Evaluation of immunologic assays to determine the effects of differential housing on immune reactivity.

The mechanism is being investigated to determine specifically how an environmental variation such as differential housing can influence the multiple components of the host defense mechanism. Male C3H/HeJ mice were housed either one or five per cage. Cells and sera from these mice were analyzed and compared by several immunologic techniques to determine in which cells or tissues the effect of differential housing was most pronounced. The individually housed mice (a) had a greater capacity to phagocytose dead cells of Candida albicans. (b) had spleens that produced more macrophage colony stimulating factor (M-CSF). (c) were more responsive to M-CSF, (d) had peritoneal macrophages that released greater quantities of interleukin-1 in vitro into the surrounding medium and that had a greater capacity to migrate toward a chemotactic stimulus, and (e) had higher titers of IgM hemagglutination antibody to sheep erythrocytes. Differential housing of mice may therefore be a highly important modulator and indicator of the nature and extent of an animal's immunologic response to an environmental stimulus.

Effect of differential housing and time on immune reactivity to sheep erythrocytes and Candida.

Male C3H/HeJ mice were housed 5 or 1 per cage for varying periods of time. Approximately 10-14 days after the animals were placed under the differential housing conditions, the reactivity of lymphocytes to concanavalin A and to intraperitoneal injection of sheep erythrocytes became greater in animals housed 1 per cage in comparison with those housed five per cage. By 3 weeks the immune reactivity was similar regardless of the number of animals housed per cage. Similarly, resistance to infection with Candida albicans was greater in animals housed one per cage at approximately 10-14 days after the animals had been isolated. By 3 weeks a difference in resistance to C. albicans was not apparent between animals housed 5 or 1 per cage. Thus, housing conditions can transiently alter immunologic reactivity, including susceptibility to an infectious agent in male C3H/HeJ mice. However, female C3H/HeJ mice and male C57BL/6J mice did not show an effect of housing conditions on immune reactions.

The effect of dietary zinc and prothymosin alpha on cellular immune responses of RF/J mice.

Mice of the RF/J strain on a normal diet are defective in some aspects of cellular immunity, as evidenced by their susceptibility to infection with Candida albicans, their failure to release detectable quantities of circulating migration inhibitory factor (MIF) in vivo, and the presence of a low rate of phagocytosis and killing by peritoneal macrophages. When the mice were fed a high-zinc diet (300 ppm) for 4 weeks and then treated daily with 160 ng prothymosin alpha, an increase occurred in resistance to infection with C. albicans, in the capacity to release MIF in vivo into the circulation and in the capacity of peritoneal macrophages to engulf (phagocytose) and kill cells of C. krusei. In addition, the number of spleen lymphocytes producing antibody to a T-dependent antigen was significantly increased in the mice fed a high-zinc diet and inoculated daily with prothymosin alpha.

Human prothymosin alpha: amino acid sequence and immunologic properties.

Prothymosin alpha has been purified from human thymus and its amino acid sequence determined, except for a 15 amino acid segment including 10 glutamyl residues near the middle of the molecule. Like prothymosin alpha from rat thymus [A. A. Haritos, R. Blacher, S. Stein, J. Caldarella, and B. L. Horecker (1985) Proc. Natl. Acad. Sci. USA 82, 343-346], human prothymosin contains the thymosin alpha 1 sequence at its NH2-terminus. It contains a total of 109-110 residues compared to 111-112 for rat prothymosin alpha, with deletions corresponding to positions Gln39 and Lys108 of the rat polypeptide. Human prothymosin alpha also differs from rat prothymosin alpha at positions corresponding to residues 87, 92, and 102 of the latter, with substitutions of alanine for proline, alanine for valine, and aspartic acid for glutamic acid, respectively. Human prothymosin is significantly less active than rat prothymosin in protecting mice against infection with Candida albicans and in stimulating release in vivo of migration inhibitory factor. Thus, the differences in amino acid sequences, present mainly the COOH-terminal half of the polypeptides, may determine species specificity in biological properties.

Parathymosin alpha: a peptide from rat tissues with structural homology to prothymosin alpha.

A peptide, parathymosin alpha, containing approximately equal to 105 amino acid residues, has been isolated from rat thymus, and the sequence of the first 30 residues at the NH2 terminus has been determined. In this region, it shows 43% structural identity with thymosin alpha 1 and prothymosin alpha. The common sequences do not include residues 2-9, which accounts for the poor reactivity of parathymosin alpha with an antibody directed against this epitope in thymosin alpha 1. Parathymosin alpha appears to modulate the action of prothymosin alpha in protecting sensitive strains of mice against opportunistic infection with Candida albicans.

Resistance and susceptibility to infection in inbred murine strains. IV. Effects of dietary zinc.

Mice of several inbred strains have been fed diets containing either large amounts of zinc (300 ppm Zn), small amounts of zinc (5 ppm Zn), or routine laboratory mouse chow. When the mice are fed on a high-zinc diet, murine strains, such as C3H/HeJ, AKR/J, and CBA/CaJ, which are normally susceptible to infection with Candida albicans and which normally release low titers of migration-inhibition factor (MIF) in vivo into the circulation, become more resistant to infection with C. albicans and release higher titers of MIF in vivo into the circulation. In addition, their capacity to elicit delayed type hypersensitivity responses may be enhanced. When the mice are maintained on a low-zinc diet, murine strains, such as C57Bl/10SNJ, which are normally resistant to infection with C. albicans and which normally release high titers of MIF in vivo into the circulation on appropriate antigenic challenge, become more susceptible to infection and release lower titers of MIF into the circulation. Under these conditions of low-zinc concentrations in the diet, their capacity to elicit delayed type hypersensitivity may be reduced. Thus, the concentration of zinc in the diet may have a pronounced effect on some in vivo parameters of cell-mediated immunity.

Resistance and susceptibility to infection in inbred murine strains. III. Effect of thymosin on cellular immune responses of alloxan diabetic mice.

Three parameters of cell-mediated immunity, namely, (a) resistance to infection with Candida albicans, (b) in vivo release of migration inhibitory factor (MIF) into the circulation and (c) delayed hypersensitivity were markedly reduced when mice of such normally resistant high responder strains as C57B1/10SNJ and C57B1/KsJ became hyperglycaemic after treatment with alloxan. When the alloxan diabetic mice were inoculated daily intraperitoneally with thymosin fraction 5, beginning 3 days before infection, resistance to infection was greatly enhanced. When the mice were administered 5 micrograms thymosin fraction 5 for 3 days before sensitization and for 3 days before challenge, the amount of MIF released in vivo into the circulation after the antigenic challenge was much greater. When the mice were treated daily with 5 micrograms thymosin fraction 5, beginning on the day of sensitization, the capacity to develop delayed footpad reactions was increased. Thus, the treatment of alloxan diabetic mice with thymosin fraction 5 enhanced the three parameters of cell-mediated immunity that were under investigation.

Thymosin alpha 11: a peptide related to thymosin alpha 1 isolated from calf thymosin fraction 5.

Two peptides related to thymosin alpha 1 have been isolated from preparations of calf thymosin fraction 5. One, lacking four amino acid residues at the COOH terminus, is designated des-(25-28)-thymosin alpha 1. The other, named thymosin alpha 11, contains seven additional amino acid residues at the COOH terminus. The sequence of this peptide is: AcSer-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu- Lys-Glu-Lys- Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-Gly-Arg-Glu-Ala-Pro-Ala-AsnOH. Thymosin alpha 11, in doses of less than 300 ng per mouse, protects susceptible inbred murine strains against opportunistic infections with Candida albicans. It is approximately equal to 30 times as potent as thymosin fraction 5 and approximately equal in potency to thymosin alpha 1.

Resistance and susceptibility to infection in inbred murine strains. II. Variations in the effect of treatment with thymosin fraction 5 on the release of lymphokines in vivo.

Of nine inbred murine strains sensitized intravenously with killed lyophilized Candida albicans and challenged 3 weeks later with a C. albicans filtrate, four strains were low responders and five were high responders in the in vivo release of migration inhibitory factor (MIF) and gamma interferon (IFN-gamma). An identical distribution of high- and low-responder strains occurred in response to sensitization with Mycobacterium bovis BCG and subsequent challenge with old tuberculin. Treatment of the murine strains with thymosin fraction 5 prior to sensitization and challenge had different effects: (a) the high-responder strains had a decrease in their release in vivo of the two lymphokines; (b) three of five of the low-responder strains had a striking increase in the in vivo release of MIF and IFN-gamma; and (c) one low-responder strain did not have its response altered. A parallelism existed between the capacity of a murine strain to release the two lymphokines in vivo on stimulation with C. albicans antigens and the capacity of that strain to resist intravenous infection with living C. albicans.

Resistance and susceptibility to infection in inbred murine strains. I. Variations in the response to thymic hormones in mice infected with Candida albicans.

Nine inbred murine strains were either highly resistant or highly susceptible to intravenous challenge with 4 X 10(4) to 1 X 10(5) cells of Candida albicans. The resistant strains had the capacity to develop delayed footpad reactions on appropriate sensitization and challenge; the susceptible strains did not have this innate capacity. Administration of thymosin fraction 5 beginning on the day of infection greatly increased the resistance of the susceptible strains to infection, but decreased the resistance of the resistant strains. In contrast, thymosin fraction 5 enhanced the delayed footpad responses of resistant-sensitized mice to specific antigen, but did not have a detectable effect on the delayed footpad reactions of the susceptible strains. Reinfection of the two types of strains had different effects, in that, depending on the strain, resistance could be increased, decreased, or not influenced at all.

Mechanisms in the in vivo release of lymphokines: relationship of high and low responsiveness to other parameters of the immune response.

Variations exist between different strains of inbred mice in the release of lymphokines into the circulation. A number of manifestations of cell-mediated immunity in mice sensitized intravenously with Mycobacterium bovis BCG were analyzed to determine their association with the in vivo release of gamma interferon (IFN-gamma) and migration inhibitory factor. Differences occurred among the strains in the proliferative responses of splenic cells to specific antigen and in the release of IFN after the challenge of BCG-sensitized mice with lipopolysaccharide. However, the capacity of an individual strain to release migration inhibitory factor and IFN-gamma into the circulation did not parallel the extent either of the proliferative responses or of the release of IFN induced by lipopolysaccharide. Not all of the strains developed marked delayed footpad reactions to challenge with PPD regardless of the extent of their responses by other parameters. Delayed footpad reactions did develop in mice sensitized via the subcutaneous route, although this sensitization did not result in the capacity to release migration inhibitory factor and IFN-gamma into the circulation of individual inbred strains.

Adjuvants in the induction of suppressor cells.

The effect of different mycobacterial adjuvants on the parameters of delayed hypersensitivity was investigated in strain 13 guinea pigs. The composition of the tubercle bacilli and the type of vehicle in which the antigen was presented determined the presence and extent of suppressor cell activity. When antigen was introduced in complete Freund adjuvant, both adherent and nonadherent cells had suppressive properties, with the suppressive effect demonstrable in vitro from 1 to 5 weeks after sensitization. Suppressor activity was indicated in vivo by a reduction of delayed footpad hypersensitivity in animals presensitized with complete Freund adjuvant.

Type I and II interferons and migration inhibitory factor: production in Mycobacterium bovis BCG-infected mice desensitized with old tuberculin or lipopolysaccharide.

Mice with delayed hypersensitivity induced by infection with Mycobacterium bovis strain BCG were desensitized by a single large dose of specific antigen (old tuberculin, OT) or a nonspecific interferon stimulus (bacterial lipopolysaccharide, LPS). Subsequent challenge of the desensitized animals revealed only a homologous hyporeactivity, that is, mice desensitized with OT showed decreased type II and migration inhibitory factor (MIF) responses to the specific antigen, which were unaffected by desensitization with LPS. Conversely, mice desensitized with LPS showed a decreased type I interferon and MIF response to LPS, which was unaffected by desensitization with OT. These results suggest that type I interferon and its accompanying low-titered MIF activity are produced by cell populations different from those that produce type II interferon and its accompanying high-titered MIF activity.

Cellular source of interferons in the circulation of mice with delayed hypersensitivity.

The cellular origins of type I and type II interferons released into the circulation of mice with delayed hypersensitivity were investigated. We determined the effect of treatment with various immunosuppressive agents, including cyclophosphamide, cycloheximide, antithymocyte serum, and whole-body X-irradiation, on the release of interferons after intravenous injection of specific (old tuberculin) or nonspecific (lipopolysaccharide) stimuli. The results suggest that (i) a heterogeneous population of lymphocytes (T and B cells) produces type II interferon, (ii) type I interferon is produced by a different cell population, and (iii) type II interferon is produced de novo after challenge with old tuberculin of mice sensitized with Mycobacterium bovis BCG.