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Biomedical subjects

D B Budzko

Publications and source records attributed to D B Budzko.

At least 19 recordsLinked to original sources

Fc receptors on the surface of Toxoplasma gondii trophozoites: a confounding factor in testing for anti-Toxoplasma antibodies by indirect immunofluorescence.

Negative sera often produce false-positive polar patterns of fluorescence in indirect immunofluorescence tests for serum anti-Toxoplasma gondii antibodies, representing a confounding factor in the diagnosis of toxoplasmosis. In this work, we studied whether T. gondii trophozoites, the antigenic material used in the immunofluorescence tests, expressed surface Fc receptors that could cause the binding of normal immunoglobulins, thus producing false-positive results. We report here that T. gondii trophozoites indeed have Fc receptors on their surface. This was shown by the direct binding of purified human Fc to the parasite, evidenced by the subsequent binding of fluorescein-labeled Fab specific for human Fc. In addition, pretreatment of the parasite with excess purified Fc to saturate the surface Fc-binding sites abrogated the formation of polar fluorescence. The trophozoites appeared to express Fc receptors with different degrees of affinity for the specific ligand, since a diffuse fluorescence pattern was observed following incubation with 1 mg of Fc per ml (10 micrograms per well), whereas with 0.2 mg (0.2 micrograms per well), a majority of parasites showed polar fluorescence. This observation suggests that the Fc receptors accumulated at the polar cap are those with higher affinity. The present findings raise intriguing questions regarding the possible biological role(s) of the Fc receptors on T. gondii but, more immediately, indicated that pretreatment of the antigenic material with Fc, a commercially available reagent, constitutes a practical, simple way to avoid false-positive immunofluorescence test results due to the binding of nonspecific immunoglobulin to the parasite.

Animals↗

Immunologic alterations in monkeys with simian acquired immunodeficiency syndrome (SAIDS).

Levels of lymphocyte responsiveness to T- and B-cell-specific mitogens and expressions of Ia, T4, T8, and T11 surface markers were monitored during the course of Simian acquired immunodeficiency syndrome (SAIDS) in four Rhesus macaques that either died or became ill and survived. The monkey that died showed progressively suppressed responses to concanavalin A (Con A), phytohemagglutinin (PHA), pokeweed mitogen (PWM), and Staphylococcus aureus Cowan I strain (SAC) through the time of death (5 1/2 weeks). For the three animals that survived, the responses of peripheral blood mononuclear cell (PBMC) to the same mitogens were decreased significantly during the period 4-6 weeks after inoculation. Levels of Ia-bearing cells in the PBMC population were markedly reduced in the moribund monkey but were not significantly decreased in the three survivors. There was no significant change in the percentage of T11-bearing cells in any of the study animals. The ratio of T4- and T8-positive cells did not vary significantly during the 18 weeks of observation in any of the animals. The infected animals showed other evidence of immunosuppression including neutropenia, lymphopenia, and depletion of lymphocytes in lymph nodes. The animal that had progressive disease and death also had Kaposi-like lesions and staphylococcal septicemia. These results indicated that in vitro evidence of immunosuppression due to SAIDS appears within a few weeks after infection and this may progress in animals that die.

Acquired Immunodeficiency Syndrome↗

Detection of herpes simplex virus in clinical specimens by an enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) for the detection of herpes simplex virus is described that can be performed in approximately four hours. The test, which does not require specialized equipment and uses relatively inexpensive, commercially available reagents, detected herpes simplex virus in 51% of specimens found to be positive by a time-consuming cell culture technic. The ELISA test compared favorably with a direct immunofluorescence method that detected HSV in only 1% of the cell culture-positive specimens. The ELISA test was readily carried out even with specimens unsuitable for cell culture and did not require cellular material as is the case with immunofluorescence technics. An advantage of the ELISA test for herpes simplex virus over the cell culture method was the detection of nonviable virus.

Antigens, Viral↗

Rapid test for detection of rabies antibodies in human serum.

A simple, sensitive, rapid method based on the principle of immunoadherence hemagglutination (IAHA) has been devised for the detection of rabies antibody. In this test, fixation of complement to complexes of rabies antigen with specific antibodies is readily detected by agglutination of human erythrocytes bearing receptors for C3. Sera from individuals undergoing preexposure rabies immunization were tested for rabies antibodies by the IAHA method and by a virus neutralization test performed in tissue culture, the rapid fluorescent focus inhibition test. IAHA titers showed a high degree of correlation with rapid fluorescent focus inhibition test titers, although it is not known whether results of the IAHA test represent the detection of neutralizing antibodies. An advantage of the IAHA test over the rapid fluorescent focus inhibition test was that results were obtained in a shorter period of time. In some instances, this can be of clinical significance in determining antibody levels to rabies virus. Furthermore, the IAHA test is most applicable as a rapid screening tool for the detection and quantitation of rabies antibodies in vaccinated subjects.

Antibodies, Viral↗

Exacerbation of Trypanosoma cruzi infection in mice treated with the immunoregulatory agent cyclosporin A.

Treatment with the immunoregulatory fungus metabolite cyclosporin A exacerbated the course of Trypanosoma cruzi infection in outbred as well as in inbred BALB/c mice. This effect was not observed in congenitally athymic, nu/nu, mice but was readily reproduced in their thymus-bearing, nu/+, littermates. These results suggest that the deleterious action of cyclosporin A on the course of experimental Chagas' disease results from activity of the drug on thymus or thymus-derived lymphocytes and emphasizes the role of T cells in host defense against T. cruzi infection.

Animals↗

Trypanosoma cruzi: deficient lymphocyte reactivity during experimental acute Chagas' disease in the absence of suppressor T cells.

Infection of mice with Trypanosoma cruzi has been shown to lead to an impaired ability of lymphocytes to proliferate in response to mitogenic stimulation which is manifested during the acute period of the disease. A possible involvement of suppressor T lymphocytes has been postulated by other authors and was investigated in this work as a part of our efforts to disclose the mechanisms underlying the immunologic deficiency. Spleen cells from acutely infected CBA/J mice readily exhibited unresponsiveness to stimulation with concanavalin A, phytohaemagglutinin or a bacterial lipopolysaccharide. However, these cells were unable to reduce the responses that normal syngeneic-mouse spleen cells mounted to these mitogens when cultured together in equal proportions. Furthermore, removal of the Lyt 2.1-bearing cells, known to include the suppressor T cell subpopulation, from infected mouse splenocyte suspensions, did not alter the deficient responsive status of the remaining cells. These results, together with the severe depletion of the T-cell compartment which occurs in the spleens of animals acutely infected with T. cruzi, do not support an important role of suppressor T lymphocytes in the noted deficiency in lymphoid cell reactivity to mitogens. Reduced numbers of responder cells, intrinsic lymphocyte alterations or suppression by cells other than T lymphocytes remain plausible explanations to be explored.

Animals↗

Nonspecific reactions in the hemagglutination inhibition test for detection of rubella antibodies.

Approximately 7% of the sera tested to determine the presence of rubella-specific antibodies by the hemagglutination inhibition test demonstrated abnormal patterns of reactivity, rendering the test unreadable. Another 3% of sera were shown to have false-positive titers as high as 1:128. When these abnormally reacting and false-positive sera were heated at 56 degrees C for 30 min after chemical treatment they always converted to negative, indicating the absence of specific rubella hemagglutination-inhibiting antibody. These results were confirmed by fractionation of the sera after sucrose gradient centrifugation. It was established that manifestation of these nonspecific results was dependent on the concentration of Ca2+ or Mn2+. The heat-labile inhibitor(s) responsible for abnormal and false-positive reactions was found not to be complement. This inhibitor(s) was detected in the light fractions of sera and when added to negative sera was capable of reproducing the abnormal patterns of reactivity. These results emphasize the necessity of heating sera for the rubella hemagglutination inhibition test after the chemical removal of nonspecific inhibitors.

Antibodies, Viral↗

Antibody-independent, natural resistance of birds to Trypanosoma cruzi infection.

Complement (C) activity present in normal human serum has been reported to lyse circulating forms of Trypanosoma cruzi following activation by specific host antibodies bound to the surface of the parasites. In view of this observation, we examined the possibility that a similar phenomenon may cause lysis of T. cruzi by avian complement, a mechanism postulated to be responsible for the natural resistance of birds to T. cruzi infection and previously described as being antibody independent. Trypomastigote forms of T. cruzi grown either in lethally irradiated mice or in cell cultures were lysed readily by the sera of agammaglobulinemic chickens. Lytic activity and titers of normal and agammaglobulinemic sera were comparable. The lytic reaction was inhibited by heat inactivation of the sera, or by addition of EDTA or cobra venom factor to the assay mixtures. Lysis of T. cruzi was observed when calcium, but no magnesium ions, were chelated with EGTA. Furthermore, a significant loss of lytic activity was observed when sera from C-depleted chickens were tested. Normal and agammaglobulinemic chickens cleared intravenously injected parasites (from either lethally irradiated mice or cell cultures) from their circulation in 7 min or less whereas C-depleted animals required 1,740 min or longer. Routine examination of the parasites from these two sources by immunofluorescence confirmed the absence of immunoglobulins on their surface. These results emphasize the lack of requirement of antibodies for, and the important role of complement in both the natural resistance that birds exhibit against T. cruzi infection and the lytic activity displayed by avain serum on virulent forms of T. cruzi.

Animals↗

The guinea-pig complement-activating lymphocyte surface component (GPCA): a mouse T-cell marker distinct from Thy-1.

Mouse lymphocytes selectively expressing sensitivity to antibody-independent complement-mediated lysis by normal guinea-pig serum have been previously shown to be identical with T cells. This correlation has raised the question addressed in this work of whether or not the complement-activating surface component and Thy-1 were the same marker. S.49 mouse lymphoblastoid cells, sensitive to killing by anti-Thy-1.2 antibodies plus a source of complement activity devoid of non-specific cytotoxicity, were not sensitive to the non-specific, antibody-independent lytic effects of normal guinea-pig serum. Furthermore, rat thymus cell suspensions containing 93%--95% Thy-1-bearing cells were only partially susceptible (20%--35%) to guinea-pig serum cytotoxicity. Young rat thymus cells virtually devoid of guinea-pig serum-sensitive cells (less than 2%) were readily lysed by antirat Thy-1 and complement (greater than 98%). While these results do not exclude the possibility that Thy-1 may constitute an indirect requirement for T cells to manifest sensitivity to GPS cytotoxicity, it is clear that this antigen does not confer such sensitivity by itself. Therefore, GPCA--the guinea-pig complement-activating marker of murine T cells--is not identical with Thy-1 and represents a distinct surface component of T lymphocytes which manifests itself in terms of a non-specific, but selective ability to activate complement.

Animals↗

Selective removal of T cell function from mouse lymphocyte suspensions by treatment with normal guinea pig serum.

Treatment of murine spleen cells with normal guinea pig serum selectively abrogated responsiveness of these cells to the T cell mitogens PHA or Con A, but failed to affect responses to LPS, i.e., a B cell-specific mitogen. Although pretreatment with GPS inhibited the in vitro immune response of mouse splenocytes to SRBC, responses were normal after restoration with T cells only, indicating that B cells had been spared by GPS. Consistent with these results, incubation with GPS resulted in the loss of reactivity of mouse lymphoid cells in MLC as well as CML systems, both of which test for T cell activities. Furthermore, parental spleen cells treated with GPS were no longer capable of inducing a GVH reaction in F1 hybrids. When compared, the effects of GPS and anti-Thy-1.2 antibodies plus C were found to be comparable. These results indicate that GPS can selectively remove a number of T cell functions from heterogeneous murine lymphoid cell suspensions. Since spleen macrophages were insensitive to GPS cytotoxicity, lack of T cell function is not likely to be due to depletion of these accessory cells.

Animals↗

Enhanced resistance against Junin virus infection induced by Corynebacterium parvum.

The effects of intraperitoneal administration of Corynebacterium parvum on the course of Junin virus infection in mice were investigated. This treatment produced enhanced resistance to the virus infection, evidenced by an increase in both survival times and the proportion of survivors. The protective effect was dependent upon the dose of C. parvum, and 280 mug/g of body weight was found to be the optimal dose. In various experiments, about 80% of the infected animals receiving this dose survived, whereas survival ranged between 0 and 20% among untreated infected mice. Maximal protection was afforded by C. parvum when administered simultaneously with the virus. A smaller but significant degree of resistance was induced by C. parvum given 3 or 6 days after infection. C. parvum injected before infection was ineffective. Viral titers measured in the brains of C. parvum-treated and untreated mice at various times after infection were found to be comparable. In addition, there were no significant differences between circulating-antibody titers measured either by neutralization tests or by complement fixation. Depression of the reticuloendothelial system by treatment with silica particles also resulted in enhanced resistance to Junin virus infection, suggesting that the protective effect of C. parvum is not likely to be due merely to its capacity to stimulate macrophages. The present data, highlighting that the presence of high titers of Junin virus and disease do not necessarily correlated, suggest that in mice this disease is not the consequence of cell damage caused directly by the virus but of a still undefined indirect mechanism induced by the virus, not necessarily mediated by macrophages.

Animals↗

Cytotoxic effects of normal sera on lymphoid cells. II. Requirements for inhibition of nonspecific serum cytotoxicity by agarose.

Agarose is known to inhibit nonspecifically the cytotoxic effects of normal sera on xenogeneic lymphoid cells. To find an explanation for this agarose effect we have studied its requirements using guinea pig and human sera as the source of activity and rat thymocytes as target cells. Control assays were performed using heat-inactivated (56 C, 30 min) normal rat serum. The inhibitory effect of agarose was readily reproduced with untreated sera and also when sodium ethyleneglycoltetraacetate, a selective chelator of calcium ions, was added to the sera together with excess magnesium. However, the agarose effect failed to occur in the presence of 0.01 M EDTA unless magnesium ions were restored. Abrogation of cytotoxicity in human serum by incubation with a large number of target cells instead of agarose was also found to be magnesium dependent. Titrations of human serum, performed after absorption with agarose in the presence of EDTA, which does not interfere with antigen-antibody binding, and subsequent restoration of divalent cations, revealed no significant change in its cytotoxic titer when compared with that of mock-absorbed serum not subjected to the agarose treatment. Incubation of human serum with either agarose or rat thymocytes resulted in the conversion of factor B, essential for complement activation via the alternative pathway, previously shown to provide the complement activity necessary for the cytotoxic reaction. These results suggest that the agarose effect is mainly attributable to complement consumption via the alternative pathway rather than to the absorption of "natural" antibodies.

Animals↗