Ablastin: Control of Trypanosoma musculi infections in mice

Trypanosoma musculi infections in CBA mice consist of a phase of increasing parasitemia during which dividing forms of the parasite are present in the blood, followed by a period when only nondividing trypomastigotes are seen. A second crisis terminates the blood infection and leaves the host immune, but small numbers of trypanosomes, including multiplicative forms, persist in the kidneys for many months. Studies were made involving infections in T-lymphocyte deprived mice, the effects of passive transfer of serum and cells, measurement of DNA synthesis by the parasite, serological responses, and in vitro effects of serum on the trypanosomes. These indicated that the initial check on the increase in blood parasitemia is due in part to two humoral factors. One of these has a trypanocidal effect (this is thought to be an IgM antibody) while the other, which may be an IgG antibody, is the ablastin that inhibits further reproduction by the parasite. Both trypanocidal and ablastic effects were demonstrable in the serum of immune mice yet the parasite was still able to survive and multiply in the kidneys.     

A culture and biochemical assay system for Trypanosoma lewisi

Trypanosoma lewisi was cultivated as forms which appeared to be physiologically similar to those found in vivo. The medium consisted of 1.0 g peptone, 1.0 g glucose, 10 ml rat serum, 10,000 units penicillin G, 10,000 μg streptomycin and 90 ml Hank's Balanced Salt Solution. It was supplemented with 8.0 × 10⁸ rat erythrocytes per milliliter. In the complete medium trypanosomes multiplied for 48–72 hr. Cultured forms were lethal to newborn rats and infective to adults.Adsorbed early immune serum inhibited the growth of the trypanosomes in vitro and the percentage of reproductives declined from 66 to 45%. The cultured trypanosomes were also susceptible to both trypanocidal antibodies.     

Trypanosoma lewisi: accumulation of antigen-specific host IgG as a component of the surface coat during the course of infection in the rat.

Naturally acquired host IgG, adsorbed to the surface of Trypanosoma lewisi during the course of infection in the rat, was labeled with fluorescein-conjugated rabbit IgG, or Fab fragments of this IgG, directed against rat IgG. The intensity of fluorescent labeling increases with time, concomitant with the increase in anti-T. lewisi activity of host plasma. Trypanosomes harvested from immunosuppressed hosts lack detectable surface IgG. Trypanosomes having little or no detectable surface IgG (harvested from immunosuppressed hosts or early in the infection from immunocompetent hosts) can adsorb IgG from serum with ablastic activity only (obtained from other infected rats between the first and second crises and adsorbed to remove trypanocidal antibodies), but not from normal serum. Therefore, the absence of detectable surface IgG on such cells is not caused by the parasites' inability to adsorb host IgG, but rather results from the immune state of the host. Hence surface IgG on T. lewisi is specific antibody. Host albumin is nonspecifically adsorbed, in contrast to IgG. Trypanosomes from immuno-suppressed and immunocompetent rats were positive and visually indistinguishable from each other when labeled with anti-rat albumin, and were equally agglutinable with anti-rat albumin serum.     

Trypanosoma lewisi: alterations in membrane function in the rat.

DEAE-cellulose-purified Trypanosoma lewisi from 4-day (dividing trypanosomes) and 7-day (non-dividing trypanosomes) infections in rats were compared for initial uptake of glucose, leucine, and potassium. Glucose entered the parasitic cells by mediated (saturable) processes, whereas leucine and K⁺ entered by mediated processes and diffusion. Glucose entry was significantly elevated in 4-day cells (V_max 4.00 ± 1.02 nmoles/ 1 × 10⁸ cells/min) with respect to 7-day cells (V_max 1.83 ± 0.62 nmoles 1 × 10⁸ cells/min). Likewise, the affinity of the glucose carrier was significantly greater in 4-day cells (K_m = 0.30 ± 0.02 mM) than in 7-day cells (K_m = 0.59 ± 0.11 mM). When leucine and K⁺ transport were compared in 4- and 7-day populations, significant elevations in the rate of entry (V_max) of both substrates were observed for 4-day cells; K_m values for leucine and K⁺ were not altered by the stage of infection. For leucine, the V_max and K_m for 4-day cells were 2.40 ± 0.50 nmoles/1 × 10⁸ cells/30 sec and 78 ± 7 μM, respectively; corresponding values in 7-day cells were 1.06 ± 0.02 nmoles/1 × 10⁸ cells/30 sec and 66 ± 11 μM. For K⁺, the V_max and K_m for 4-day cells were 15.97 ± 0.38 nmoles/1 × 10⁸ cells/min and 1.2 mM, respectively; corresponding values in 7-day cells were 4.76 ± 1.82 nmoles/1 × 10⁸ cells/min and 1.05 mM. The observed increase in the rate of K⁺ entry into 4-day cells was attributable to enhanced influx; no significant difference in the rate of K⁺ efflux was noted when 4- and 7-day cells were compared ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of K⁺ leak for 4- and 7-day cells were 68.1 ± 9.3 and 67.9 ± 15.2 min, respectively). Potassium influx was ouabain insensitive. Membrane function in 7-day cells was not uniformly inhibited. No significant difference in the activity of the membrane-bound enzyme, 5′-nucleotidase, was observed when 4- and 7-day cells were compared.     

Trypanosoma lewisi: antibody classes which mediate precipitation, agglutination, and the inhibition of reproduction

Sera from Trypanosoma lewisi-infected and uninfected rats were applied to Protein A-Sepharose CL-4B columns. The absorbed fractions of antisera which contained only IgG molecules were reacted in microimmunodiffusion analyses with the exoantigens of T. lewisi in plasma collected from irradiated infected rats, and formed one precipitin line. These sera were also applied to T. lewisi extract immunoabsorbent columns and bound proteins were eluted and analyzed by immunodiffusion against antisera specific for rat immunoglobulins. IgG₁, IgG_2a, IgG_2b, IgG_2c, and IgM were absorbed by the immuno-absorbent columns. Absorption of the rat antisera with anti-rat IgG or anti-rat IgM removed one of the two precipitin lines against extracts prepared from parasites collected from irradiated infected animals. The absorbed IgG fractions and nonabsorbed fractions of antisera which were collected after Protein A-Sepharose CL-4B column chromatography agglutinated trypanosomes. After treatment of antisera with 2-mercaptoethanol, the agglutinin titers were lower than those of the control antisera suggesting both IgG and IgM are involved in the agglutination. The ablastic activity of the fractions eluted from Protein A-Sepharose CL-4B Chromatographic columns was assayed in cultures of bloodstream forms ofT. lewisi. Ablastic activity of proteins of antisera absorbed by the columns was demonstrated indicating they belonged to the IgG class of antibodies.     

Immunosuppression and ablastin

Ablastin formed by animals in response to infections by rodent trypanosomes possesses the characteristics of an antibody. Partial resistance to Trypanosoma lewisi is demonstrable in animals previously injected with live Trypanosoma musculi. Antisera from T. musculi infected mice do not inhibit reproduction by T. lewisi bloodstream forms in vitro as efficiently as homologous antisera collected at similar times during infections, indicating a degree of specificity. Ablastin activity in antisera is not altered by treatment with 2-mercaptoethanol or by heatng at 60 °C for 3 hr. Sephadex G-200 gel filtration of early and late antisera from T. lewisi infected rats and assays with bloodstream forms cultured at 37 °C detect ablastin activity in the second major fraction eluted from the columns. Ablastin appears to be an antibody of the immunoglobulin G species.Immunosuppressant procedures utilized in studies of the host responses to rodent trypanosomes are reviewed and include: chemical agents, irradiation, splenectomy, reticuloendothelia blockade and thymectomy, and treatment with antilymphocyte and antithymocyte sera. Evaluation of the results of the application of these procedures to rodent parasite systems indicates ablastin is an antibody and supports the concept that the inhibition of trypanosome reproduction is separate and distinct from the first trypanocidal event responsible for the decreasing parasitemias observed during the infections. Recent studies concur and suggest that the first crisis in the infections is mediated by the combined actions of a thymus-dependent ablastin and a thymus-independent trypanocidal antibody.     

Trypanosoma lewisi: Avidity and adsorbability of ablastin,the rat antibody inhibiting parasite reproduction

The relation of naturally acquired host IgG in the surface coat of bloodstream forms of Trypanosoma lewisi to ablastin was studied to determine whether, contrary to a long-held conclusion, the antibody is avid and adsorbable. It was found by immunofluorescence and agglutination tests with monospecific antisera to rat IgG that bloodstream forms collected from immunosuppressed hosts, in contrast to those from immunocompetent hosts, have little or no detectable surface IgO. Specificity of adsorption was also demonstrated in other immunofluorescence experiments in which bloodstream forms from immunosuppressed hosts adsorbed IgG from immune serum with ablastic activity only (previously adsorbed with trypanosomes from immunocompetent hosts to remove the trypanocidal antibodies), but did not adsorb IgG from normal rat serum. To determine whether this specific adsorption of IgG by the parasite could be correlated with a reduction in ablastic activity, immune sera were adsorbed with bloodstream forms from immunosuppressed hosts at packed cell/serum ratios of either 1.2 or 2.0, and the adsorbed sera were then tested for ablastic activity in vitro. With both cell/serum ratios, ablastic activity was reduced by 50%. In comparison, similar adsorptions of immune sera with trypanosomes from immunocompetent hosts resulted in reductions of ablastic activity of only about 9 and 27% with the low and high cell/serum ratios, respectively. It is concluded that the failure to effect significant adsorption of ablastin in earlier studies resulted from the use of ablastinsensitized trypanosomes from immunocompetent hosts.     

Ablastin: The phenomenon

A brief report is made of a conference held on the subject of ablastic immunity. Included are the background of the meeting, its organization, and its purpose, which was to summarize our knowledge of the subject and to consider future research plans. A list of some of the research problems discussed by the participants and possible experimental approaches for their resolution are presented. The report also serves as an introduction to a series of papers by the participants of the conference published concurrently.     

The ablastin phenomenon: inhibition of membrane function.

Autoradiography of Trypanosoma lewisi labeled in vivo with ³H-thymidine (³HTdR) shows that the shortest doubling time for labeled organisms is 8 hr in intact and immunosuppressed rats. The parasite doubling time increases progressively after the fourth day of infection to 12 hr in immunosuppressed rats and to 24 hr or more in intact rats. The number of days following infection during which the trypanosomes reproduce is prolonged in immunosuppressed rats. In vitro studies of ablastin using ³HTdR-labeled trypanosomes confirmed that cell reproduction halts in the presence of ablastin, but resumes when the parasites are removed from the antibody. Several lines of evidence have been obtained, indicating that the primary effects of ablastin may be on membrane function. Thus, the saturable component for glucose transport in reproducing and ablastin inhibited trypanosomes has an average K_m value of 2.8 × 10^?4M, but the average V_max values for glucose transport are reduced from 3.15 nmole/min/1.25 × 10⁷ reproducing parasites to an average of 1.8 nmole/min/1.25 × 10⁷ nonreproducing forms. Glucose transport is competitively inhibited by 2-deoxyd-glucose (2DOG). The exit and counterflow of ¹⁶C-2DOG from previously loaded trypanosomes is restricted in the presence of antiserum.     

Nutritional and immunological factors in the control of infections with Trypanosoma lewisi

This paper is an attempt to view the infection with T. lewisi as a balance of host nutritional factors to which the parasite is attuned and the host defensive system, which recognizes and attempts to subdue him. The host nutritional factors seem to bring parasite numbers to a high level operating through many log units of growth. The host defense system has far narrower limits and can be overwhelmed by much smaller modifications. An essential feature of the host defense is an exceedingly active lymphomyeloid interaction, so far visualized in the spleen. Here the very proximity of plasma cells and reticular cells affords great opportunities for direct transfer of immunoglobulins without evidence of these molecules as agglutinating antibodies in the blood. This mechanism of active trypanosome venting foci with selectivity for younger forms seems more satisfactory than proposing a unique and unusual antibody system.