Enumeration of Trypanosoma cruzi-specific antibody-secreting cells by solid-phase immunoenzymatic technique.

A specific and sensitive enzyme-linked immunoassay is applied for enumeration of cells secreting specific antibodies to Trypanosoma cruzi antigens. Spleen cells from immunized mice are incubated in antigen-coated plastic tissue culture plates. Individual antibody-producing cells secrete antibody which binds to the antigen at close vicinity of the cells. The areas of bound antibodies are demonstrated by an enzyme-linked antibody assay as dark round spots, which can be easily enumerated. This assay allows the enumeration of specific antibody-secreting cells to parasite antigens, overcoming the failures to develop conventional plaque-forming cells assays to complex antigens.

Cross-reactivity between Trypanosoma cruzi and insect trypanosomatids as a basis for the diagnosos of Chagas' disease.

Immunological cross-reactivity between Trypanosoma cruzi and insect trypanosomatids was demonstrated by immunofluorescence and confirmed by complement fixation, direct agglutination and cross-absorption experiments. As antigens, the following organisms were surveyed: Crithidia deanei, Crithidia fasciculata, Crithidia luciliae, Herpetomonas samuelpessoai, Herpetomonas megaseliae, Herpetomonas muscarum muscarum, Leptomonas seymouri and Blastocrithidia culicis. Sera from patients with Chagas' disease or sera from rabbits immunized against various trypanosomatids were used as sources of antibodies. The demonstration of cross-reactivity was followed by a survey of 500 human sera (from normal persons or Chagas' disease patients) by immunofluorescence using insect trypanosomatids (H. muscarum muscarum, C. fasciculata and L. seymouri) as antigens. With H. muscarum muscarum 98.7% coincident positive results and 100% of coincident negative results were obtained. These findings may validate the use of insect trypanosomatids as an alternative source of antigen in the serodiagnosis of Chagas' disease by indirect immunofluorescence.

Staphylococci adherence to trypanosomes exposed to immune sera as a method for the diagnosis of Chagas' disease.

A new method, the staphylococci adherence test (SAT), for the serological diagnosis of Chagas' disease is described; it is based on the affinity of staphylococcal Protein A for IgG globulins and uses epimastigotes of Trypanosoma cruzi which are fixed on to glass slides and incubated stepwise with probing sera and a Staphylococcus aureus A suspension. After Giemsa staining, epimastigote preparations which have been incubated with positive sera appear covered by cocci. Results using indirect immunofluorescence, complement fixation and SAT were in agreement in 98.37% of 860 human sera. A slightly modified SAT (SAT') may also be utilized for the diagnosis of acute Chagas' disease. The simplicity of the method may allow its adaptation for field work. Other possible uses were investigated.

Egg deposition is the major stimulus for the production of Th2 cytokines in murine schistosomiasis mansoni.

To characterize Th cell populations induced by helminth infection, spleen cells from mice infected with Schistosoma mansoni were stimulated with parasite (worm or egg Ag) or mitogen (Con A) and the supernatants assayed for the Th1-specific cytokines IFN-gamma and IL-2 and the Th2-specific cytokines IL-4 and IL-5. Th2 cytokine production was not detected in substantial quantity until the 6 to 8th wk of infection and after reaching peak levels at 8 to 12 wk declined slowly thereafter. The time courses of IL-4 and IL-5 production, whereas differing from each other, closely resembled corresponding published data on IgE and peripheral blood eosinophil levels during murine schistosome infection. In contrast, Th1 cytokine responses occurred only during the first 6 wk of infection and were virtually absent during the peak period of Th2 production. To assess the role of egg deposition in the observed pattern of Th response, cytokine production was assayed in mice carrying unisexual schistosome infections in which parasite eggs are absent. Splenocytes from these animals displayed only marginal Th2 cytokine synthesis but greater Th1 cytokine responses than the corresponding cells from mice with bisexual infections. Moreover, cultures of liver tissue or isolated granulomas from infected mice constitutively produced high levels of IL-4 and IL-5 but failed to synthesize significant amounts of IL-2 and IFN-gamma even when stimulated with egg Ag or mitogen. Taken together the data indicate that egg deposition is the major stimulus of Th2 cytokine response in S. mansoni-infected mice and suggest that T cells belonging to this subset must play a major role in egg granuloma formation.