Trypanosoma musculi infection in B-cell-deficient mice

The course of infection with Trypanosoma musculi was assessed in mice deprived of B-lymphocytes by administration, from birth, of rabbit antiserum to mouse immunoglobulin M (IgM). Initial control of parasitemia leading to the first crisis and establishment of the plateau phase was unaffected by lack of B-lymphocyte function, although multiplicative forms persisted throughout the infection in anti-IgM-treated mice, instead of disappearing after the first crisis as in intact mice. Elimination of trypanosomes after the second crisis was not observed in anti-IgM-treated mice, which maintained high numbers of parasites in the blood and peritoneal cavity, resulting in some mortality. A temporary reduction in parasitemia was achieved in anti-IgM-treated mice by transfusion of immune plasma. Immunodepression, as measured by splenic mitogen responsiveness, and splenomegaly were both observed in anti-IgM-treated as well as in intact mice, indicating that these features of murine trypanosomiasis are independent of B-lymphocyte function. Since in T. musculi infection parasitemia can be controlled initially but not eliminated in mice lacking B-cell function, the only crucial protection provided by antibody would appear to be in curing the infection after the second crisis.     

Characterization of antibodies mediating protection and cure of Trypanosoma musculi infection in mice

Plasma samples were collected from mice infected with Trypanosoma musculi at different times postinfection and administered to naive recipient mice either before or during T. musculi infection. The protective and curative activities of these plasma samples were shown to increase as the time of collection postinfection increased; plasma collected at 14 days postinfection was partially protective and partially curative, whereas that collected at 28 days postinfection was completely protective and curative. The curative activity was labile to heat treatment (30 min at 56 degrees C), whereas the protective activity was heat stable. Additional kinetic parameters relating to the efficacy of protection were investigated. Evidence is presented that both activities are immunoglobulin in nature. Protein A-Sepharose chromatography indicated that the activities are associated with the immunoglobulin G2a or immunoglobulin G3 subclasses of immunoglobulin G. The curative antibody appears to be intrinsically heat labile, since heat treatment of a purified immunoglobulin preparation abolished the ability to cure. Studies on the mechanism of parasite elimination from blood suggest that the process not only requires antibody but is also complement dependent.     

Macrophage function during Trypanosoma musculi infection in mice.

Quantitative and functional changes in peritoneal macrophages from mice infected with Trypanosoma musculi were investigated. Increase in the number and size and in the protein content of peritoneal macrophages and the presence of parasites in the peritoneal activity were observed during the course of parasitemia. Induced releases of H2O2 by macrophages were increased during parasitemia, but H2O2 release returned to normal after infection. A correlation was also observed between parasitemia and 99mTc colloidal uptake by peritoneal macrophages. These changes in macrophage function may indicate an activation of these cells.     

Chronic infection with Trypanosoma musculi in congenitally athymic nude mice.

Trypanosoma musculi produces a chronic infection with a consistently elevated parasitemia in nude mice. Thymic reconstitution of nude mice restores immunity to the infection.     

Histopathological and immunocytochemical studies of Trypanosoma musculi infection of mice.

Mice infected with Trypanosoma musculi developed hyperplasia of the spleen, lymph nodes, and liver; in contrast, their thymuses displayed transient involution. All organs returned to normal in a month or less. There was modest anemia, lasting until the parasites were cleared from the bloodstream, followed by a rapid influx of erythrocytes into the blood and a subsequent return to normal erythrocyte numbers. During the first 2 weeks, trypanosomes and trypanosome-derived substances were found in the livers and, in moderate amounts, in the red pulp of the spleens; thereafter, trypanosomes and trypanosome-derived substances gradually decreased in these organs. The lymphoreticular hyperplasia involved a large increase of immunoglobulin G (IgG)-containing cells in the spleens and lymph nodes at 2 weeks of infection. Hyperplasia of immunoglobulin-producing cells correlated with elevation of serum immunoglobulins, especially IgG. Cells producing IgG in the spleens proliferated mainly around the central arterioles of the white pulp, i.e., in the T-cell-dependent areas. The decline of trypanosome-derived substances in the livers and spleens was associated with marked hyperplasia of IgG-containing cells in the spleens and lymph nodes. These results suggest that trypanosome-mediated depression of murine immune responses is attributable to proliferation and terminal differentiation of more-mature lymphoid cells and temporary inhibition of normal maturation of less-mature precursor cells.     

In vitro assay for curative activity in blood of mice infected with Trypanosoma musculi.

An in vitro assay for curative antibody present in plasma of mice cured of Trypanosoma musculi is described. The assay involves the addition of plasma to a sample of infected blood, followed by hourly monitoring of the parasite count therein. Immune plasma effected a significant reduction in parasite number, whereas normal mouse plasma did not. Heat treatment of immune plasma for 30 min at 56 degrees C abolished its ability to effect any reduction. The assay is simple, rapid, and economical and correlates well with results of all in vivo studies performed to date.     

Differences in resistance to Trypanosoma musculi infection among strains of inbred mice.

Inbred strains of mice were inoculated with Trypanosoma musculi, and the course of the ensuing parasitemia was followed. The mouse strains fell into three groups: those displaying high and moderate (fivefold less) parasitemia and C57BL/6 (B/6) mice which had exceptionally low infections. To gain insight concerning the mechanisms responsible for interstrain variations in infections, several types of experiments were performed. Comparison of the ability of spleen cells from the various strains to provide the growth-promoting substances required by T. musculi for growth in culture revealed that B/6 cells were deficient; this suggested one mechanism for regulating parasite infections. Exposure of C3H (high parasitemia) and B/6 mice to graded levels of ionizing radiation revealed that B/6 mice have much greater innate resistance to infection than do C3H mice. The effects of treating mice with silica dust or mercaptoethanol indicated that relative resistance to infection is not primarily associated with macrophage activity or limited growth-promoting substances. We conclude that variations in immune responsiveness to parasite antigens (probably not associated with the H-2 complex), possibly in concert with variations in a non-immunological mechanism, account for interstrain variation in resistance to T. musculi infections.     

Cure of Trypanosoma musculi infection by heat-labile activity in immune plasma

Passive transfer of plasma from a mouse cured of parasitemia to a Trypanosoma musculi-infected host rapidly eliminates parasitemia; this curative activity, presumably mediated by an immunoglobulin, is sensitive to heat treatment (56 degrees C, 30 min). In addition, pretreatment with immune plasma, even after heat treatment, prevents the development of a patent parasitemia in a naive host (protective activity).     

Lipopolysaccharide hyperreactivity of animals infected with Trypanosoma lewisi or Trypanosoma musculi.

Rats and mice infected with Trypanosoma lewisi and Trypanosoma musculi, respectively, showed hyperreactivity to lipopolysaccharide (LPS) from gram-negative bacteria. Fatal shock could be precipitated with a dose of LPS 100 to 1,000 times less in infected compared with noninfected animals. In trypanosome-infected rats and mice, extensive liver damage was evident after LPS challenge. These animals showed a pronounced hypoglycemia, marked elevation of blood aspartate transaminase level, and diffuse severe degeneration and total depletion of glycogen in hepatocytes. Only minor changes were observed in noninfected animals given the same dose of LPS. No mononuclear phagocytic cell infiltration was observed in the liver of infected animals. The most striking change was the great increase in size and the probable increase in phagocytic activity and number of sinusoidal Kupffer cells. We suggest that elevated Kupffer cell activity in trypanosome-infected animals may play a role in LPS-induced hepatotoxicity.     

Hepatic cells' mitotic and peritoneal macrophage phagocytic activities during Trypanosoma musculi infection in zinc-deficient mice.

The effects of zinc deficiency on hepatic cell mitotic and peritoneal macrophage phagocytic activities were examined in mice infected with Trypanosoma musculi or immunized with parasitic products. On a full-complement or pair-fed diet, infected and homogenate-inoculated mice showed mitotic activity gains of 7.9% to 80.3% and 6.5% to 99.0%, respectively. Infected and homogenate-inoculated mice on a zinc-deficient diet showed 21.8% to 95.7% and 17.2% to 65.2%, respectively, more dividing liver cells compared with controls. In comparison to controls, macrophages isolated from infected and homogenate-immunized mice on full-complement or pair-fed diets had phagocytized 13.4% to 31.4% more latex particles from day 50 to 80. In the zinc-deficient group, macrophages isolated from infected mice had significant numbers of phagocytized latex particles (1.8% to 38.5%) from day 20 to day 80 compared with controls. The homogenate-immunized mice also had increased numbers (18.6 to 30.8%) of phagocytized latex particles.     

The immunosuppressive and mitogenic effects of Trypanosoma musculi.

In CD-1 mice infected with Trypanosoma musculi, the production of IgM and IgG antibodies in response to sheep erythrocytes (SRBC) was significantly suppressed when mice were immunized with SRBC once high parasitaemias had developed. In infected mice which were not immunized with SRBC, background plaque-forming responses of spleen cells to SRBC were significantly higher than in uninfected, unimmunized mice. Factors derived from T. musculi were found to be mitogenic in vitro for spleen cells taken from CD-1 mice. The mitogenic response to these factors by spleen cells from athymic mice was highly significant, whereas the response of spleen cells taken from CD-1 mice which had been pre-treated with cyclophosphamide was much less, suggesting that the B cell was the major target of the trypanosome-derived mitogen. In this paper we discuss the possible relationship of T. musculi-induced mitogenesis to the immunosuppression and non-specific antibody formation associated with T. musculi infections.     

Natural killer activity in mice infected with Trypanosoma musculi.

Reproducing forms of Trypanosoma lewisi isolated from X-irradiated rats and adult forms from intact rats were not lysed by fresh mammalian sera. Treating parasites with trypsin or chymotrypsin, but not with neuraminidase, under conditions which did not impair viability rendered the parasites sensitive to lysis by rat, mouse, rabbit, and human sera. Serum from animal strains or humans genetically deficient in complement component C3, C5, or C6 did not lyse protease-treated parasites. The lytic factors in serum displayed the heat sensitivity and the Mg2+ requirement characteristic of the alternate complement pathway. Lysis was resolved into two phases, Mg2+-dependent binding of serum factors to parasites and subsequent C5-dependent, Mg2+-independent lysis. Allowing protease-treated parasites to readsorb host proteins did not block lysis by serum. Protease-treated parasites regenerated components which prevented complement-mediated lysis during 2 h in culture at 37 degrees C. This regeneration was inhibited by cycloheximide but not by tunicamycin. Ten major components were resolved in radioautographs of sodium dodecyl sulfate-polyacrylamide gels of extracts of radioiodinated intact cells. Protease treatment before radioiodination reduced the amount of radioactivity associated with these components disproportionately. Components with apparent molecular weights of 102,000, 88,000, and 47,000 were strongly labeled in intact cells, poorly labeled after enzyme treatment, and again labeled in cells that were cultured at 37 degrees C after enzyme treatment. Cycloheximide blocked the reappearance of these components on cultured cells. The presence of these three components was therefore correlated with resistance to complement-mediated lysis.     

T lymphocytes and the transfer of immunity to Trypanosoma musculi in mice.

Trypanosoma musculi produces a resolving infection in mice and the immune response is thymus dependent. Spleen cells from immune and from uninfected mice were transferred to T cell-deprived mice and restored their ability to control the infection, the immune cells being effective most rapidly. Treatment of the cells in vitro with anti-theta serum did not impair their ability to restore immunocompetence and it is proposed that, though T-cell dependent, the immune response is effected by theta- cells.     

Changes in immunoglobulin levels in zinc-deficient mice infected with Trypanosoma musculi.

A metabolic imbalance technique was used to study the effects of zinc deficiency on immunoglobulin levels in mice infected with Trypanosoma musculi or immunized with parasite products. Zinc-deficient mice developed higher numbers of parasitemia earlier and exhibited prolonged infection. Irrespective of the diet, higher IgG1, IgG2b, and IgM levels, lower IgG2a and IgA levels, and uniform IgG3 levels were exhibited primarily by mice infected with T musculi and to a lesser extent by mice immunized with parasite products. Zinc-deficient mice showed smaller increases in IgG1 and IgM, but larger gains in IgG2b compared with mice on full-complement and pair-fed diets. However, IgG2a decreased significantly in zinc-deficient mice.     

Immune and nonimmune regulation of the population of Trypanosoma musculi in infected host mice.

A clear understanding of the population dynamics of trypanosome infections is lacking. In the case of murine Trypanosoma musculi infections, there are no answers to questions concerning (i) the nature of the prolonged plateau phase during which the number of parasites present in the host remains nearly constant (is it a static or dynamic steady state?); (ii) the origin of new parasites, if the plateau is a dynamic steady state, given the relatively early disappearance of generative forms from the bloodstream; and (iii) the role, if any, of a putative ablastin (reproduction-restricting antibody) in regulating the population dynamics of T. musculi infections. We describe here the results of studies of the number and distribution of mature and reproductive forms (RF) in the blood and peritoneal space of both immunocompetent and cyclophosphamide-treated mice throughout the course of infection. While the RF disappeared from the blood within a few days after parasite inoculation, a high fraction (20 to 30%) of the parasites in the peritoneal space were RF throughout the course of infection and, thus, represented a source of new parasites. If an ablastin is responsible for inhibiting RF in the blood, it appeared to have no effect on RF in the peritoneal space. The results of this investigation support the conclusion that the control of the dynamics of T. musculi infections is largely nonimmunological (until cure of the infection) and probably is exercised by the supply of nutrients and reproduction-inhibiting (nonimmunological) and maturation-promoting factors that affect the generative fraction of the population.     

The immunological response of CBA mice to Trypanosoma musculi. I. Initial control of the infection and the effect of T-cell deprivation

Trypanosoma musculi produced a self-limiting infection in CBA mice which was characterized by a phase of increasing parasitaemia, during which dividing forms of the parasite were present in the blood, and a more stable plateau phase, when only non-dividing `adult' parasites are seen. Blood parasitaemia then rapidly regressed and subsequently blood was non-infective on sub-inoculation. Infection of normal mice in this manner apparently conferred a strong and lasting immunity. Fluorescent antibody titres rose rapidly during infection and IgM, IgGl and IgG2 antibodies were synthesized simultaneously. Total immunoglobulin and IgG2 antibody titres fell following recovery from infection but relatively high and constant antibody levels were detectable for many months.     

Further characterization of the fate of human monoclonal antibodies in rhesus monkeys

We have shown previously that human monoclonal antibodies are not very immunogenic in rhesus monkeys, with only one monkey out of five mounting an anti-monoclonal antibody response. Two additional monkeys have been injected multiple times with much larger amounts of one human monoclonal antibody. No anti-antibody response has been detected in these monkeys. Structural changes that occur in the monoclonal antibodies over time in vivo have been investigated by Western Blots using anti-idiotypic antisera. Sodium dodecyl sulfate gel electrophoresis reveals that very little antibody has altered molecular weight. Isoelectric focusing patterns change more dramatically. Forms of the antibodies with lower isoelectric points appear in the serum. These forms have a similar in vivo half-life as the freshly prepared antibody. Very low pI forms of the monoclonal antibodies are not detected in the serum. Isoelectric focusing can be used to determine the in vivo or in vitro condition of a monoclonal antibody preparation. Finally, the monkey anti-human IgG that arose in the single monkey studied previously has been further characterized.