Toxoplasma-infected Mice Research Articles

Macrophage-mediated suppression of immune responses in Toxoplasma-infected mice : II. Both H-2-linked and -nonlinked control of induction of suppressor macrophages

The suppressive effect of Toxoplasma infection on initiation of memory cells to dinitrophenylated keyhole limpet hemocyanin (DNP-KLH) was drastically different among inbred strains of mice. C57BL/6 (B6), C57BL/10 (B10), and SJL mice showed markedly suppressed secondary anti-DNP responses when infected. In contrast, the suppression did not occur in BALB/c mice. The infected DBA/2 and C3H/He mice produced moderately suppressed responses. In B6 mice, an injection with 1 × 10 2 organisms of T. gondii induced a suppressed elicitation of the memory cells to DNP-KLH. However, in BALB/c mice, the responses were not affected even by inoculation with 1 × 10 4 organisms. The difference in the suppressive effect of infection between B6 and BALB/c mice was also observed in the primary anti-DNP antibody responses to DNP-KLH. Both H-2-linked and -nonlinked genes appeared to be responsible for the regulation of the immunosuppression, since the suppressive effect of infection in B10.D2 mice, which have the B10 background and the same H-2 haplotype as BALB/c, was weaker than that of B10 mice, but stronger than in BALB/c mice. In vitro studies using a primary antisheep erythrocytes (SRBC) antibody response system demonstrated that the activation of plastic-adherent suppressor cells by Toxoplasma infection, in which suppressor macrophages have been proved to be the responsible cells for the suppressive activity, was controlled by both H-2-linked and -nonlinked genes.

Antitumor effect of intralesional injection with formalin-fixed toxoplasma gondii organisms on lewis lung carcinoma in toxoplasma-infected mice

The antitumor effect of formalin-fixed Toxoplasma organisms (f-Tp) as an immunostimulant was examined in Toxoplasma-infected female C57BL/6 mice using a syngeneic Lewis lung carcinoma (3LL). Toxoplasma-infected mice, intradermally inoculated with the tumor cells mixed with 10(5), 10(6) or 10(7) f-Tp, developed a marked antitumor effect, inhibition of tumor growth and prolongation of life-span, in direct relation to the strength of the delayed-type hypersensitivity (DTH) reaction induced by f-Tp. The antitumor effect could also be observed even if an intralesional injection with f-Tp was performed 1, 3 or 5 days after the tumor inoculation. In a control, the injection with 2.5 X 10(6) live Mycobacterium bovis (BCG) in BCG-sensitized mice induced a significant antitumor effect, only when the BCG was injected in the mixture of tumor cells. These results demonstrate that the injection with f-Tp can induce a potent antitumor activity in mice with Toxoplasma infection.

Influence of Toxoplasma on manifestations of Moloney virus infections

Considerable evidence documents the importance of co-factors, including the immune response, in expression of oncogenicity of tumour viruses. To determine whether a common protozoal infection that can depress lymphocyte function alters manifestations of oncogenic virus infection, a mouse model of Toxoplasma infection with depressed T lymphocyte function was developed. In this model, Toxoplasma depressed blastogenic transformation to the T-cell mitogen Concanavalin A and primary antibody response to sheep red blood cells which requires T cell help. Uninfected and Toxoplasma-infected mice were then infected with Moloney leukaemia or Moloney sarcoma viruses and development of lymphoma and sarcoma were evaluated. Toxoplasma infection, which induced depression of T-cell function, decreased the incidence of Moloney sarcoma virus induced rhabdomyosarcomas but did not alter progression or regression of tumour in those mice that developed tumour. Conjoint infection with Toxplasma and Moloney leukaemia virus did not increase incidence of lymphoma when compared with incidence of lymphoma in mice infected with Moloney leukaemia virus alone.

Macrophage-mediated suppression of immune responses in Toxoplasma-infected mice : I. Inhibition of proliferation of lymphocytes in primary antibody responses

The suppressor cells induced by Toxoplasma infection were shown to be macrophages, since they adhered to plastic, and their suppressive activity in anti-sheep erythrocytes (SRBC) antibody responses was abrogated by treatment with silica or carrageenan, which are selectively cytotoxic for macrophages. The suppressor macrophages strongly inhibited the uptake of tritiated thymidine ([ 3H]TdR) by normal mouse spleen cells in the responses to SRBC and Toxoplasma antigens. Supernatant fluids from the suppressor macrophages could not passively transfer the suppressive effect on anti-SRBC antibody responses. Furthermore, when the suppressor macrophages were isolated by a cell-impermeable membrane from normal mouse spleen cells, the antibody responses of normal spleen cells were not suppressed. These results indicate that suppression of antibody responses in Toxoplasma-infected mice is caused by an inhibitory effect of the suppressor macrophages upon proliferation of lymphocytes via direct contact with responder target cells. The suppressive effect of the macrophages was not counteracted by indomethacin, a potent inhibitor of prostaglandin synthesis, or catalase, a catabolic enzyme for hydrogen peroxide (H 2O 2).

Regulation of natural killer cell activity by anti-I-region monoclonal antibodies

The effects of a monoclonal antibody directed against immune response gene products on mouse NK activity were examined. In vivo administration of an anti-I-A k antibody to C3H/He (H-2 k) mice modulated their peritoneal cell (PC) and spleen cell (SC) natural killer (NK) activity against YAC-1 lymphoma target cells in vitro. No such effect was observed when BALB/c (H-2 d) mice were treated with this antibody. Administration of anti-I-A k antibody to mice before and after infection with Toxoplasma or treatment with poly(I:C) leads to suppression of NK activity in comparison to NK activity of mice infected with Toxoplasma or injected with poly(I:C) alone. A similar treatment regimen with M5/114 antibody which reacts with I-A b, I-A d, I-E d, and I-E k molecules resulted in decreased NK activity in B10.D2 (H-2 d) but not in B10.BR (H-2 k) mice. Serum and cell culture supernatant interferon (IFN) concentrations were not altered as a result of anti-I-A k treatment. Removal of adherent cells did not restore NK activity of anti-I-A k-treated Toxoplasma-infected mice to levels obtained with mice infected with Toxoplasma. In contrast, depletion of Ly 2.1 + cells from nylon-wool nonadherent SC of mice treated with anti-I-A k antibody, before and after infection with Toxoplasma, resulted in restoration of NK activity to the same level as that observed in Toxoptasma-infected mice.

Congenital toxoplasmic retinochoroiditis in a mouse model.

A study of the eyes of adult mice infected in utero with Toxoplasma gondii is reported. The histopathological features of the ocular inflammatory response in the infected mice ranged from minimal damage to complete destruction of the retinal tissue. Notable features such as retinal vasculitis and an almost uniform and highly selective destruction of the photoreceptor layer of the retina suggest a similarity between experimental autoimmune retinitis and the disease process in the retinas of our Toxoplasma-infected mice. We suggest that our mouse model could provide a simple and inexpensive tool for the investigation of immuno-pathological processes in the retina resulting from congenital Toxoplasma infection. The model has the advantage of low post-natal mortality coupled with high ocular morbidity. Furthermore, its aetiology is probably analogous to that of human ocular toxoplasmosis, in that the foetus becomes infected in utero via a mother whose primary infection is acquired during gestation.

The effect of congenital and adult-acquired Toxoplasma infections on activity and responsiveness to novel stimulation in mice.

Activity and responsiveness to novel stimulation were assessed in three groups of mice infected with Toxoplasma. One group was infected when adult; two groups were infected congenitally, one born to dams infected during gestation, the other to dams chronically infected prior to mating. Each mouse was tested in a box, the floor of which was marked off into 16 equal squares, and its activity was measured over ten minutes by counting the number of times the mouse entered each square. Infected mice were more active. In addition, infected mice showed a smaller relative preference for the more novel central area of the box, especially towards the end of the observation period. These differences were independent of emotionality (as measured by defaecation counts), general health (as measured by subjective health ratings and body weight) and the number of Toxoplasma tissue cysts in specified brain regions. We suggest that differences arise from pathological changes caused by proliferating toxoplasms in the brains of the infected mice; an immunopathological reaction due to the presence of tissue cysts in the brain may also be involved. Other possible factors contributing to observed deficits in behaviour are also discussed. We suggest that such deficits may render Toxoplasma-infected mice more susceptible to predation by the domestic cat, the definitive host of Toxoplasma.

The effect of congenital and adult-acquired Toxoplasma infections on the motor performance of mice.

Motor performance was assessed in three groups of mice infected with Toxoplasma. One group was infected when adult. Two groups were infected congenitally: the first was born to dams infected during gestation and the second to dams which were chronically infected prior to mating. All mice were placed individually on a rotating cylinder and the number of falls from it noted over a two-minute period. Infected mice fell significantly more often than uninfected controls. The difference was independent of emotionality (as measured by defaecation) and general body health (as measured by body weight and a subjectively assessed health rating). There was no significant difference in motor performance between the two congenitally infected groups. However, the offspring of mice infected during pregnancy fell significantly more often than mice infected when adult. There were no significant correlations between motor performance and the actual number of Toxoplasma tissue cysts in the brains (or in separate defined sectors of the brains) of infected mice. We suggest that differences between infected and uninfected mice result from pathological changes caused by proliferating toxoplasms in the brains of infected mice. An immunopathological reaction due to the presence of the tissue cysts may also be involved. Other possible factors contributing to observed deficits in motor performance of infected mice are discussed. We suggest that such interference with the motor performance of Toxoplasma infected mice may render them more susceptible to predation by the domestic cat, the definitive host of Toxoplasma.

Detection of soluble antigens of Toxoplasma gondii by a four-layer modification of an enzyme immunoassay.

A sensitive four-layer modification of an enzyme immunoassay for the detection of soluble antigens of Toxoplasma gondii is described. Microtiter plates were sensitized with rabbit anti-toxoplasma immunoglobulins (6 micrograms/ml) used as the primary antibodies; guinea pig anti-toxoplasma immunoglobulins (6 micrograms/ml) were used as the secondary trapping antibodies. Horseradish peroxidase-conjugated anti-guinea pig immunoglobulins were used as the indicator antibodies. The specificity of the antigen assay was confirmed by using guinea pig immunoglobulins from preimmunization sera. The sensitivity of the antigen assay was found to be at least 10 ng of antigen protein per ml. The suitability of the method for detecting antigens of T. gondii in different specimens was studied by experimental toxoplasma infection in mice. Antigenic components of T. gondii could be detected in different tissue specimens from infected animals from the first day after infection onwards. Toxoplasma antigen in serum and urine samples from infected mice reached detectable levels on day 2 after infection followed by a linear increase in antigen concentration in succeeding samples. This method might offer a valuable aid for a rapid etiological diagnosis also in human cases of acute toxoplasmosis.

Suppression of unprimed T and B cells in antibody responses by irradiation-resistant and plastic-adherent suppressor cells in Toxoplasma gondii-infected mice

In the acute phase of Toxoplasma infection, the function of both helper T and B cells was suppressed in primary antibody responses to dinitrophenol (DNP)-conjugated protein antigens. During the course of infection, the suppressive effect on T cells seems to continue longer than that on B cells, since suppression in responses to sheep erythrocytes, a T-dependent antigen, persisted longer than those to DNP-Ficoll, a T-independent antigen. Plastic-adherent cells from the spleens of Toxoplasma-infected and X-irradiated (400 rads) mice had strong suppressor activity in primary anti-sheep erythrocyte antibody responses of normal mouse spleen cells in vitro. These data suggest that the activation of irradiation-resistant and plastic-adherent suppressor cells causes the suppression of both T and B cells in Toxoplasma-infected mice.

Effect of Immunomodulation on the Fate of Tumor Cells in the Central Nervous System and Systemic Organs of Mice. Distribution of [<sup>125</sup>I]5-lodo-2′-deoxyuridine-Labeled KHT Tumor Cells After Left Intracardial Injection<xref ref-type="fn" rid="fn2">2</xref>

The effect of systemic immunomodulation on tumor cell arrest and retention in the central nervous system was studied by following radioactively labeled tumor cells. KHT mouse sarcoma tumor cells were labeled in vitro with [125I]IdUrd, and 1 X 10(5) tumor cells were injected into the left side of the hearts of syngeneic C3H mice. Experimental groups consisted of untreated normal mice, mice pretreated iv with Corynebacterium parvum, and mice chronically infected with Toxoplasma gondii; in this model both groups of immunomodulated mice are protected from developing systemic metastatic tumor, but only Toxoplasma-infected mice have protection against metastatic brain tumor. At time intervals from 1 to 96 hours, groups of mice from each experimental group were killed, and the brain and other organs were monitored for radioactivity to determine the number of viable tumor cells that had been present at the time of death. Normal mice demonstrated significant retention of tumor cells in the brain and kidneys plus adrenals at 96 hours. By contrast, in both groups of immunomodulated mice tumor cells were rapidly eliminated from systemic organs, but tumor cells were significantly retained in the central nervous system even at 96 hours after tumor cell injections. The results indicated that generalized immunomodulation had more effect in elimination of tumor cells from systemic organs than from the brain and that the elimination of tumor cells from the brain in Toxoplasma-infected mice was a delayed phenomenon.

The course of Plasmodium berghei infection in mice latently infected with Toxoplasma gondii.

The course of infection with 2 different virulent strains of Plasmodium berghei was investigated in mice latently infected with Toxoplasma gondii. When given the highly virulent ANKA strain of P. berghei all Toxoplasma-infected mice died but the survival time was prolonged. After infection with the less virulent strain K 173 mice could survive the subsequent infection. In these cases levels of parasitemia depended upon the duration of the T. gondii infection. Mice infected for about 6 weeks with T. gondii showed maximum protection.

Nonspecific suppression of initiation of memory cells in Toxoplasma gondii-infected mice.

The effect of Toxoplasma infection on initiation and expression of memory cells to dinitrophenol (DNP)-conjugated protein antigens in humoral immune responses was studied in mice. Marked suppression in the initiation of memory cells to DNP-conjugated keyhole limpet hemocyanin occurred in the acute phase of infection. However, once the memory cells were induced before infection, expression of the memory cells was not affected. Moreover, the suppression of priming occurred on both T and B cells. The suppressive effect was observed in all immunoglobulin classes tested, i.e., immunoglobulin M (IgM), IgG1, IgG2, and IgE, regardless of the four kinds of DNP-conjugated protein antigens. This nonspecific suppression could be induced only by living toxoplasmas, by either the peroral or parenteral route, but not by lysed organisms. A transfer of normal spleen cells could not restore the ability of infected mice to initiate memory cells. Furthermore, the suppressive effect of the infected mice was not removed by 400 R of gamma-irradiation. These results suggested that irradiation-resistant suppressor cells cause nonspecific suppression of the initiation of memory cells in Toxoplasma-infected mice.

Nonspecific suppression of primary antibody responses and presence of plastic-adherent suppressor cells in Toxoplasma gondii-infected mice.

The effect of Toxoplasma infection on primary antibody responses to both T-dependent and T-independent antigens was examined in mice. Drastic suppression of primary responses to sheep erythrocytes (SRBC) occurred when mice were immunized 7 days after infection. The suppression was observed in both 2-mercaptoethanol-sensitive and -resistant hemagglutinin responses. Anti-dinitrophenol (DNP) immunoglobulin E and G1 responses to DNP-conjugated keyhole limpet hemocyanin were also suppressed by infection. It was suggested that the suppressive effect is nonspecific for the antigens and immunoglobulin classes produced. Anti-DNP responses to DNP-Ficoll, a T-independent antigen, were suppressed by infection, but the suppressive effect was weaker than that on the responses to SRBC. This suggests that both T and B cells are suppressed by infection. In vitro responses of infected mouse spleen cells to SRBC and DNP-Ficoll confirmed the results of in vivo experiments. In addition, the presence of plastic-adherent suppressor cells was demonstrated in the spleen cells of infected mice, which suppressed the ability of normal mouse spleen cells to mount an SRBC-specific plaque-forming cell response. These plastic-adherent suppressor cells appeared to be a major cause of nonspecific suppression of primary antibody responses in Toxoplasma-infected mice.

Production and properties of immune interferon from spleen cell cultures of Toxoplasma-infected mice.

In response to antigenic stimulation, spleen cells from Toxoplasma-infected mice produce a factor showing inhibitory activity against vesicular stomatitis virus infection in L cell cultures. When BALB/C and ICR mice were inoculated intraperitoneally with the low-virulent S-273 strain of T. gondii, such activity was first detected in 4 and 7 days and reached maximum levels at 10 and 14 days respectively, and retained these levels for at least three weeks. However, BALB/C mice, which are considerably more sensitive to Toxoplasma infection than ICR mice, produced significantly smaller amounts of interferon (IF) after challenge with the high virulent strain. The IF produced in this system possessed certain known properties of immune (type II) IF and was not neutralized by rabbit antiserum against mouse type I IF. The immune IF preparation also inhibited multiplication of Toxoplasma within nonphagocytic L cells in an IF-like fashion, whereas Newcastle disease virus-induced (type I) IF had no effect on this parasite. The antiviral and anti-Toxoplasma activity in immune IF preparations could not be distinguished solely on the bases of their molecular weight and isoelectric point. The experiments with anti-theta serum plus complement and with nylon wool column effluent cells strongly suggest that immune IF was produced by T lymphocytes and required the assistance of macrophages.

Chronic Toxoplasma infections and motor performance in the mouse.

Motor performance in Toxoplasma-infected mice and uninfected controls was assessed by placing them, individually, on a rotating cylinder. The infected mice fell significantly more often than the controls. The difference was independent of differences in fearfulness (as measured by defaecation counts) and general bodily health (as measured by body weight). Although Toxoplasmatissue cysts were present in the brains of all of the infected animals, there was no significant correlation between cyst counts (either in the cerebellum or brain minus cerebellum) and number of falls within the infected group. It is suggested that the absence of significant correlations may have been due to the restricted range and variability in tissue cyst counts.

Behavioural abnormalities in Toxoplasma-infected mice.

Observational methods, using video recordings and computer-assisted data analysis, were used to investigete the behaviour of Toxoplasma-infected mice. Infection had a selective effect, increasing the amount of general movement but decreasing the amounts of rearing and digging. In addition infection affected the pattern of bouts of behaviour, increasing the number of shorter bouts, and this was found to underlie a variety of specific behavioural changes. The results indicate that Toxoplasma infection probably affects the animal's response to its environment and the stimulation arising from it, and may even affect endogenous regulatory processes in the brain.

Chronic Toxoplasma infections and familiarity-novelty discrimination in the mouse.

Male mice were exposed to a black arm and the stem of a Y-maze; entrance to a white arm was blocked by a transparent door. Toxoplasma-infected mice were significantly less active and tended to produce fewer faecal boluses than uninfected controls. In a subsequent free-choice trial, in which both arms were black, the uninfected mice spent significantly more time in the novel (previously blocked) arm; the infected mice showed no preference for either choice arm. Possible explanations are discussed. In particular, it seems that Toxoplasma-infected mice may be less responsive to novel stimuli. This suggestion has important implications for our understanding of one of the major ways in which Toxoplasma passes from host to host. If Toxoplasma infections impair responsiveness to novel stimuli, then infected mice are more likely to be taken by predators.

Lack of immunoglobulin M suppression by immunoglobulin G antibody in thymectomized, irradiated, and bone marrow-reconstituted mice infected with Toxoplasma gondii.

Thymectomized, irradiated, bone marrow-reconstituted (T-deprived) mie infected with an avirulent strain of Toxoplasma gondii produced antibody titers comparable to those produced in intact syngeneic mice. Both immunoglobulin M (IgM) and IgG antibodies were produced in T-deprived animals; however, the IgM antibody remained constant in the presence of increasing amounts of IgG. In the intact animals, IgM became undetectable by day 50 postinfection as expected. Feedback inhibition of IgM by IgG seems to be dependent upon T-cells in Toxoplasma-infected mice.

The immunocytochemical localization of GFA protein in experimental murine CNS tumors.

Immunocytochemical localization of GFA protein in formalin-fixed, paraffin-embedded tissue sections by the peroxidase-antiperoxidase method of Sternberger was used to study experimental murine CNS tumors. Transplacental tumor induction in rats by ethylnitrosourea produced oligodendrogliomas and mixed gliomas in the cerebrum and spinal cord, and malignant Schwannomas of the trigeminal nerve. A methylcholanthrene-induced mouse "ependymoblastoma" inoculated intracerebrally in normal and in toxoplasma-infected mice was also studied. A positive reaction of GFA protein antibody was seen in the astrocytic portion of the mixed gliomas; the oligodendrogliomas, the malignant Schwannomas and the mouse "ependymoblastoma" were negative. Staining for GFA protein delineated the astrocytic reaction of neural tissue adjacent to the tumors. The reaction was markedly intensified in the brains of mice infected with toxoplasma. Additionally, ependymal cells near the tumors stained positively for GFA protein; normal ependyma at a distance from tumor remained negative. The technique, which combines a high degree of specificity with great sensitivity and is readily adaptable to routinely processed tissue, should prove a valuable tool in experimental oncology of the central nervous system.