Anti-P antibodies present in sera from patients with chronic Chagas heart disease (cChHD) recognize peptide R13, EEEDDDMGFGLFD, which encompasses the C-terminal region of the Trypanosoma cruzi ribosomal P1 and P2 proteins. This peptide shares homology with the Cterminal region (peptide H13 EESDDDMGFGLFD) of the human ribosomal P proteins, which is in turn the target of anti-P autoantibodies in systemic lupus erythematosus (SLE), and with the acidic epitope, AESDE, of the second extracellular loop of the ␤ 1 -adrenergic receptor. Anti-P antibodies from chagasic patients showed a marked preference for recombinant parasite ribosomal P proteins and peptides, whereas anti-P autoantibodies from SLE reacted with human and parasite ribosomal P proteins and peptides to the same extent. A semi-quantitative estimation of the binding of cChHD anti-P antibodies to R13 and H13 using biosensor technology indicated that the average affinity constant was about 5 times higher for R13 than for H13. Competitive enzyme immunoassays demonstrated that cChHD anti-P antibodies bind to the acidic portions of peptide H13, as well as to peptide H26R, encompassing the second extracellular loop of the ␤ 1 adrenoreceptor. Anti-P antibodies isolated from cChHD patients exert a positive chronotropic effect in vitro on cardiomyocytes from neonatal rats, which resembles closely that of anti-␤ 1 receptor antibodies isolated from the same patient. In contrast, SLE anti-P autoantibodies have no functional effect. Our results suggest that the adrenergicstimulating activity of anti-P antibodies may be implicated in the induction of functional myocardial impairments observed in cChHD.

Our results demonstrate a strong correlation between anti-betaRAbs and ventricular arrhythmias and anti-M2RAbs and sinus node dysfunction. Anti-betaRAbs increase and anti-M2RAbs inhibit cAMP production. These findings offer new insight into the etiology and pathophysiology of cardiac arrhythmias, with therapeutic implications.

SummarySera from chagasic patients possess antibodies recognizing the carboxy-terminal part of the ribosomal P0 protein of Trypanosoma cruzi and the second extracellular loop of the human fll-adrenergic receptor. Comparison of both peptides showed that they contain a pentapeptide with very high homology (AESEE in P0 and AESDE in the human fll-adrenergic receptor). Using a competitive immunoenzyme assay, recognition of the peptide corresponding to the second extracellular loop (H26R) was inhibited by both P0-14i (AAAESEEEDDDDDF) and P0-fl (AESEE). Concomitantly, recognition of P0-fl was inhibited with the H26R peptide. Recognition of P0 in Western blots was inhibited by P0-14i, P0-fl, and H26R, but not by a peptide corresponding to the second extracellular loop of the human fl2-adrenergic receptor or by an unrelated peptide. Autoantibodies affinity purified with the immobilized H26R peptide were shown to exert a positive chronotropic effect in vitro on cardiomyocytes from neonatal rats. This effect was blocked by both the specific fll blocker bisoprolol and the peptide P0-fl. These results unambiguously prove that T. cruzi is able to induce a functional autoimmune response against the cardiovascular human fll-adrenergic receptor through a molecular mimicry mechanism.

Antibodies of chronic chagasic patients have been shown to interfere with electric and mechanical activities of cardiac embryonic myocytes in culture and with whole mammalian hearts. A mechanism proposed for this effect involves interaction of the antibodies with G-protein-linked membrane receptors, thus leading to activation of beta adrenergic and muscarinic receptors; more specifically, IgG of chagasic patients would interact with the negatively charged regions of the second extracellular loop of these receptors. We performed competition experiments to test this hypothesis. We evaluated the effect of sera/IgG from patients previously known to depress electrogenesis and/or atrioventricular conduction in isolated rabbit hearts after incubation with live and lysed parasites, the peptide corresponding to the second extracellular loop (O2) of the M2 receptor, and different peptides derived from two ribosomal proteins of T. cruzi: P0 and P2beta. Our results indicate that 1) the antigenic factor inducing the functionally active IgGs in the chagasic patients is probably an intracellular T. cruzi antigen; 2) IgG/serum is interacting with the O2 region of the M2 receptor in the rabbit heart; and 3) the negative charges present in the ribosomal proteins of T. cruzi are important in mediating the interaction between the patients' serum/IgG and the receptor.

Strategies to construct the physical map of the

The rationally designed novel anti-HIV drug candidate Stampidine exhibited (a) remarkable subnanomolar to low nanomolar in vitro ARV potency against genotypically and phenotypically NRTI-resistant primary clinical HIV isolates, non-nucleoside reverse transcriptase inhibitor (NNRTI)-resistant HIV-1 isolates, clinical non-B subtype HIV-1 isolates (subtypes A, C, F, and G) originating from South America, Asia, and sub-Saharan Africa with resistance to stavudine, adefovir and tenofovir, as well as recombinant HIV clones containing common patterns of RT mutations responsible for NRTI resistance such as multiple TAMs plus M184V, multiple TAMs plus T69 insertion, and Q151 complex (b) favorable, safety profile in mice, rats, dogs, and cats, and (c) promising prophylactic in vivo anti-retroviral activity in Hu-PBL-SCID mice as well as therapeutic anti-retroviral activity in FIV-infected domestic cats. Notably, in a placebo-controlled Phase I study involving 30 therapy-naïve adult HIV-infected adult patients, formulated GMPgrade oral Stampidine capsules did not cause dose-limiting toxicity at single dose levels ranging from 5 to 25 mg/kg. Taken together, the presented favorable preclinical and early clinical safety/activity profile of Stampidine warrants its further development as a new anti-HIV drug candidate. J o ur nal o f A ID S & Cli n ic a l R es earc h

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