Activity of the alternative complement pathway during mycobacterial infections in inbred mice

The relationship between kinetic activity of the alternative pathway (AP) of complement and susceptibility to Mycobacterium bovis (BCG) and M. fortuitum was examined in inbred mice. After subcutaneous injection of BCG, the organisms were mostly contained by the draining lymph nodes, with minimal effects on spleen and no apparent relationship with serum AP. After intravenous injection of BCG or M. fortuitum, male mice, which had a more effective AP than female mice, showed lower spleen bacterial counts. AP kinetics became faster in mice with high spleen bacterial counts and slower in mice with low counts, suggesting that infection or inflammatory processes affected AP. These experiments suggest that if tuberculosis is confined to tissues and draining lymph nodes AP plays no part in pathogenesis or host resistance, but AP might reduce the infectivity of low numbers of organisms spreading by blood or lymph from a primary focus of infection.     

Susceptibility and resistance to Trypanosoma congolense infections

We have put emphasis on recent findings in experimental Trypanosoma congolense infections in highly susceptible BALB/c and relatively resistant C57Bl/6 mice. Based on various analyses, it has been shown that a major difference in resistance to T. congolense infections is expressed early in infection at the macrophage level. A novel plastic-adherent Thy1.2(+) suppressor lymphocyte, which in absolute synergy with a Thy 1.2(-) cell exerts its suppression via interleukin-10 and interferon-gamma opens up an exciting new field of research.     

Trypanosoma congolense: inheritance of susceptibility to infection in inbred strains of mice

Inbred strains of mice have shown marked differences in susceptibility to infection with Trypanosoma congolense, as judged by survival and levels of parasitemia. The underlying genetic basis of the susceptibility was examined with F1 hybrids and backcrosses derived from mouse strains of high and low susceptibility. The influence of H-2 haplotype on susceptibility was studied using H-2 congenic resistant strains of mice. F1 hybrids between the most susceptible strain (A/J) and the least susceptible strain (C57Bl/6) showed similar survival to that of the C57Bl/6 parent. This was reflected in a similar undulating pattern of parasitemia, although the level of parasitemia was consistently higher in the F1 hybrids than in the C57Bl/6. Backcrosses of the F1 hybrids with C57Bl/6 also had a similar pattern of parasitemia although there was a greater scatter in survival times so that a few animals survived longer than either of the parental strains. Backcrosses of F1 hybrids with A/J showed a range of survival times; approximately 25% of these animals died during the period when the A/J mice died, approximately 25% had a similar survival to that of C57Bl/6, while the remaining animals showed an intermediate duration of survival. All these backcrosses had a high initial peak of parasitemia; in about 70% of the mice the early parasitemia showed a distinct undulating pattern. F1 hybrids of A/J and C57Bl/6 with C3H/He mice, which are known to be of intermediate susceptibility, were also examined. The degree of dominance for low susceptibility was much less pronounced in these hybrid combinations than in the A/J × C57Bl/6 hybrids. The H-2 congenic resistant strains, all of which were on a C57Bl/10 genetic background, showed a similar pattern of parasitemia and survival. However, although the majority of all these strains survived for more than 100 days, there was a significant difference in survival between the C57Bl/10 mice and the H-2 congenic resistant strains. It was concluded that susceptibility of mice to T. congolense infection is likely to be under complex genetic control and that, at least in C57Bl/mice, H-2 haplotype has little influence on susceptibility.     

Non-specific induction of increased resistance in mice to Trypanosoma congolense and Trypanosoma brucei by immunostimulants.

Administration of the immunostimulants Corynebacterium parvum, Bacillus Calmette-Guérin (BCG) or Bordetella pertussis prior to, or at the same time as, challenge with Trypanosoma congolense significantly increased survival times in mice, both of trypano-susceptible (A/J) and trypano-resistant (C57Bl) strains. The increased survival time was associated with significant alterations in parasitaemia, which included lengthening of the pre-patent period, a delay in the time taken to reach the first peak of parasitaemia and a reduction in the level of parasitaemia. Similar results were obtained when these strains of mice were challenged with Trypanosoma brucei following pre-treatment with C. parvum. Thus, by the use of immunostimulants it was possible to reduce the susceptibility of mice to trypanosomiasis and the hope is that this can also be achieved with domestic livestock.     

Mechanism of resistance to lysis by the alternative complement pathway in Trypanosoma cruzi trypomastigotes: effect of specific monoclonal antibody

Trypanosoma cruzi G strain epimastigotes were lysed by normal human serum (NHS) through activation of the alternative complement pathway (ACP), whereas metacyclic trypomastigotes were resistant to lysis. Epimastigotes and metacyclics with equivalent amounts of C3b deposited on their surface bound factor B with similar affinities. In contrast, factor H bound with higher affinity to metacyclics than to epimastigotes. Both T. cruzi forms with bound C3b were extensively (60 to 80%) lysed after formation of surface C3-convertase and the addition of a C3-C9 complement source. In the presence of factors H and I, or incubation with NHS with EDTA, the percentage of lysis of metacyclics decreased faster than that of epimastigotes with increasing incubation times. These data suggest, as a possible mechanism of resistance to lysis in metacyclic trypomastigotes, the higher binding affinity of factor H to C3b and the inactivation of the latter by serum regulatory proteins. Metacyclics were lysed by NHS, through ACP, in the presence of human immune serum to T. cruzi or anti-T. cruzi monoclonal antibody, but not with the Fab fragment of the latter, which recognizes a 90,000 m.w. antigen from T. cruzi metacyclics. Protection of parasite-bound C3b from serum control proteins was observed when parasites were incubated, before C3 deposition, with the lytic monoclonal antibody but not with its Fab fragment or a nonrelated IgG control. When C3b was deposited on metacyclics before antibody binding, C3b inactivation occurred. In the lysis of metacyclics, through ACP activation, binding of antibody apparently creates new acceptor sites which prevent the activity of serum regulatory proteins.     

Eosinophil granule cationic proteins regulate complement. I. Activity on the alternative pathway.

Eosinophil granules contain several cationic proteins that mediate tissue damage in allergic disease. The present study examined the capacity and mechanisms by which these cationic proteins regulate activity of the alternative pathway of C. Eosinophil peroxidase and eosinophil cationic protein inhibited formation of cell-bound alternative pathway C3 convertase, causing 50% inhibition of lysis at about 0.19 and 0.75 microgram/10(7) cellular intermediates, respectively. Major basic protein inhibited alternative pathway C3 activity by only 19% at 1.5 micrograms/10(7) cellular intermediates. Eosinophil-derived neurotoxin had no activity on the alternative pathway. The eosinophil granule proteins were examined for the mechanism by which they inhibited alternative pathway activity. Eosinophil peroxidase and major basic protein inhibited fluid phase factor B consumption in a reaction mixture that also contained factors D and C3b, eosinophil-derived neurotoxin had no activity on factor B consumption, and eosinophil cationic protein consumed factor B in the absence of C3b and factor D. Both eosinophil cationic protein and eosinophil peroxidase enhanced the decay of preformed alternative pathway convertase. Lysis of EAC4b,3b cellular intermediates formed to contain a low surface amount of C3b was more inhibited than was lysis of cells formed with a standard amount of C3b on the surface. This suggests that these eosinophil proteins acted predominantly on C3b to regulate alternative pathway activity. We also found that none of the eosinophil granule cationic proteins had any effect on later events after the formation of the C3 convertase. We conclude that although eosinophil-derived neurotoxin (isoelectric pH value (pI) = 8.9) does not regulate alternative pathway activity, the more highly charged eosinophil granule cationic proteins--major basic protein (pI = 10.9), eosinophil cationic protein (pI = 10.8), and eosinophil peroxidase (pI = 10.8)--do share the capacity to regulate C activity and may exert this activity in vivo.     

Mechanisms of self-cure from Trypanosoma congolense infection in mice

The mechanism(s) of resistance to African trypanosomiasis caused by Trypanosoma congolense was investigated by using the Dinderesso/80/CRTA/3 isolate to which C57B1/6 are resistant (low parasitemia and self-cure) and BALB/c sensitive (high parasitemia and death). The resistance of C57B1/6 is similar to that found in some natural hosts of African trypanosomes such as certain indigenous West African cattle and wild Bovidae. The antibody response to epitopes exposed on the variant surface glycoprotein of a clone obtained from the Dinderesso/80/CRTA/3 isolate was measured by a complement-mediated lysis assay in C57B1/6 and BALB/c. After infections with 10(4), 10(5), or 10(7) motile organisms, antibody appeared in C57B1/6 4 to 8 days earlier than in BALB/c. Peak antibody titers were similar in both strains but were reached about 4 days earlier in C57B1/6. In this strain, antibody appeared during and controlled the first wave of parasitemia, whereas in BALB/c, parasitemia reached a plateau above 10(8) organisms per ml before antibody could be detected, and at this time the animals were dying. At peak antibody response, the proportion of immunoglobulin (Ig) M and IgG antibody was the same in both strains. The antibody response had the same kinetics in C57B1/6 and BALB/c after injection of 10(4), 10(5), and 10(7) lethally irradiated but intact parasites, but the peak titers were 10(3) to 10(4) times lower than after live challenge. The response to nonirradiated trypanosomes appeared to be T cell independent, because the antibody titers were the same in congenitally athymic nu/nu and normal C57B1/6, and no evidence for the induction of T cell activity could be demonstrated in the infected nude mice. A role for trypanolytic serum factors in resistance could not be demonstrated. The extent of immunosuppression after infection with nonirradiated organisms was compared in the two strains by measuring the in vitro response of their splenic lymphocytes to concanavalin A, pokeweed mitogen, and allogeneic cells and their ability to mount an in vivo response to an unrelated trypanosome challenge. Both strains were partially immunosuppressed during rising parasitemia, but as C57B1/6 controlled parasitemia, immunosuppression was gradually reversed, whereas in BALB/c it became worse. Several explanations might account for the resistance of C57B1/6 to the Dinderesso/80/CRTA/3 isolate of T. congolense. It appears that an early immune response is a decisive factor in this resistance.     

Cryptosporidium infections in inbred strains of mice.

Cryptosporidium, a protozoan parasite of man and animals, is an important etiological agent of diarrhea throughout the world, particularly in children and immunocompromised individuals such as AIDS patients. Unfortunately, because of the lack of both in vivo laboratory models and reliable in vitro parasite culture systems, virtually nothing is known about the immunological events occurring during disease. In order to identify reliable animal models for infection, we studied C. parvum infections in 19 different strains of mice representing 12 H-2 haplotypes: A/J, AKR/J, B10.D2/J, B10.M/J, C3H/HeJ, C57BL/65, C57BL/6J-bgJ, CBA/NJ, DBA/1J, DBA/2J, HRS/J, HTG/J, NZB/B1NJ, NZW/J, P/J, RIII/J, SJL/J, SWR/J, and WB/ReJ, and in one gerbil: Meriones unguiculatus. Fecal samples and histological sections of the intestine taken on day 7 post-Cryptosporidium inoculation indicated that only the beige mouse (C57BL/6J-bgJ) harbored significant numbers of parasites compared to the other strains. The numbers of parasites harbored in these NK cell-deficient beige mice were, however, considerably lower than those seen in neonatal mice. Adult inbred mouse strains susceptible to Cryptosporidium infections are discussed.     

Trypanosoma cruzi: role of the immune response in the natural resistance of inbred strains of mice.

Nine inbred strains of mice were challenged with 10⁴ or 10⁵ trypomastigotes of the Brazil strain of Trypanosoma cruzi. A spectrum of resistance was evident ranging from highly susceptible strains, e.g., C3H, which developed high parasitemias and died within 3 to 4 weeks, to resistant strains, e.g., C57BL/10, which developed low parasitemias and survived. Impairment of the immune system in resistant C57BL/10 mice by X-irradiation, splenectomy, or treatment with silica led to high, often fatal parasitemias. Athymic nude mice, in particular, attained exceptionally high parasitemias before dying. The immune response appears to be necessary for survival and to play a role in the natural resistance of some mouse strains by effectively eliminating parasites and minimizing parasitemia. Using congenic strains of mice, it was shown that the principal genetic determinant of resistance is not associated with their H-2 haplotype.     

Selective pressure can influence the resistance of Trypanosoma congolense to normal human serum

Resistance and sensitivity to normal human serum (NHS) of Trypanosoma congolense, a parasite believed to cause disease in animals only, were investigated in vivo as well as in vitro. Our results indicate that like Trypanosoma brucei, T. congolense can be grouped into three different phenotypes according to its resistance to NHS. Some strains are completely resistant to NHS, like Trypanosoma brucei gambiense and the resistant form of Trypanosoma brucei rhodesiense. Other strains show a very low degree of resistance comparable to the sensitive form of T. b. rhodesiense, and some are completely sensitive to NHS. Continuous passaging in mice in the presence or absence of NHS shows that the resistance and sensitivity of T. congolense can be reversed like in T. b. rhodesiense. Our data suggest that T. congolense might be able to infect man in regions where animals may serve as reservoirs for the infection.     

Trypanosoma congolense: drug resistance during cyclical transmissions in tsetse flies and syringe passages in mice

A drug-resistant Trypanosoma congolense strain with predetermined curative doses (CD50 and CD90) of samorin at 13.9 +/- 1.02 and 20.3 +/- 1.13 mg/kg body weight, respectively, was cyclically transmitted through tsetse flies and by syringe passages in mice in the absence of drug pressure. The changing levels of drug sensitivity were determined after every 3rd cyclic and 5th syringe passage intervals. It was noted that when the strain was maintained in tsetse flies through 12 cyclical transmissions, the CD50 and CD90 dropped slightly from 13.9 to 11.9 +/- 1.06 and from 20.3 to 18.0 +/- 1.08 mg/kg body weight, respectively. This decrease in the level of resistance was not significant (P greater than 0.05). However, when the trypanosomes were maintained by syringe passages in mice, there was a significant reduction (P less than 0.05) in the degree of resistance (CD50 from 13.9 to 11.4 +/- 1.07 and CD90 from 20.3 to 16.7 +/- 1.16 mg/kg), by the 15th syringe passage.     

In vitro binding of Trypanosoma congolense to erythrocytes.

Trypanosoma congolense Broden, an intravascular parasite, binds to vessel walls and erythrocytes of infected hosts. In an attempt to characterize T. congolense adhesion to host cells, an in vitro assay was devised. It was shown in the in vitro experiments that T. congolense binds to bovine, sheep, and goat erythrocytes, but not always to erythrocytes of rats, mice, rabbits, horses or humans. Only the anterior part of live trypanosomes adheres to erythrocytes, and the attachment site on the trypanosomes is destroyed by trypsin and chymotrypsin-trypanosomes did not adhere to bovine erythrocytes that had been incubated with neuraminidase, sodium periodate and poly-L-lysine. The foregoing experiments suggest that the surface of T. congolense contains a protein-associated site which binds to sialic acid of some host cells. This surface site is most likely responsible for attachment to blood vessels in vivo.     

Elevation of morphological abnormalities of spermatozoa in the semen of Zebu bulls consequent to Trypanosoma vivax and Trypanosoma congolense infections

Twenty-four Zebu bulls were used in a 12-wk long study. Eight bulls were infected with T. vivax , eight others with T. congolense and eight bulls served as controls. All the infected bulls developed chronic trypanosomiasis. Mean percentage base-line values prior to infection for acrosomal, sperm-head, detached heads, proximal cytoplasmic droplets, distal cytoplasmic droplets, sperm-tail, midpiece and total sperm morphological abnormalities ranged between 0.1+/-0.1 for acrosomal and 8.7+/-3.4 for total morphological abnormalities in the semen of the bulls. These values were very low and within the range of those for fertile bulls. Following infection, there was a progressive increase in the mean values of all the abnormalities. Peak percentage mean values recorded for total sperm morphological abnormalities in the course of the investigation in the bulls infected with T. vivax and T. congolense and in the controls were 95+/-7.2, 100+/-0 and 7.9+/-5.0, respectively. Mean percentage values throughout the duration of the investigation for control bulls were low and within the normal range for fertile bulls. These values differed (P<0.001) from the elevated values of the infected bulls. The results indicate that trypanosomiasis due to either T. vivax or T. congolense infections can cause a marked increase in morphological abnormalities of spermatozoa which can, in turn reduce the fertility of breeding bulls.     

Innate resistance to Trypanosoma congolense infections: differential production of nitric oxide by macrophages from susceptible BALB/c and resistant C57Bl/6 mice.

BALB/c and C57B1/6 mice differ in resistance to Trypanosoma congolense infections. Evidence suggests that macrophages play a central role in the resistance to trypanosomiasis. Nitric oxide (NO) produced by macrophages in response to various stimuli or pathogens is one of the important arms of nonspecific immunity. We investigated the production of NO by the peritoneal macrophages and bone marrow-derived macrophages (BMDM) from trypanosome-resistant C57B1/6 and -susceptible BALB/c mice following stimulation with T. congolense whole cell extract (WCE) or following phagocytosis of T. congolense mediated by anti-variant surface glycoprotein (VSG) antibodies of IgM or IgG2a isotype. C57B1/6 peritoneal macrophages as well as BMDM produced significantly more NO than similar BALB/c macrophages in response to T. congolense lysate and IFN-gamma. In both BALB/c and C57B1/6 BMDM cultures, phagocytosis of T. congolense mediated by anti-VSG antibodies of IgG2a isotype in the presence of IFNgamma induced two- to ninefold more NO than phagocytosis mediated by IgM antibodies and C57B1/6 BMDM produced significantly higher amounts of NO than BALB/c BMDM under these conditions. NO produced by BMDM was found to exert trypanostatic effect on T. congolense in vitro, but was not found to influence the in vivo infectivity of these treated parasites under the conditions used in this study.     

Trypanosoma cruzi: early parasite proliferation and host resistance in inbred strains of mice

The extent of parasite proliferation following completion of the first cycle of intracellular replication was significantly higher in CD-1 nu/nu mice and in irradiated mice compared to other, including highly susceptible, mouse strains. A control of parasite proliferation thus occurs in normal mice as early as the first cycle of intracellular replication. The thymus dependency and radiation sensitivity of the early control of proliferation of Trypanosoma cruzi suggest that an immune response to the parasite is involved in the early control of proliferation. The BXH-2 recombinant inbred strain demonstrated an inability to control early proliferation and, 4-5 days after infection, had parasitemias several times higher than those observed in susceptible mouse strains. The BXH-2 strain appears to lack the early control mechanism. When the extent of proliferation of T. cruzi at completion of the first cycle of intracellular replication was compared in inbred strains of mice having varying levels of resistance to the parasite, the extent of proliferation correlated with host resistance, being lowest in the most resistant strains (C57BL/6, SJL) and highest in the most susceptible strains (C3H, A). It is suggested that the mechanism(s) controlling early parasite proliferation may be of primary importance as the basis for host resistance.     

Activation of complement via the alternative pathway

Activation of complement via the alternative pathway represents one means of natural resistance to infection because it is capable of neutralizing a wide variety of potential pathogens in the total absence of antibody. The pathway involves six serum proteins and possesses a unique amplification system capable of depositing large numbers of C3b molecules on the surfaces of activating particles. C3b deposition enhances phagocytosis and results in activation of the membrane attack pathway of complement. C3b attachment is covalent, arising from a reaction between an intramolecular thiolester bond in nascent C3b and nucleophiles such as hydroxyl groups on surface carbohydrates. The reactions that initiate C3b attachment are not specific interactions like those initiating other biological cascade systems, but involve slow, spontaneous hydrolysis of the thiolester bond in C3 and subsequent random deposition of C3b onto all nearby surfaces. Once bound, C3b is capable of discriminating between host-derived cells and activating particles. Recognition is evidenced by a lower affinity between activator-bound C3b and the complement control protein factor H. Measurements of the association constant between unbound, soluble C3b and factor H suggest that activator-bound C3b recognizes structures on activators that inhibit factor H binding.     

Trypanosoma lewisi: restriction of alternative complement pathway C3/C5 convertase activity

The rat parasite Trypanosoma lewisi was incubated in vitro with rat or human serum, washed, and extracted in detergent. Extracts were fractionated by electrophoresis in denaturing gels, transferred to nitrocellulose, allowed to renature, then immunoblotted with polyclonal antibodies to rat complement component C3 and human complement components C3, C5, and factor B. Molecules that reacted with these antibodies were detected in the extracts. Fragments of rat C3 were detected in extracts of parasites that had not been exposed to serum in vitro. Additional complement deposition occurred during in vitro incubations; human complement components deposited in vitro could be distinguished from rat components deposited in vivo. Complement deposition in vitro required magnesium ions and did not occur when heat inactivated serum was used. Components reacting with antibodies to human C3 included a group of bands with molecular weights higher than C3 alpha or beta chains. Blotting with affinity purified, chain specific antibodies demonstrated that a 68 kDa component on parasites is C3 beta and that a 44 kDa molecule is derived from C3 alpha. A 73 kDa component that was difficult to resolve from C3 beta is probably also a C3 alpha fragment. This suggests that an inactive iC3b-like molecule is present on parasites. Kinetic studies showed that cleavage of C3 alpha is rapid and that the amount of C3 alpha fragments and C3 beta on intact parasites reached a steady state after 15 min. When parasites were trypsinized prior to incubation in C5 or C6 deficient serum, the rate and extent of C3 and C5 deposition increased. Unprocessed C3 alpha' and C5 alpha' chains were detected. Trypsinized parasites were lysed by the alternative complement pathway in normal serum. Intact parasites could be lysed by complement in the presence of antibody. The data support our previous suggestion that trypsin sensitive surface proteins on intact T. lewisi limit alternative pathway activity by restricting C3/C5 convertase activity.     

Trypanosoma congolense bloodstream forms evade complement lysis in vitro by shedding of immune complexes

In the presence of antibodies against the variant surface glycoprotein (VSG) and guinea pig complement, Trypanosoma congolense bloodstream forms were lysed. Parasites, which had been preincubated with antibodies at 37 degrees C before addition of complement, escaped from complement lysis in a time- and temperature-dependent process. Preincubation caused removal of the antibodies from the cell surface by formation of filopodia and accumulation of the immune complexes between aggregated cells. Addition of secondary antibodies or of complement component C1q did not enhance this effect. In order to eliminate effects due to cell aggregation, single living trypanosomes, which had been immobilized by attachment to formvar-coated glass slides, were incubated under equivalent conditions. Immunofluorescence showed that in these experiments, anti-VSG antibodies were neither capped nor shed from the surface unless coincubation with secondary antibodies or C1q was performed. Fixation of the cells after incubation with anti-VSG prevented patching and capping of the antibodies. Removal of immune complexes apparently required no secondary cross-linker: removal from the surface of T. congolense obviously occurred during cell aggregation. This mechanism could therefore be of significance also in vivo.