An immunodominant MHC class II-restricted tumor antigen is conformation dependent and binds to the endoplasmic reticulum chaperone, calreticulin.

There is accumulating evidence that CD4(+) T cell responses are important in antitumor immunity. Accordingly, we generated CD4(+) T cells against the murine CT26 colon cancer. Three of three independent CT26-specific CD4(+) hybridomas were found to recognize the high m.w. precursor of the env gene product gp90. The CD4(+) response was completely tumor specific in that the same glycoprotein expressed by other tumors was not recognized by the CT26-specific hybridomas. The recognition of gp90 by the hybridomas was strictly dependent on the conformation of gp90. Different procedures that disrupted the conformation of the glycoprotein, such as disulfide bond reduction and thermal denaturation, completely abrogated recognition of gp90 by all three hybridomas. In CT26 cells, but not in other tumor cells tested, a large proportion of gp90 was retained in the endoplasmic reticulum, mostly bound to the endoplasmic reticulum chaperone, calreticulin. Although calreticulin was not essential for the stimulation of the gp90-specific hybridomas, most of the antigenic form of gp90 was bound to it. The antigenicity of gp90 correlated well with calreticulin binding, reflecting the fact that specificity of binding of calreticulin to its substrate required posttranslational modifications that were also necessary for the generation of this tumor-specific CD4(+) epitope.

Major lectin of alligator liver is specific for mannose/L-fucose.

We surveyed the calcium-requiring (C-type) lectins in alligator liver. The major lectin purified by affinity chromatography was termed alligator hepatic lectin (AHL) and was found to be specific for mannose/L-fucose. AHL contained approximately equal amounts of 21- and 23-kDa bands upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Binding characteristics of AHL were similar to those of hepatic lectins of other classes in that 1) only terminal monosaccharide was recognized, and 2) the affinity increased exponentially when neoglycoproteins containing increasing numbers of mannose or L-fucose were used as ligand. However, unlike mammalian and chicken hepatic lectins, which exist as hexamers in Triton-containing solutions, AHL was present mainly as monomers, although small amounts of dimer and higher oligomers were present in equilibrium. Mannose-binding proteins and mannose-specific lectins of macrophages bind N-acetylmannosamine, glucose, and N-acetylglucosamine in addition to mannose, indicating that the nature and orientation of the C-2 substituent are not important to these lectins. In contrast, AHL shows a strict requirement for the presence of an axial hydroxyl group at the C-2 position (i.e. mannose).

Galactofuranose-containing glycoconjugates of epimastigote and trypomastigote forms of Trypanosoma cruzi.

Antiserum to LPPG, a lipopeptidophosphoglycan originally described on the surface of Trypanosoma cruzi epimastigotes of the Y strain, and antibodies to furanoic galactose (galf) were obtained in rabbits. A micromethod for the extraction and purification of LPPG from a limited amount of parasites is described. Analysis by Western blots of the purified glycoconjugate probed with both antisera confirmed the presence of galf-containing LPPG-like molecules in 10 different strains and clones of T. cruzi. An analogous approach indicated that trypomastigotes also contain LPPG-like components. Quantitation experiments allowed to calculate an average value of 1.0 x 10(7) LPPG molecules per epimastigote cell and 0.16 x 10(7) LPPG-like molecules per trypomastigote cell. Immunoelectron microscopy has shown a homogenous distribution of LPPG on the surface of epimastigotes. The trypomastigote population, however, is highly heterogenous with no more than 15% of the parasites being labeled by the anti-LPPG serum. Intense labeling has also been found in vesicles inside the epimastigote and trypomastigote forms. The distribution of galf epitopes among glycoconjugates of epimastigotes and trypomastigotes was further investigated. It was shown that galf units in epimastigotes are bound to low molecular mass compounds which co-migrate with LPPG whereas in trypomastigotes they have been found in both low molecular mass LPPG-like molecules and glycoproteins of 80-90 kDa. Direct chemical evidence for the presence of galf residues in the N-linked oligosaccharide chains of these surface glycoproteins has been obtained. Finally, the natural antigenicity of LPPG and galf in chronic Chagas' disease was investigated. It was found that all chronic chagasic sera investigated recognize this glycoconjugate and that an important part of such recognition can be attributed to galf residues. Furthermore, no correlation among reactivity to LPPG, strain zymodeme and clinical forms of the disease was found.

Molecular approaches to diagnosis of Chagas' disease: use of defined antigens and a target ribosomal RNA sequence.

We evaluated the performance of two defined antigens in the serological diagnosis of Chagas' disease. One of them is a recombinant protein named B13 isolated from a genomic library of Trypanosoma cruzi in the expression vector lambda gtll. We show that the gene corresponding to B13 is conserved in the evolutive stages of the two "polar" strains of T. cruzi. The protein epitopes cloned in B13 are represented in 140 kDa, 116 kDa and 35 kDa polypeptides of trypomastigotes. The other antigen chosen for serodiagnosis is a lipopeptidophosphoglycan (LPPG). This glycoconjugate is also widely distributed in T. cruzi strains. The use of a rabbit serum to LPPG allowed the demonstration that this molecule bears epitopes in common to LPPG-like components and to 80-90 kDa glycoproteins of trypomastigotes. Both B13 and LPPG were evaluated in serodiagnosis by ELISA and RIA using a panel of normal human, Chagasic and Leishmaniasis sera. It was observed that B13 presents high sensitivity and specificity for Chagasic sera. For LPPG it was also concluded that this reagent discriminates between individuals infected and non-infected with T. cruzi. A heterogeneity in the level of antibodies to LPPG in Chagasic patients was detected. No correlation was found between the clinical form of Chagas' disease and the preferential reactivity to B13 or LPPG. We also report preliminary studies towards the characterization of a 100 bp sequence of the 24S alpha rRNA as a target for DNA-based detection systems for diagnosis. We show that polymerase chain reaction of total DNA of different trypanosomatids lead to the specific amplification of a 100 bp fragment only for T. cruzi. Northern blots confirmed the presence of the target region in the mature 24S alpha rRNA. Titration experiments based on the direct amplification of RNA with Taq DNA polymerase allowed the detection of 50 parasites. Studies are in progress to increase the sensitivity of the proposed system.

The human immune response to defined immunogens of Schistosoma mansoni: elevated antibody levels to paramyosin in stool-negative individuals from two endemic areas in Brazil.

Sera from individuals living in 2 areas endemic for Schistosoma mansoni in Minas Gerais, Brazil were assayed for the presence of antibodies against paramyosin and glutathione-S-transferase (GST), molecules previously implicated as vaccine immunogens from studies in laboratory hosts. A group was identified consisting of subjects who were stool-negative and had no record of previous infection but who were seropositive by enzyme-linked immunosorbent assay against crude adult worm antigen (SWAP). These individuals had anti-paramyosin antibody levels which were dramatically elevated with respect to those measured in infected (stool-positive) individuals living in the same endemic area. In contrast, the same 2 groups of stool-positive and stool-negative subjects could not be distinguished on the basis of their seroreactivity to either GST or SWAP. After chemotherapy, anti-paramyosin antibodies rose above pre-treatment levels and remained elevated in those individuals who became stool-negative. In contrast, anti-paramyosin antibodies decreased to pretreatment values in drug-treated individuals who failed to show complete parasitological cure. These results suggest that the immune response of humans to paramyosin may play a role in natural resistance to schistosome infection, and that an elevated antibody level against this antigen may be a useful correlate of drug-induced cure.