The role of T-cell subpopulations in experimental Chagas' disease.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
R L Tarleton
Publications and source records attributed to R L Tarleton.
Medium-scale production and purification of monoclonal antibodies in protein-free medium.
Hybridoma cell lines can be adapted to grow in a totally protein-free tissue culture medium and cultured in spinner flasks to generate moderate-to-high quantities of monoclonal antibodies. Such antibodies are easily purified by ammonium sulfate precipitation. This system was shown to be useful for growth of 23 different hybridoma cell lines from different sources to yield an average of 40 mg of highly purified antibody per liter of tissue culture medium.
Animals↗
Depletion of CD8+ T cells increases susceptibility and reverses vaccine-induced immunity in mice infected with Trypanosoma cruzi.
The role of CD8+ T cells in immune control of Trypanosoma cruzi infection in mice was examined by using in vivo depletion of CD8+ T cells with antibodies. Both the resistant C57BL/6J and the highly susceptible C3H/HeSnJ developed higher parasitemias and increased or earlier mortality when depleted of CD8+ T cells before infection with the Brazil strain of T. cruzi. CD8 depletion also affected the induction and expression of immunity to T. cruzi after vaccination with the avirulent Corpus Christi strain of T. cruzi. C57BL/6J mice depleted of CD8+ T cells either before or after vaccination showed a near total loss of vaccine induced protection. C3H mice were only partially protected by the vaccination procedure but the protection was totally reversed by anti-CD8 treatment. Chronically infected mice that had survived the acute infection were unaffected by CD8 depletion and showed neither a recrudescence of parasitemia nor an increased susceptibility to reinfection. These results suggest that CD8+ T cells play a role in immunity to T. cruzi in the acute phase but possibly not during the chronic phase of infection. Also, immunization with Corpus Christi strain T. cruzi induces an immunity that is distinct from that induced by survival of the acute phase of infection. The mechanism by which CD8+ T cells contribute to control of T. cruzi infection is not known. However, CD8 depletion had no effect on suppressed immune responses, suggesting their function in T. cruzi-infected mice is related more to the cytotoxic activity or cytokine-producing capacity of this subpopulation of T cells.
Animals↗
Endotoxin-induced tumor necrosis factor activity production by equine peritoneal macrophages.
A study was performed to determine whether equine macrophages produce tumor necrosis factor (TNF) activity in vitro in response to endotoxin and to study the effects of endotoxin concentration and incubation time on the amount of TNF produced. Equine peritoneal macrophages were isolated and cultured in vitro for 2, 6, 12, or 24 hr in tissue culture media containing 1) no additive (nonstimulated control), 2) endotoxin (0.5 ng/ml, 5 ng/ml, or 5 micrograms/ml), or 3) the calcium ionophore A23187 (0.95 microM). The supernatant media concentrations of TNF activity were determined by an in vitro cytotoxicity bioassay using the murine fibrosarcoma cell line WEHI 164 clone 13. At all incubation times, macrophages exposed to endotoxin produced significantly more TNF than did nonstimulated control cells (P less than 0.05). Among the three doses of endotoxin tested, there were no differences in concentrations of TNF activity in the supernatant media. The mean concentration of TNF activity in media from nonstimulated macrophages was significantly less after 24 hr incubation than after 2, 6 or 12 hr. Incubation time had no effect on TNF production by macrophages exposed to 0.5 ng/ml and 5 micrograms/ml endotoxin, but macrophages exposed to 5 ng/ml endotoxin produced significantly more TNF after 6 and 12 hr than after 2 hr. To address the reason for the decrease in TNF activity in media from nonstimulated macrophages over time, a second study was designed to determine the effect of incubation on the activity of TNF under tissue culture conditions. Human recombinant TNF was added to media that was incubated 2-24 hr.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Diagnosis of recent Toxoplasma gondii infection in cats by use of an enzyme-linked immunosorbent assay for immunoglobulin M.
Subclinical Toxoplasma gondii infection was induced in young and adult cats by oral administration of tissue cysts. An antibody-capture ELISA to detect anti-Toxoplasma IgM-class antibodies in the serum of cats was developed. The serologic response to experimental infection was followed in the 2 groups of cats by use of anti-Toxoplasma IgM and IgG detection. This study shows that anti-Toxoplasma IgM-class antibody titers develop early in the course of experimental infection in cats and that the combination of IgM- and IgG-class antibody titer measurement can aid in the detection of recent subclinical toxoplasmosis.
Animals↗
Enzyme-linked immunosorbent assay for the detection of circulating antigens of Toxoplasma gondii in the serum of cats.
An ELISA was developed to detect circulating antigens of Toxoplasma gondii in the serum of cats. For the experiment, toxoplasmosis was induced in a group of cats by oral administration of bradyzoites. An ELISA that detects anti-Toxoplasma IgG, an ELISA to detect circulating antigens, and fecal examinations were performed on samples from each cat for 1 year after inoculation. When coupled with IgG-class antibody measurement, antigen detection can aid in the diagnosis of some cases of subclinical feline toxoplasmosis.
Animals↗
Trypanosoma cruzi-induced suppression of IL-2 production. I. Evidence for the presence of IL-2-producing cells.
Mice infected with the protozoan parasite Trypanosoma cruzi, the causative agent of human Chagas' disease, are profoundly immunodepressed in their response to various Ag and mitogens. A key factor in this immunosuppression is the essential inability to produce the T cell growth factor IL-2. In this study we demonstrate that this failure to produce IL-2 in response to mitogen stimulation is not the result of the absence of production of soluble or membrane-bound IL-1 by macrophages. Limiting dilution analysis of the precursor frequency of IL-2 producers suggests that an adequate number of precursors for IL-2 production are present in the spleens of infected mice, but that their activity may be regulated by suppressor cells. The presence of precursor cells for IL-2 production is supported by experiments showing that the combination of calcium ionophores and PMA elicits IL-2 production by spleen cells from both normal and T. cruzi-infected mice. Although Con A can provide either of the signals necessary for IL-2 production, calcium flux or protein kinase C activation, to T cells from normal mice, Con A in combination with either calcium ionophore or phorbol ester failed to activate T cells from infected mice to produce IL-2. Preculture of spleen cells from infected mice for 48 to 72 h before addition of Con A results in near normal production of IL-2. This recovery of the capacity to produce IL-2 does not occur if parasite Ag is present during the preculture period. These results suggest that the inability of T cells from T. cruzi-infected mice to produce IL-2 in vitro in response to Con A is not due to the lack of IL-2-producing cells, but may be the result of the maturational state of the T cells or to the presence of a suppressor population.
Animals↗
Trypanosoma cruzi-induced suppression of IL-2 production. II. Evidence for a role for suppressor cells.
Suppression of IL-2 production during experimental Chagas' disease accounts at least in part for the overall depressed state of the immune system in infected mice. The failure to produce IL-2 in response to mitogen stimulation is not the result of the lack of cells capable of producing IL-2, but appears to be due to regulation of IL-2 production by suppressor cells. This conclusion is supported by cell-mixing experiments where the ability of cells from infected mice to suppress normal spleen cell IL-2 production is evident. Although depletion of plastic and Sephadex G-10 adherent cells results in modest increases in IL-2 production by spleen cells from infected mice, even in the presence of normal adherent cells as a source of IL-1 producers, IL-2 production does not approach normal levels. Also, isolated macrophages are not by themselves suppressive for normal spleen cell IL-2 production, whereas plastic and G-10 nonadherent cells from infected mice are. Depletion of Thy-1+ and Ly-2+ cells not only completely abrogates the ability of spleen cells from infected mice to suppress normal IL-2 production, but results in a cell preparation which actually enhances IL-2 production. Anti-Ly-2 and C treatment of infected spleen cells also markedly enhances their ability to produce IL-2. These results indicate a major role for Ts cells in the regulation of IL-2 production, and a relatively minor role of macrophages as direct effector cells of suppression in this response. The ability to enhance IL-2 production in this system with PG synthesis inhibitors suggests a role for PG-producing cells such as macrophages in the suppressor mechanism, perhaps as inducers of the suppressor effector cells.
Animals↗
Interleukin 2 production in patients with Chagas' disease: correlation with anti-parasite antibody responses.
Peripheral blood mononuclear cells (PBMC) isolated from chronic chagasic patients and control individuals in Recife, Brazil were examined for the ability to produce IL-2 in response to concanavalin A (Con A) stimulation. Overall, there was little difference in the range of the response (IL-2 production) between the chagasic and control groups. Within the chagasic group, however, there was a high negative correlation between IL-2 production and the level of anti-parasite antibody. This correlation is thought to be a reflection of the fact that individuals with more recent or more vigorous infections exhibit higher anti-parasite antibody responses but also display a greater degree of immunosuppression, as reflected in depressed IL-2 production.
Adult↗
A monoclonal antibody to alpha tubulin recognizes host cell and Trypanosoma cruzi tubulins.
A mouse monoclonal anti-alpha-tubulin antibody was used to investigate the disposition of the cytoskeletal microtubules of three tissue culture cell lines--J774 macrophages, BSC-1, and Vero cells--infected with the Brazil strain of Trypanosoma cruzi. Indirect immunofluorescence light microscopy was used to demonstrate the antigenic response in host cells and parasites, simultaneously. In all morphotypes of T. cruzi, the monoclonal antibody reacted with all subpopulations of microtubules, inclusively, the subpellicular, flagellar, cytopharyngeal, and mitotic. The host cell cytoskeletal microtubule framework was revealed and the redistribution and destruction of the microtubular lattice in response to parasite infection over a 120 h period recorded. Our results show that after the initial inoculation of tissue cultures with trypomastigotes, the parasites penetrate the cells and locate in the perinuclear region of the cell where they multiply. The number and distribution of host cell microtubules were altered during the infection. The normal radial distribution of microtubules extending from the center of the cell to the periphery was destroyed. The remaining microtubules were observed at the periphery encircling, but well removed from the proliferating parasites. The complete transformation of the parasites was monitored throughout the infection with the end result being the liberation of parasites and the near complete destruction of the microtubular framework of the host cell. A residual population of dividing spheromastigotes was observed in cells liberating trypomastigotes. Colloidal gold labeling of thin sections as seen in the electron microscope affirmed the specificity of our monoclonal antibody to all subpopulations of microtubules in T. cruzi.
Animals↗
Regulation of lymphokine production in experimental and human Chagas' disease.
Explore the source record for details and available documents.
Animals↗
Tumour necrosis factor (cachectin) production during experimental Chagas' disease.
Mice infected with the protozoan parasite Trypanosoma cruzi are primed for the production of tumour necrosis factor/cachectin. Active infection is not required for stimulation of TNF production as formalin-fixed, but not heat-killed, parasites can act as a priming stimulus. Both epimastigote and trypomastigote stages of the parasite can prime for TNF production but on a per cell basis, trypomastigotes are more potent stimulators than epimastigotes and live parasites prime more efficiently than killed preparations. Although the priming effect of epimastigotes and trypomastigotes is dose-dependent when assayed 10 days after parasite injection, parasitemia levels in the infected mice do not correlate with the level of TNF production. Spleen cells from infected mice are also primed for the production in vitro of TNF in response to LPS or T. cruzi. These results suggest that TNF may be constitutively produced in vivo and that the priming for TNF production, or the production itself, may be regulated during the course of the infection in mice.
Animals↗
Initial induction of immunity, followed by suppression of responses to parasite antigens during Trypanosoma cruzi infection of mice.
Infection of a relatively resistant strain of mice (C57BL/6J) with the protozoan parasite Trypanosoma cruzi results in both the induction of parasite-specific T-helper cells and nonspecific suppressor cells. A time course study of the activation of help and suppression revealed that parasite-specific T-helper cell activity increases very early in infection (less than 12 days) at a time when suppression of non-parasite-specific responses and suppressor cell activity is increasing. Between 12 and 14 days of infection, the T-helper cell response to T. cruzi, as measured by the antibody response to hapten-T. cruzi in vitro, is suddenly and dramatically regulated. As reported previously, plastic and G-10 adherent cells appear to be responsible for the regulation of antibody responses to heterologous antigen during T. cruzi infection. These adherent suppressor cells are also responsible for the suppression of antibody responses to hapten-T. cruzi following the first 2 weeks of infection. Suppressor cells continue to regulate the parasite-specific response well into chronic infection even though the response to hapten-T. cruzi appears to return to normal levels. These results are the first to directly implicate nonspecific suppressor cells in the regulation of anti-T. cruzi humoral immune responses.
Acute Disease↗
Measurement of parasite-specific immune responses in vitro: evidence for suppression of the antibody response to Trypanosoma cruzi.
Using the Mishell and Dutton culture system, we have developed an assay for eliciting and quantifying parasite-specific immune responses in vitro. The ability of spleen cells from noninfected and Trypanosoma cruzi-infected mice to respond to parasite-associated antigens was assessed by examining the primary plaque-forming cell response to trinitrophenylated T. cruzi (TNP-TC). The response to TNP-TC of both normal, noninfected, unprimed mice and mice infected with T. cruzi is T cell dependent and appeared to involve recognition of parasite antigens by T. cruzi-specific T cells. In most experiments, spleen cells from infected mice respond to TNP-TC at levels equal to, or below, that of spleen cells from normal mice. This near "normal" response is in apparent contrast to the suppressed response of spleen cells from infected mice to another antigen (sheep red blood cells) or TNP on a different carrier (TNP-chicken erythrocytes). Demonstration that the response of spleen cells from infected mice to TNP-TC can be potentiated by addition of interleukin 2-containing supernatants or by depletion of plastic and Sephadex G-10-adherent cells suggests that the mechanisms which control the response of infected mice to nonparasite antigens may also limit parasite-specific immune responses.
Animals↗
Diagnosis of Chagas' disease in humans using a biotin-3H-avidin radioimmunoassay.
A radioimmunoassay (RIA) originally designed to measure antibody responses to Trypanosoma cruzi in mice was adapted for use in the immunodiagnosis of Chagas' disease in humans. The assay utilizes biotinylated antibodies and 3H-avidin as the tracer molecules, and has proven to be both safe and sensitive. Results using the RIA and those from direct agglutination and indirect fluorescent antibody tests were comparable in most cases. Using the RIA, we were able to discriminate between mice infected with T. cruzi and Trypanosoma rangeli. Also, sera from Leishmania-infected individuals do not have detectable levels of antibodies capable of binding to T. cruzi. Intact, fixed epimastigotes of T. cruzi are used as the detecting antigen in the RIA and give results comparable to those obtained with intact trypomastigotes.
Adult↗
Loss of suppressor activity in the serum of mice infected with Trypanosoma cruzi.
Previous work from this laboratory documented the presence of a suppressor substance in the serum of mice infected with Trypanosoma cruzi. Currently, however, we have been unable to demonstrate the presence of this suppressor factor, although infected mice were still profoundly suppressed in their ability to respond to SRBC, and suppressor cells were present in the spleens of these animals. Strains of T. cruzi other than the one presently in use in our laboratory gave similar results. Several possible explanations for our inability to demonstrate the presence of the suppressor factor in sera are discussed.
Animals↗
Restoration of in vitro immune responses of spleen cells from mice infected with Trypanosoma cruzi by supernatants containing interleukin 2.
During the course of Trypanosoma cruzi infection, mice become suppressed in the ability to produce IL 2. Addition of supernatants from concanavalin A-stimulated normal spleen cells or supernatants from EL 4 cells enhanced the suppressed in vitro response of infected spleen cells to sheep erythrocytes. This enhancement was demonstrable at various times after infection and was shown not to be the result of Con A stimulation alone. Spleen cells from infected mice respond to IL 2-rich supernatants in Mishell-Dutton cultures in a dose-dependent fashion. Absorption of IL 2 activity from EL-4 supernatants by CTLL-2 cells results in a decrease in the enhancing effect of the supernatants on the in vitro antibody responses of infected spleen cells. Also, the reversal of suppression occurs only if IL 2-rich supernatants are added within 24 hr of the initiation of cultures. These results indicate that defective IL 2 production may play a role in the inability of mice infected with T. cruzi to respond appropriately to neoantigens.
Absorption↗
Measurement of parasite-specific antibody responses using a tritiated avidin-solid phase radioimmunoassay.
A solid phase radioimmunoassay (RIA) to measure parasite-specific antibody responses has been developed. The assay utilizes 3H-labeled avidin as the indicator molecule and represents a marked improvement in detection of specific antibodies to Trypanosoma cruzi over previously used RIA. Avidin is easily labeled to high efficiency (3.6 X 10(5) cpm/micrograms) under very mild conditions using N-succinimidyl-[2,3-3H]propionate, and the high efficiency of avidin-biotin binding coupled with the increased safety and longer half-life of the labeled reagent give this method advantage over currently used 125I-labeled Ab RIAs.
Animals↗