Lytic antibodies elicited by Trypanosoma cruzi infection recognize epitopes present on both bloodstream trypomastigote and epimastigote forms of parasite

Sera of Chaga's disease patients containing anti-T. cruzi lytic antibodies were submitted to affinity chromatography using Sepharose 4B conjugated with antigen extracted from epimasiigote or trypomasiigote forms of the parasite. Epimastigotes were obtained from culture at the exponential growth phase and the trypomastigotes from blood of infected and immunosuppressed mice. Antigen of both parasite forms was obtained by sonication of the parasites followed by centrifugation. Both antigens were then conjugated to activated Sepharose 4B. Affinity chromatography was performed by passing sera from chagasic patients through an immunoadsorbent column containing either epimasiigote or trypomasiigote antigens. Antibodies bound to the column were eluted with cold 0,2 M glycine buffer pH 2,8. The eluted antibodies were analysed regarding their isotype and lytic activity. The results showed that anti-T. cruzi lytic antibodies present in sera from chagasic patients are mainly located in the IgG isotype and recognize epitopes present in both trypomasiigote and epimastigote forms. A brief report of this work has already been published12.

Quantitative toxoplasma gondii oocyst detection by a modified Kato Katz test using Kinyoun staining (KKK) in ME49 strain experimentally infected cats

We detected Toxoplasma gondii oocysts in feces of experimentally infected cats, using a Kato Katz approach with subsequent Kinyoun staining. Animals serologically negative to T. gondii were infected orally with 5x10² mice brain cysts of ME49 strain. Feces were collected daily from the 3rd to the 30th day after challenge. Oocysts were detected by qualitative sugar flotation and the quantitative modified Kato Katz stained by Kinyoun (KKK). In the experimentally infected cats, oocysts were detected from the 7th to 15th day through sugar flotation technique, but oocysts were found in KKK from the 6th to 16th day, being sensitive for a larger period, with permanent documentation. The peak of oocysts excretion occurred between the 8th to 11th days after challenge, before any serological positive result. KKK could be used in the screening and quantification of oocysts excretion in feces of suspected animals, with reduced handling of infective material, decreasing the possibility of environmental and operator contamination.

Characterization of protective and non-protective surface membrane carbohydrate epitopes of Schistosoma mansoni

We have produced a number of monoclonal antibodies, protective and non-protective, which recognize a complex of schistosomula antigens, including the 38 kDa antigen. Eight different protective and non-protective monoclonal antibodies, varying in isotypes, were used in the binding assays. Lectin inhibition studies suggested that the monoclonal antibodies probably recognized carbohydrate epitopes on the antigen(s). Immunoprecipitation studies showed that at least two of the monoclonal antibodies recognized different epitopes on the same molecule. Additionally, we tested for monoclonal antibody binding after the antigens were treated with; 1) proteases, 2) periodate, 3) various exo- and endoglycosidases, 4) mild acid hydrolysis. We also tested for binding of the antibodies to keyhole limpet hemocyanin (KLH). Using the 8 monoclonal antibodies as probes, we were able to define at least 4 different carbohydrate epitopes related to the protective monoclonal antibodies, and at least one epitope which is seen by the non-protective antibodies. The epitope seen by the non-protective antibodies was shown to be cross-reactive with epitopes on KLH. These results demonstrate the importance of epitope mapping studies for any defined vaccine.

Antitumor activity of chemical modified natural compounds

Search of new activity substances starting from chemotherapeutic agents, continously appears in international literature. Perhaps this search has been done more frequently in the field of anti-tumor chemotherapy on account of the unsuccess in saving advanced stage patients. The new point in this matter during the last decade was computer aid in planning more rational drugs. In near future "the accessibility of supercomputers and emergence of computer net systems, willopen new avenues to rational drug design" (Portoghese, P. S. J. Med. Chem. 1989, 32, 1). Unknown pharmacological active compounds synthetized by plants can be found even without this eletronic devices, as tradicional medicine has pointed out in many contries, and give rise to a new drug. These compounds used as found in nature or after chemical modifications have produced successful experimental medicaments as FAA, "flavone acetic acid" with good results as inibitors of slow growing animal tumors currently in preclinical evaluation for human treatment. In this lecture some international contributions in the field of chemical modified compounds as antineoplasic drugs will be examined, particularly those done by Brazilian researches.

A modified assay for measuring thymocyte co-stimulatory activity

The observation that murine thymocytes increase their proliferation to interleukin 1 (IL-1) in the presence of phytohemagglutinin (PHA) when pre-incubated with interleukin 2 (IL-2) allowed the introduction of a modified assay for the measurement of IL-1 or the search of thymocyte-inducing proliferative activities in biological samples. Pre-incubation of thymocytes for 24 hr with 50 u/ml IL-2, followed by washings, elicited their maximal response to IL-1 in the usual lymphocyte activating factor (LAF) assay. This suggests that sequential events lead to thymocyte activation. The responsiveness is three to five fold greater than, and the total time of assay is the same as that of the LAF assay. Interestingly, pre-incubation with IL-2 renders thymocytes more sensitive than responsive to crude monocyte conditioned media. The use of the MTT colorimetric method for the assessment of thymocyte proliferation, and of the lectin jacalin as a co-mitogen are suggested as alternatives to be used in co-stimulatory assays.

Immunogenicity of multiple antigen peptides containing Plasmodium vivax CS epitopes in BALB/c mice

Multiple antigen peptide systems (MAPs) allow the incorporation of various epitopes in to a single synthetic peptide immunogen. We have characterized the immune response of BALB/c mice to a series of MAPs assembled with different B and T cell epitopes derived from the Plasmodium vivax circumsporozoite (CS) protein. A B-cell epitope from the central repeat domain and two T-cell epitopes from the amino and carboxyl flanking regions were used to assembled eight different MAPs. An additional universal T cell epitope (ptt-30) from tetanus toxin protein was included. Immunogenicity in terms of antibody responses and in vitro T lymphocyte proliferation was evaluated. MAPs containing B and T cell epitopes induced high titers of anti-peptides antibodies, which recognized the native protein on sporozoites as determined by IFAT. The antibody specificity was also determined by a competitive inhibition assay with different MAPs. A MAP containing the B cell epitope (p11) and the universal epitope ptt-30 together with another composed of p11 and the promiscuous T cell epitope (p25) proved to be the most immunogenic. The strong antibody response and specificity for the cognate protein indicates that further studies designed to assess the potential of these proteins as human malaria vaccine candidates are warranted.

Protective CD8+ T cell responses against the pre-erythrocytic stages of malaria parasites: an overview

CD8+ T cells have been implicated as critical effector cells in protection against the pre-erythrocytic stage of malaria in mice and humans following irradiated sporozoite immunization. Immunization experiments in animal models by several investigators have suggested different strategies for vaccination against malaria and many of the targets from liver stage malaria antigens have been shown to be immunogenic and to protect mice from the sporozoite challenge. Several prime/boost protocols with replicating vectors, such as vaccinia/influenza, with non-replicating vectors, such as recombinant particles derived from yeast transposon (Ty-particles) and modified vaccinia virus Ankara, and DNA, significantly enhanced CD8+ T cell immunogenicity and also the protective efficacy against the circumsporosoite protein of Plasmodium berghei and P. yeti. Based on these experimental results the development of a CD8+ T cell inducing vaccine has moved forward from epitope identification to planning stages of safety and immunogenicity trials of candidate vaccines.

Laboratory diagnosis of Schistosomiasis in areas of low transmission: a review of a line of research

After 57 years of successful control of schistosomiasis in Venezuela, the prevalence and intensity of infection have declined. Approximately 80% of the individuals eliminate less than 100 eggs/g of stools, therefore morbidity is mild and the majority are asymptomatic. The sensitivity of Kato-Katz decreases to approximately 60%. Available serological methods for the detection of circulating antigens only reach a 70% of sensitivity. Tests based on the detection of antibodies by immunoenzymatic assays have been improved. The circumoval precipitine test has shown a high sensitivity (97%), specificity (100%), and correlation with oviposition, being considered the best confirmatory diagnostic test. Additionally to the classical immunoenzymatic assays, the development of the alkaline phosphatase immunoassay, allowed to reach a 100% specificity with an 89% sensitivity. Recently, we have developed a modified ELISA in which the soluble egg antigen is treated with sodium metaperiodate (SMP-ELISA) in order to eliminate the glycosilated epitopes responsible for the false positive reactions. The specificity and sensitivity reaches 97% and 99%, respectively. Synthetic peptides from the excretory-secretory enzymes, cathepsin B (Sm31) legumain (Sm32) and cathepsin D (Sm45), have been synthesized. The combination of two peptides derived from the Sm31 have been evaluated, reaching a sensitivity of 96% when analyzed independently and with a 100% specificity. Antibodies raised in rabbits against peptides derived from the Sm31 and Sm32 are currently evaluated in two different antigen-capture-based assays. The development of a simple, cheap and reliable test that correlates with parasite activity is a major goal.