F. Kierszenbaum

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.

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.

Immunological properties of a trypanosomal lipopolysaccharide and its effects on the infection with Trypanosoma (Schizotrypanum) cruzi.

A lipopolysaccharide, TCLP, was obtained from culture form of T.(S) cruzi by extraction with phenol-water at 68° C. Unlike the lipopolysac-charides obtained from Gramnegative bacteria, TCLP lacked adjuvant action on antibody formation, did not react with complement and was incapable of sensitizing rabbits for or eliciting the Shwartzman phenomenon, Mice given TCLP showed an initial short period of increased reticuloen-dothelial clearance of colloidal carbon, followed by a 2-day period of significantly depressed clearance.TCLP had an adverse effect on mice infected with virulent forms of T. (S.) cruzi. Peak parasitemias were significantly increased over those of control animals (given only the parasite). Early mortality also ensued. These effects were found to be independent of the sequence of administration of TCLP and the parasite.Prior immunization with TCLP did not prevent either the disease or death in mice challenged with virulent T.(S.) cruzi

Endotoxin-induced protection against experimental Junín virus infection

The effects of the administration of a bacterial endotoxin on the course of the Junín viral infection in the mouse were studied. The results showed that maximal protection against the disease was produced when both endotoxin and the virus were given on the day of birth. If endotoxin was administered first (on the day of birth) and was followed by the virus 24 hours later, a lesser degree of protection was obtained, but with a 48-hours interval there was no protection. If the virus was administered first (on the day of birth), followed by endotoxin 1 to 8 days later, the mortality was the same as with virus alone. If the interval between the administration of the virus and the endotoxin was 9 to 10 days, a very high mortality was observed in 24 hours, possibly due to an increased sensitivity of the infected mouse to the lethal effect of endotoxin. Viral titrations showed that the brains of endotoxin-protected mice contained enough virus to kill normal mice, indicating that the mere presence of the virus is not sufficient to produce death in the infected animals. Circulating antibodies to the virus were detectable 28 days after infection, i.e., about 13 days after the signs of the disease had disappeared. This fact and the failure of endotoxin-protected mice to survive a new challenge with Junín virus suggest that the protective phenomenon is not antibody-related. Since endotoxin fails to protect when given to mice either simultaneously with the virus 2 days after birth or one or more days after infection on the day of birth, it is very unlikely that an enhanced release of interferon might be responsible for the observed protection. Thymic function and its possible alteration by endotoxin is discussed.