Elicitation of specific, Th1-biased immune response precludes skeletal muscle damage in cruzipain-vaccinated mice.

Cruzipain (Cz), the major cystein proteinase of Trypanosoma cruzi, is able to induce protective immunity against parasite challenge. However, some concern has arisen regarding its potential to elicit pathogenic autoimmune reactivity. To determine whether the adverse myopathic effects of Cz-based immunization could be prevented, we evaluated the co-administration of Cz with different adjuvants. Mice were immunized with Cz adjuvantized by alum (Cz+alum), oligodeoxynucleotides containing CpG motifs (Cz+ODN-CpG) or Freund's preparation (Cz+CFA). Cz triggered a vigorous specific humoral response, irrespective of the adjuvant used. Alum mainly drove response towards Th2 phenotype, characterized by specific IgG1 antibodies and IL-10 induction, whereas Cz+ODN-CpG mice exhibited Th1-dominant immunity, with antibodies of the IgG2a isotype and enhanced IFN-gamma production. Histological examination of cardiac tissue demonstrated lesions in Cz+CFA but not in Cz+alum nor Cz+ODN-CpG immunized animals, suggesting that CFA is critical for Cz-mediated injury. Analysis of skeletal muscle revealed that mice receiving Cz+CFA exhibited disrupted and hyalinized myofibers, whereas [Cz+alum]-immunized animals showed hyalinization, architecture modifications and small inflammatory foci. Conversely, no abnormalities were observed in the striated muscle from the Cz+ODN-CpG group. Hence, generation of specific immune response skewed towards Th1, as that recorded for the ODN-CpG adjuvant, may preclude triggering of Cz-mediated muscle tissue damage.

Bombesin induces cyclooxygenase-2 expression through the activation of the nuclear factor of activated T cells and enhances cell migration in Caco-2 colon carcinoma cells.

Cyclooxygenase-2 (Cox-2), the gastrin-release peptide (GRP) and its cognate receptor (GRP-R) are overexpressed in a significant percentage of colorectal carcinomas and are associated with cell growth, invasiveness and tumor progression. However, a molecular link between all of them in adenocarcinomas has not been established. Here, we show that bombesin (BBS), a GRP homolog, stimulates the expression of Cox-2 mRNA and protein in human colon adenocarcinoma Caco-2 cells, resulting in enhanced release of prostaglandin E(2). These effects were markedly inhibited by the specific BBS antagonist RC-3940-II. BBS promotes the activation of the nuclear factor of activated T cells (NFAT) through a Ca(2+)/calcineurin (Cn)-linked pathway. Upon BBS stimulation, the NFATc1 isoform translocates into the nucleus with a concomitant increase in NFATc1 binding to two specific recognition sites in the promoter region of the Cox-2 gene. Furthermore, inhibition of Cn activity by the immunosuppressive drug cyclosporin A impaired NFAT activation and diminished Cox-2 expression in BBS-stimulated cells. Interestingly, BBS pretreatment strongly enhances the invasive capacity of carcinoma cells, effect which was inhibited by a Cox-2-specific inhibitor. These findings provide the first evidence for the involvement of the Ca(2+)/Cn/NFAT pathway in BBS-mediated induction of genes involved in colon carcinoma invasiveness such as Cox-2.

Adamantylamide dipeptide as effective immunoadjuvant in rabbits and mice.

In the search for more potent and less toxic immunomodulators, adamantylamide dipeptide (AdDP) was synthesized by the covalent union of amantadine with the L-alanyl-D-isoglutamine residue of muramyldipeptide (MDP). The present experiments demonstrate the ability of AdDP, co-administered with a protein immunogen, to raise or enhance a humoral response in immunized animals. BALB/c mice were immunized either by the intraperitoneal (ip) or oral route with ovalbumin (Ova) alone or combined with either AdDP or CpG oligonucleotide (ODN-CpG), a proved adjuvant. A clear adjuvant dose-response relationship was observed on the increment of Ova-specific serum antibody titers when AdDP was used as adjuvant, irrespectively of the administration route. The IgG isotype analysis showed that AdDP promotes a consistent increment in IgG1 antibodies associated with a dominant Th2 response pattern. When administered by the oral route, AdDP was at least as efficient as ODN-CpG as adjuvant. Similar results were obtained in rabbits immunized by the oral route, suggesting that the adjuvanticity of AdDP is not restricted to the murine system. In conclusion, AdDP was shown to be a powerful and non-toxic adjuvant at both systemic and mucosal levels, which makes it a promising tool for vaccine development.

DNA from protozoan parasites Babesia bovis, Trypanosoma cruzi, and T. brucei is mitogenic for B lymphocytes and stimulates macrophage expression of interleukin-12, tumor necrosis factor alpha, and nitric oxide.

The activation of innate immune responses by genomic DNA from bacteria and several nonvertebrate organisms represents a novel mechanism of pathogen recognition. We recently demonstrated the CpG-dependent mitogenic activity of DNA from the protozoan parasite Babesia bovis for bovine B lymphocytes (W. C. Brown, D. M. Estes, S. E. Chantler, K. A. Kegerreis, and C. E. Suarez, Infect. Immun. 66:5423-5432, 1998). However, activation of macrophages by DNA from protozoan parasites has not been demonstrated. The present study was therefore conducted to determine whether DNA from the protozan parasites B. bovis, Trypanosoma cruzi, and T. brucei activates macrophages to secrete inflammatory mediators associated with protective immunity. DNA from Escherichia coli and all three parasites stimulated B-lymphocyte proliferation and increased macrophage production of interleukin-12 (IL-12), tumor necrosis factor alpha (TNF-alpha), and nitric oxide (NO). Regulation of IL-12 and NO production occurred at the level of transcription. The amounts of IL-12, TNF-alpha, and NO induced by E. coli and protozoal DNA were strongly correlated (r2 > 0.9) with the frequency of CG dinucleotides in the genome, and immunostimulation by DNA occurred in the order E. coli > or = T. cruzi > T. brucei > B. bovis. Induction of inflammatory mediators by E. coli, T. brucei, and B. bovis DNA was dependent on the presence of unmethylated CpG dinucleotides. However, at high concentrations, E. coli and T. cruzi DNA-mediated macrophage activation was not inhibited following methylation. The recognition of protozoal DNA by B lymphocytes and macrophages may provide an important innate defense mechanism to control parasite replication and promote persistent infection.

CpG DNA as a Th1-promoting adjuvant in immunization against Trypanosoma cruzi.

Th1-type immune response plays a critical role in resistance to Trypanosoma cruzi infection. We asked whether a synthetic oligodeoxynucleotide that contains immunostimulatory CpG motifs (CpG ODN), known to promote a Th1 response, could act as an adjuvant in immunization with parasite antigens. Mice immunized with a whole homogenate (WH) of T. cruzi antigens co-administered with CpG ODN presented high titers of T. cruzi antibodies (IgG2a isotype), strong delayed type hypersensitivity and a Th1-dominated (IFN-gamma and IL-12) cytokine profile. Furthermore, WH plus CpG ODN protected mice from challenge with an otherwise lethal dose of bloodstream trypomastigotes. As reported for leishmaniasis and malaria, CpG ODN holds considerable promise as an adjuvant for future vaccines against T. cruzi.

Trypanosoma cruzi tubulin eliminated in the urine of the infected host.

In previous studies we have identified and characterized an 80-kDa Trypanosoma cruzi urinary antigen (UAg) eliminated during acute infection. Polyclonal antibodies raised against this antigen revealed by western blotting and immunoprecipitation analyses showed the existence of another antigenic component of 50-55 kDa in the UAg preparation. The antiserum was also used for screening of a T. cruzi expression library. Sequencing of inserts from selected cDNA clones showed high homology with the 3' end of the T.cruzi beta-tubulin gene sequence encoding for the C-terminus of the protein. The presence of T. cruzi tubulin in the UAg was confirmed by immunoprecipitation of a 50-55-kDa protein from 125I-labeled UAg with monoclonal antibodies (MAbs) to human alpha/beta-tubulin. Interestingly, MAbs recognized radiolabeled T. cruzi tubulin eliminated in the urine of infected mice 24 hr postinoculation of [35S]methionine-labeled viable trypomastigotes. Tubulin found in the urine proved to be of T. cruzi origin because this protein could not be identified in urinary specimens from uninfected animals or mice acutely infected with Leishmania infantum or Toxoplasma gondii. We conclude that tubulin is one of the parasite antigens eliminated in the urine of T. cruzi-infected hosts. This finding may be used to develop a noninvasive procedure for early diagnosis of Chagas' disease.

Presence of IgM antibodies to Trypanosoma cruzi urinary antigen in sera from patients with acute Chagas' disease.

An 80-kilodalton Trypanosoma cruzi antigen is eliminated in the urine of infected hosts during the acute stage of Chagas' disease. We show that affinity-purified urinary antigen is recognised by IgM antibodies in the sera from acute chagasic patients. Comparing our urinary antigen assay with that using a whole T. cruzi lysate antigen for IgM antibody detection, we demonstrated that ELISA with urinary antigen increases the diagnostic sensitivity and specificity of IgM serology in recent chagasic infection. Twenty-six of 30 patients with acute T. cruzi infection had serum IgM antibodies that reacted with urinary antigen by ELISA, while lysate antigen IgM was detected in 24 sera. When sera from patients suffering other parasitoses were tested, strong cross-reactions occurred in ELISA with T. cruzi lysate antigen, whereas ELISA with urinary antigen proved to better discriminate acute chagasic patients. Human antibodies to urinary antigen immunoprecipitated this T. cruzi urinary antigen and also inhibited the binding of monoclonal antibody to urinary antigen in an inhibition assay. These findings suggest that urinary antigen may be useful for the development of serodiagnostic procedures for acute T. cruzi infection.

Detection and characterization of antigens in urine of patients with acute, congenital, and chronic Chagas' disease.

Monoclonal antibodies raised against purified Trypanosoma cruzi urinary antigens were used in an enzyme-linked immunosorbent assay (ELISA) capture test for parasite antigens present in urine specimens of Argentinean and Brazilian patients with Chagas' disease. At diagnosis, antigenuria was demonstrated by ELISA in all acutely and congenitally infected infants studied. Moreover, T. cruzi urinary antigens were detected in samples from three of five patients with acute infections and four of five patients with congenital infections following chemotherapy. At least one ELISA-positive urine specimen from each individual was recorded in a longitudinal survey of 12 chronic chagasic patients. The same parasitic antigens (90 to 80 kDa, pI 5.7 to 6.0; 70 to 65 kDa, pI 4.9 to 4.5; 50 to 45 kDa, pI 5.3 to 5.1; and 40 to 35 kDa, pI 4.8 to 4.5) were identified by immunoprecipitation and two-dimensional polyacrylamide gel electrophoresis analysis of urine samples from patients with different forms of chagasic infection. The 90- to 80-kDa urinary protein resembles a trypomastigote-shed antigen. Determination of antigenuria proved valuable for early diagnosis of Chagas' disease and also for diagnosis of chronic cases with conflicting serology.

An iron-binding Trypanosoma cruzi urinary antigen.

An 80-kDa Trypanosoma cruzi urinary antigen (UAg) was affinity-purified from the urine of infected dogs. We demonstrated that UAg is structurally and functionally related to proteins belonging to the transferrin family, as shown by amino acid sequence and iron binding experiments. Nevertheless, monoclonal antibodies raised against UAg specifically and selectively recognized this parasite's circulating antigen. The existence of an 80-kDa T. cruzi antigen co-migrating with UAg could be confirmed when epimastigotes were metabolically labelled with [35S] methionine and then immunoprecipitated with the above mentioned antibodies. We conclude that UAg is an iron-binding T. cruzi component eliminated in the urine of the infected host.

Effect of anti-gamma-interferon and anti-interleukin-4 administration on the resistance of mice against infection with reticulotropic and myotropic strains of Trypanosoma cruzi.

We studied the effect of in vivo administration of anti-gamma-IFN and anti-IL-4 monoclonal antibodies on the resistance of mice against myotropic and reticulotropic strains of Trypanosoma cruzi. Anti-gamma-IFN treatment augmented the susceptibility of mice when infected with the reticulotropic RA and Tulahuén strains of T. cruzi but did not alter the course of infection with the myotropic CA-I strain of the parasite. In vivo administration of anti-IL-4 enhanced the resistance of mice when infected with either Tulahuén or RA strains but did not affect the course of parasitemia when infected with CA-I. The possible biological relevance of these observations is discussed.

Detection of soluble exoantigens of Trypanosoma cruzi by a dot-immunobinding assay.

A simple and highly sensitive assay for the detection of Trypanosoma cruzi antigens by spotting samples on nitrocellulose membrane filters is described. The immobilized antigens are analyzed by subsequent binding of specific anti-T. cruzi immunoglobulins and secondary enzyme-labeled antibodies. Trypomastigote exoantigens were determined by this technique in the supernatant from cell cultures two days after infection. The T. cruzi circulating antigens present in serum specimens collected from mice infected with the parasite were demonstrable by day 3 of infection, when the parasitemia was still low. Antigenemia was also demonstrable by the dot-immunobinding assay in 14 of 16 congenitally infected patients. No cross-reactivity was observed using sera from healthy individuals, as well as from patients with other related parasitoses. The assay proved to be easy to perform and requires only small volumes of samples.

Purification and characterization of an 80-kilodalton Trypanosoma cruzi urinary antigen.

A Trypanosoma cruzi antigen eliminated in the urine of experimentally infected dogs was detected by enzyme-linked immunosorbent assay between 9 and 28 days after infection. The parasite urinary antigen (UAg) was purified by affinity chromatography with polyclonal antibodies to T. cruzi. The eluate of the antibody column was subjected to high-performance liquid chromatography and showed a single peak of A280. This antigen was the only parasite component found in the urine of infected dogs during the course of acute T. cruzi infection. Antigen characterization was performed by two-dimensional gel electrophoresis, lectin affinity chromatography, proteolytic digestion, and Western blotting (immunoblotting). The isolated UAg exhibited a relative molecular size of 80 kilodaltons (kDa), an isoelectric point of 6.2 to 6.8, binding to concanavalin A, and sensitivity to trypsin. The parasite antigen was electroeluted from polyacrylamide gels and subjected to acid hydrolysis and amino acid analysis by reverse-phase high-performance liquid chromatography. The 80-kDa glycoprotein was recognized by serum antibodies from a wide variety of T. cruzi-infected hosts. The UAg proved to be a highly antigenic component present in different strains of T. cruzi. This 80-kDa polypeptide resembles one of the parasite antigens previously found in the urine of patients with acute Chagas' disease.

Antigenuria in infants with acute and congenital Chagas' disease.

Detection and partial characterization of Trypanosoma cruzi soluble antigens (SAg) in urine, as well as demonstration of parasite circulating antigens (CAg) in serum from pediatric patients with acute (10 patients) and congenital (10 patients) Chagas' disease, are reported. Classical techniques for parasite detection and antibody serology were also conducted in both groups. Samples collected before the onset of parasiticidal drug treatment were tested by an enzyme-linked immunosorbent assay for SAg and CAg demonstration. The control population consisted of 6 children with acute toxoplasmosis, 6 with cutaneous leishmaniasis, and 20 healthy individuals. Patients with acute cases were 100% positive for both SAg and CAg, whereas patients with congenital disease were 80% CAg positive and 100% SAg positive. Controls yielded negative results in all cases. Partial characterization of SAg from two patients with acute disease was performed by iodination, affinity chromatography, immunoprecipitation, and two-dimensional gel electrophoresis. Two different antigenic glycoproteins (80 kilodaltons, pI 6 to 6.5 and 55 kilodaltons, pI 6.5 to 7) were identified by these methods. Traditional serology and classical parasitologic tests failed, each in a different way, to provide an accurate diagnosis in the total of our patients. The enzyme-linked immunosorbent assay for SAg detection proved to be the most effective procedure for achieving early and precise proof of infection in acute and congenital cases of Chagas' disease.