An enzyme-release assay for the assessment of the lytic activities of complement or antimicrobial peptides on extracellular Toxoplasma gondii.

A method is described which allows the evaluation of the membrane lytic activity of either complement or antimicrobial peptides against the extracellular stage of the human protozoan parasite Toxoplasma gondii. The assay is based on lacZ transgenic parasites, determining the activity of released cytoplasmic beta-galactosidase into the culture supernatant upon membrane disintegration. This method was used to evaluate the lytic activities of (i) complement which is a natural defense mechanism in infected hosts against extracellular parasites, and (ii) antimicrobial peptides which have not been evaluated against T. gondii before. The results show that the assay provides a simple and convenient way to assess the membrane lytic activity of such compounds and that T. gondii, like other protozoan parasites, is vulnerable to the membrane-lytic effect of antimicrobial peptides.

Localization of T and B cell stimulating domains of the immunodominant 33-kDa protein of Onchocerca volvulus (Ov33).

The localization of T and B cell epitopes on a well characterized 33-kDa protein of the filarial nematode Onchocerca volvulus (Ov33) was studied using peripheral blood mononuclear cells (PBMC) and sera from a total of 52 onchocerciasis patients with the generalized form of infection. A proportion of the PBMC samples proliferated in response to recombinant Ov33-GST fusion protein and to fusion free Ov33-6xHis. Proliferative responses of patient PBMC to seven truncated Ov33-6xHis polypeptides and to three synthetic peptides revealed at least one major and two minor T cell epitopes in the protein. The dominant T cell stimulating domain was localized between amino acids 113 and 143. ELISA studies with the Ov33-GST fusion protein revealed that patient sera contained Ov33-specific IgG1, IgG4, IgE, and IgM antibodies. Analysis of the IgG4 response with 10 truncated Ov33 polypeptides identified four B cell stimulating domains in the N-terminal, central, and C-terminal region of the molecule. The B cell domain recognized by the majority of sera was localized between amino acids 113 and 143. The data indicate that this region of the protein is the major T and B cell stimulating domain of Ov33 and might be relevant for vaccine development and for improved immunodiagnosis of onchocerciasis.

Reactivation of chronic toxoplasmosis: is there a link to strain-specific differences in the parasite?

The protozoan parasite Toxoplasma gondii comprises three clonal lineages that are associated with the clinical outcome in infected individuals. Whereas group C strains are mainly found in animals, group A and B strains are associated with human disease (Howe and Sibley, 1995). An increased level of transcripts of the tachyzoite-specifically expressed gene SAG1 could be identified in group A T. gondii strains compared to group B strains. Since SAG1-mediated host-cell invasion seems to be important for parasite replication, the observed higher replication rate in group A T. gondii strains might explain the association with clinically overt symptoms at the acute stage in patients who are infected with this group of parasite strains. The presence of external stress factors, such as interferon-gamma (IFN-gamma)-mediated nitric oxide (NO) formation has been identified to stabilize the cyst stage, most likely by activation of promoter(s) which drive the expression of genes encoding bradyzoite-specific antigens. Reactivation of chronic toxoplasmosis thus might occur in the absence of external stress factors, as has been observed in AIDS patients with decreases levels of IFN-gamma. Since group B T. gondii strains might form more cysts in infected individuals due to an increased potential to convert into bradyzoites, reactivation with resulting toxoplasmic encephalitis could be a more common event in those AIDS patients who were infected with persistent cysts of this group of parasite strains.

Characterization of a recombinant T cell and B cell reactive polypeptide of Onchocerca volvulus.

To identify potentially protective Ag of the filarial nematode Onchocerca volvulus on the molecular level we screened a cDNA library of O. volvulus with a human serum raised against radiation-attenuated infective larvae of O. volvulus. A cDNA clone of 218 bp (OvL3-1) was selected for further studies. It was expressed in Escherichia coli and affinity purified recombinant polypeptide was tested for its ability to stimulate in vitro PBMC from African onchocerciasis patients and PBMC from chimpanzees experimentally infected with O. volvulus. An enhanced cell proliferation by PBMC was observed in many patients after stimulation with the recombinant OvL3-1 polypeptide. In addition, some patients' PBMC responded to OvL3-1 stimulation with enhanced IL-2 production. Infected chimpanzees also showed an increase in T cell proliferation. Onchocerciasis patients had variable levels of specific antibodies directed to the recombinant polypeptide when sera were tested by ELISA. A mAb directed against the recombinant protein located the native target Ag in the muscles of the adult worm. The molecular mass of native OvL3-1 was found to be 50 kDa on immunoblots. Polymerase chain reaction analysis of RNA from different life stages of the parasite showed that OvL3-1 is transcribed in all parasite stages within the mammalian host. A homologous gene is also present in other filarial parasites. The protein corresponding to OvL3-1, therefore, represents an immunogen present during the whole life-span of the parasite, and because of its B and T cell stimulatory properties, it may be a candidate for a protective Ag in human filariasis.