Gene affecting superoxide dismutase activity linked to the histocompatibility complex in H-2 congenic mice.

The activity of cyanide-sensitive, Cu-Zn superoxide dismutase (SOD) was studied in liver sytosols from H-2 congenic strains of mice. Higher SOD activity was found in livers of mice having H-2b/A.BY, B10, and C3H.SW/haplotypes than in those of H-2a, H-2k and H-2d haplotypes. Segregation studies supported these correlations. In H-2 recombinant strains of mice, the genes influencing the liver SOD activity occur, as ascertained by mapping techniques, at or near the H-2d region of the major histocompatibility complex.

Differentiation of adenovirus hexon epitopes with monoclonal antibodies by gel diffusion assays.

Nine monoclonal antibodies (MAbs) directed against crystallized human adenovirus type 1 (Ad h 1) hexon were tested with purified homologous and heterologous hexon preparations by gel diffusion. Six MAbs formed a single line with the homologous hexon in a 2-well pattern, and 3 MAbs formed lines only in biclonal combinations with an appropriate MAb. All of the 6 precipitating MAbs formed a continuous line of complete identity when tested simultaneously against homologous and different heterologous hexons. With Ad h 1 hexon a line of double partial identity (double spur) was formed when some pair combinations of 2 MAbs were placed in 2 juxtaposed wells. Other MAbs in the adjacent wells formed a line of identity. The MAbs could be divided into 2 antibody groups (groups A and B) based on this phenomenon. Members of antibody groups A and B apparently identified 2 sterically distinct epitopes: one of them is presumably the genus-specific epitope of the hexons (group A) and the other(s) should be intertype-specific epitope(s). Thus, the gel diffusion method can be used for selecting pairs of MAbs for their specificity to sterically independent epitopes. Mixtures of 2 MAbs belonging to the different antibody groups formed double lines with Ad h 1 hexons. Members of group A showed some helper effect to the members of group B for their precipitin line formation.

Multiple copies of identical epitopes on the adenovirus hexon.

A double monoclonal antibody (MAb) sandwich enzyme-linked immunosorbent assay (double MAb ELISA), which uses the same MAb as solid-phase immunosorbent (capture MAb) and as detector MAb (peroxidase-labeled), was developed to quantify the specific epitopes of adenovirus hexon. Four MAbs directed against crystallized adenovirus type 1 (Ad h 1) hexon were tested by this assay with homologous and different heterologous hexons. The lowest reacting concn with the homologous and heterologous hexon types both in direct and double MAb ELISA was determined and compared. At least two copies of four different epitopes were identified by the MAbs. Evidence is presented that more than one copy of identical or closely related epitopes exist on the homologous as well as on the heterologous hexon molecules. However, their presence could be detected only in higher concn of hexon preparations of subgenera A, B and D.

Superoxide dismutase, catalase and lipid peroxidation in liver of young mice of different ages.

The lipid peroxidation and activities of main enzymes involved in the peroxide metabolism were measured at resting conditions and after completion of a 2-month treatment with H2O2. At the start of the experiments 1- and 3-month-old female BALB/c mice received either a high dose of H2O2 (0.5% H2O2 in drinking water; intake 12.5 x 10(-3) g/day/mouse) or a low dose of H2O2 (0.5 ml of 0.5% H2O2 by esophageal canula on every alternate day; intake 1.0 x 10(-3) g/day/mouse). The following conclusions can be drawn: a relative higher increase in activity of catalase could be induced at the age of 3 months than at the age of 5 months by a high dose of H2O2. The activity of superoxide dismutase (SOD) was not changed by a high dose of H2O2. A moderate increase in the activity of catalase and a remarkable decrease in SOD-activity resulted from a low-dose H2O2 treatment at the age of 5 months. The level of lipid peroxidation was increased by the low-dose and not influenced by the high-dose H2O2 treatment in 5-month-old-mice.

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.

A simple, quick one-step ELISA assay for the determination of complex plasma factor XIII (A2B2).

A new highly sensitive sandwich ELISA assay was developed for the determination of plasma factor XIII (FXIII). Plasma FXIII is a tetrameric complex of two types of subunits (A2B2). A biotinylated monoclonal capture-antibody against the B-subunit and a peroxidase-labelled monoclonal tag-antibody against the A-subunit were added to the plasma dilution and the amount of the complex attached to streptavidin-coated microplate was quantitated by measuring peroxidase activity. Only the tetrameric plasma FXIII reacted in the assay, non-complexed A or B subunits showed no reaction. The assay is linear up-to 40 microg/L of FXIII in the assay mixture. It is a quick one-step assay which can be performed within two hours. At normal and low FXIII concentration within batch reproducibility was 2.0% and 3.3%, day to day variation was 5.5% and 8.7%, respectively. Its high sensitivity allows reliable measurement at FXIII concentrations below 1% of normal average. Plasma samples can be stored for the assay at -20 degrees C for at least one month. Plasma levels of healthy individuals were normally distributed and no gender difference was observed. A reference interval of 14-28 mg/L (67-133%) was established.

Fibronectin in bronchoalveolar lavage fluid and plasma of dogs with acute inflammation of the lungs.

Bronchoalveolar inflammation, which was generated in dogs by Broncho-Vaxom instilled into the right lower lobe, was characterized first of all by an increased influx of macrophages. In this non-purulent acute-subacute inflammatory reaction, the lavage fibronectin decreased rapidly three hours after the incubation and then a marked gradual elevation was observed, which persisted throughout the whole two-week process, while plasma fibronectin concentrations were not altered significantly. Changes in the levels of lavage fibronectin may be an important sign for the control of the inflammatory reaction activity in the lungs.

Development of ELISA and enzyme-linked immunofiltration assay (ELIFA) methods for monitoring cyclodextrin glycosyltransferase (CGTase) production and bacterial growth in Bacillus macerans batch cultures.

Immunochemical methods were developed for monitoring cyclodextrin (CD) glycosyltransferase (CGTase) production and growth of an industrial CD-producing Bacillus macerans strain. Extracellular concentrations of CGTase released into a non-transparent culture medium during a 44 h long fermentation were detected by an indirect antigen inhibition enzyme-linked immunosorbent assay (ELISA). The ELISA was sensitive (minimal detection level 6 ng ml-1) and highly reproducible (coefficients of variation < or = 1.2 and 5.9%, within-runs and between-runs, respectively) compared to assays of CGTase activity (coefficients of variation < or = 4.2 and 7.0%, respectively). The ELISA, in combination with enzyme activity measurements, was useful to detect the decrease in the specific CGTase activities after 36 h of incubation, which was clearly indicative of the proteolytic degradation of CGTase. B. macerans cell numbers were estimated using an enzyme-linked immunofilter assay (ELIFA). The assay took less than 1 h and the coefficients of variation within and between-runs (2.9-6.4%) were considerably less than for viable counting (10.6-15.4%). In the exponential phase of growth, ELIFA results correlated more closely with the cell counting based on total protein than with viable counts. Nevertheless, in the phase of cell lysis, the bacterial cell number was systematically underestimated by ELIFA in comparison to both viable cell number and total protein determinations. Thus cell antigens detected with immunological procedures might be lost during the transition from vegetative cells to spores. On the other hand, the ELIFA procedure was specific for B. macerans cells and was a better indicator of the onset of the different growth phases than the cell numbers calculated from the protein assay.

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.

A sensitive method for detection of polyclonal and monoclonal antibodies against the adenovirus hexon.

Chromic chloride and tannic acid methods were elaborated to bind purified adenovirus hexons to sheep red blood cells (SRBC). Either method gave very sensitive haemagglutination (HA) with adenovirus antisera, but treatment with tannic acid was more sensitive. Using this method, specific antibodies present in polyclonal immune sera against adenoviruses and adenovirus hexons, and the reactivity of monoclonal antibodies directed against adenovirus hexon type 1 were tested to 11 adenovirus types. With certain types, the high haemagglutination titres exceeded those obtained with ELISA, while with other types they remained slightly below the ELISA titres. The elaborated passive HA method with tannic acid is easily accomplished and may serve as an effective and useful tool in the study of both polyclonal and monoclonal antihexon antibodies.

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.

Immunoenhancement and suppression induced by adenovirus in chicken.

Chickens injected intravenously (i.v.) with human adenovirus type 6 (Ad6) reveal a 2-17-fold increase in the number of plaque-forming cells producing antibody (Ab) against sheep red blood cells (SRBC) 2-6 days after virus infection. Further, polyclonal B-cell activation has been demonstrated by the quantitation of immunoglobulin-producing cells (IgPC) and cells producing immunoglobulin (Ig) of IgM isotype (IgPC mu) in the spleen of chicken inoculated with Ad6. Ad6 infection in chicken results in immunosuppression against SRBC when this unrelated antigen is given after virus infection. It seems that coincidence occurs between the B-cell mitogenic activation and the immunosuppression caused by Ad6, as the most pronounced change in both activities appears on the fourth day following virus infection. These findings suggest that the B-cell mitogenicity of the virus contributes to the impairment of the humoral immune response to SRBC.

Superoxide anion, superoxide dismutase and lipid peroxidation in the murine macrophage cell line C4M phi.

A remarkable increase in the production of superoxide radicals and SOD activity was measured in suspension of the murine macrophage cell line C4M phi treated with Lentinan (4-10 X 10(3) micrograms/5 X 10(6) cells). In activated macrophages the decrease of lipid peroxidation could be interpreted as a consequence of enhanced SOD activity.

Genetic control of two different types of antibody responses to oxazolone.

The genetic control of antibody responses induced by contact sensitization with oxazolone and immunization with an oxazolone-mouse serum albumin conjugate was studied in inbred, H-2 congenic and nude mice. Both types of antibody responses are T cell-dependent and regulated by immune response genes mapping at the I-B subregion of the H-2 complex. The relative magnitude of antibody responses induced by the two methods of immunization is governed by a gene or genes mapping at the I-B subregion of the H-2 complex. Presentation of antigen by these two methods gives H-2-dependent distributions of high and low responsiveness which are exactly opposite. Non-H-2 genes affect the overall level of antibody response but not the relative magnitude of the responses.

Genetic control of contact sensitivity to oxazolone in inbred, H-2 congenic and intra-H-2 recombinant strains of mice.

Delayed-type hypersensitivity (DTH) to 2-phenyl-4-ethoxymethylene-5-oxazolone (oxazolone) was found to be under multigenic control in inbred, H-2 congenic and intra-H-2 recombinant strains of mice. A high response was associated with haplotypes H-2d,a,k and low response with haplotype H-2b. DTH to oxazolone was high or intermediate in different F1 hybrids of high and low responder mice. In F2 and backcross generations a higher response was associated with the "dd", than with "bb" phenotype, while intermediate response was found in the heterozygote "db" mice. A study of H-2 recombinant strains suggests that a gene controlling the DTH response maps in the I-B subregion of the H-2 complex. The response was significantly modified by gene(s) which are not linked to the H-2 complex and have not been mapped. Since congenitally athymic nude (nu/nu) mice did not respond to oxazolone, this contact sensitivity is belived to be a T cell-dependent immune response.