. Mouse erythrocytes activated only human serum, whereas rabbit erythrocytes activated the sera of all three species. Based on the observation that rabbit erythrocytes activate the murine alternative C pathway a method for estimation of alternative C pathway activity (AP50 value) in mouse serum was devised analogous to that used for human AP50 determination. The method is not very sensitive to ageing or to batch variation of the indicator cells. The AP50 value of mouse serum measured by this method is of the same order as for human and guinea pig serum. Mouse serum AP50 activity is partly determined by natural antirabbit erythrocyte antibodies and is sensitive to heating (15′ at 48°C and 4′ at 56°C), and to the actions of cobra venom factor, zymosan and cysteine. Strain and sex differences with respect to AP50 activities of mouse sera were observed.

2'5' oligo(A) polymerase activity in serum of mice infected with EMC virus or treated with interferon.

Interferon-treated cells show an increase in two double-stranded RNA (dsRNA)-dependent enzymatic activities involving an oligoadenylate polymerase and a protein kinase (ref. 1 and refs therein). The polymerase converts ATP into a series of oligonucleotides characterized by 2'5'-phosphodiester bonds, designated 2'5'-oligo(A) or 2-5A (ref. 1). These oligonucleotides activate an endoribonuclease that degrades RNA in extracts of control and interferon-treated cells. These observations have been made in tissue culture cells and no informatin is yet available on these enzymatic activities in animals with elevated interferon levels. We report here on 2-5A synthesis in tissue homogenates and serum of mice infected with encephalomyocarditis virus (EMCV); this virus induces interferon synthesis when injected intraperitoneally into mice. Significant synthesis of 2-5A was detected in extracts of spleen and lungs, but also, surprisingly, in the serum of these mice. Subsequent experiments showed synthesis of 2-5A in serum of mice treated with the interferon inducer poly(I) x poly(C) (ref. 3) or with mouse fibroblast interferon.

Antigen-specific helper activity in serum of mice primed with sheep red cells: I. Definition of the test system and comparison with other systems

An adoptive transfer system is described to measure serum helper activity in the primary antibody response to sheep red blood cells (SRBC). Mice injected with a high dose of cyclophosphamide and reconstituted with rabbit anti-thymocyte serum-treated spleen cells were used as recipients. Serum obtained 9 hr after ip injection of normal mice with 2 × 10 8 SRBC (S(SRBC)) injected i.v. in the recipients caused a significant enhancement of the antibody response to 2 × 10 7 SRBC. The serum helper activity was not generated in thymectomized animals and could be absorbed from S(SRBC) by normal and formalinized SRBC. The SRBC-specific serum helper activity (SSHA) is heat labile (30 min 56 °C) and shows allogeneic restriction. Another test system described in literature for measuring T-cell help in vivo was less suited to measure SSHA in the response to 2 × 10 7 SRBC. A system using normal mice injected with 10 5 SRBC for determining specific immune response-enhancing factor (SIREF), demonstrated SIREF activity in S(SRBC). It did, however, not measure SSHA, as absorption of S(SRBC) with formalinized SRBC did not abolish the activity in that system.

A microassay for the determination of hemolytic complement activity in mouse serum

A 51Cr release microhemolytic complement assay is described to detect hemolytic complement activity in mouse serum. 51Cr-labeled sheep erythrocytes (SRBC) which have been sensitized with a subagglutinating amount of the 7S IgG fraction of rabbit anti-SRBC serum are placed in microtiter plates and further antibody added to each well prior to the addition of the complement (C) source. The IgG antibody was found to be more efficient in the lytic assay than the 19S IgM antibody. The assay is simple to perform, reproducible, and requires small volumes of mouse serum. Comparative hemolytic values were established for serum from a variety of murine strains using pooled BALB/c serum as the C reference. No apparent relationship was noted between the H-2 type of inbred mouse strains and the presence or absence of hemolytic complement.

Antigenic Modulation in Vitro.

Abstract The acquisition of resistance by TL antibody-sensitized mouse RADA1 leukemia cells and A/J thymocytes to cytolysis in the presence of fresh TL antiserum and guinea pig C (TL antigenic modulation) required a heat-labile mouse serum factor or factors. Most mouse strains had high levels of serum-modulating activity, but the B6 strain was extremely low, and the AKR/J strain moderately low, in activity. The modulating capacity of mouse serum was also destroyed by treatment with zymosan, an immune complex, and cobra venom factor, and was partially restored to heated serum by freeze-thawing. Human C component C3, and to a lesser extent C4, promoted modulation of cells presentitized with heated TL antiserum and cobra venom factor-treated normal serum. Similar sensitivity of modulating activity of human C3 and of mouse serum to temperatures below 37°C indicated that an analogous mouse C component, mouse C3, was required for modulation. Successful modulation by C2-deficient human serum and susceptibility of mouse serum activity to zymosan, immune complex, and cobra venom factor treatment indicated involvement of the alternative pathway of C activation. All modulating activity in mouse serum and in human C3 preparations could be absorbed onto and eluted from RADA1 cells and thymocytes. Immunofluorescence analysis indicated specific binding of mouse or human C3 to the cell surface only under conditions promoting modulation. Modulating activity of human C3, unlike that of mouse serum, was not destroyed by heating, and modulation was achieved with the IgG fraction of TL antiserum and heated human C3, suggesting a contribution of factor B-like, C3-cleaving activity by the cells, resulting in deposition of C3 on the cell surface. Intercalation of C3 into aggregated TL antigen-antibody complexes may be required to achieve modulation.

Quantification of mouse macrophage chemotaxis in vitro: role of C5 for the production of chemotactic activity.

Delineation of the mechanisms of macrophage accumulation at local tissue sites will further our understanding of immunologically mediated host resistance to infectious and neoplastic diseases. Since mice are frequently used for the study of immune function, we developed a method for the quantification of mouse macrophage chemotaxis in vitro. By this method it was found that the fifth component of complement is necessary for the production of chemotactic activity in mouse serum by inflammatory agents such as endotoxin or aggregated gamma globulin. The majority of macrophage chemotactic activity produced by these agents in mouse serum can be attributed to a low-molecular-weight (ca. 15,000) chemotactic factor. The data suggest that this factor is the biologically active cleavage product of the fifth component of complement, C5a.