Common to advanced human tumors is a marked decrease in, even the complete disappearance of, the expression of Fas receptor (Apo-1; CD95) on the cell surface, where it is required to initiate the apoptosis cascade following its association with Fas ligand (FasL). Effective Fas-mediated death signaling requires the recruitment of FADD and caspase-8 to the Fas receptor, thereby assembling the death-inducing signaling complex (24, 31). It has been shown that in some cases the loss of Fas surface expression can be attributed to transcriptional silencing of the Fas promoter by cooperation between Stat3 and c-Jun (19) and that such cooperation is regulated by PI3K signaling (22). However, it has been noted that a portion of tumors that express Fas retain it within cytoplasmic pools rather than on the cell surface (8,16). Here, we demonstrate that Fas trafficking is regulated by Fas-associated phosphatase 1 (FAP-1; synonyms, protein tyrosine phosphatase nonreceptor type 13 [PTPN13] and hPTP1E/PTPL1/PTP-BAS).FAP-1 is a 270-kDa protein containing an ezrin-like cytoskeleton binding domain, a leucine zipper motif, and six PSD95/Dlg/Z-1 homology (PDZ) domains which is widely expressed in almost all tissues (7,29,34,36). FAP-1 has been implicated in the suppression of Fas-mediated apoptosis (28, 36), which requires functional interaction of either the third or the fifth PDZ domain of FAP-1 with the extreme C-terminal domain of Fas (35,36,47). However, the mechanisms by which FAP-1 inhibits Fas-mediated death signaling are still unknown.Colocalization of FAP-1 with Fas protein within the Golgi apparatus has been observed in pancreatic cancer cells, implying that FAP-1 may be involved in regulating Fas processing (45). Interestingly, increased Fas translocation from the Golgi apparatus to the cell membrane can be induced upon cellular treatment, as seen in hepatocytes that were exposed to bile salts (41). An adenovirus infection that leads to forced degradation of Fas by the adenovirus E3 proteins also results in down-regulation of Fas surface expression (14,43).Elevated levels of FAP-1 expression have been reported in various types of human cancer, including colon, pancreatic, hepatocellular, hematological, and ovarian cancers (4,13,26,27,28,30,42,45). Overall, the level of FAP-1 expression positively correlates with the resistance of human tumors to FasL-mediated apoptosis (28, 30).As a protein tyrosine phosphatase, FAP-1 is expected to alter tyrosine phosphorylation of its associated substrates. Although such data are not yet available for human cells, the mouse homologue of FAP-1 (PTP-BL phosphatase) dephosphorylates phosphoephrin B ligands following their phosphorylation by Src kinases (46). In the case of Fas, information about its own phosphorylation is very limited, although several studies have implicated Fas phosphorylation as an important regulatory event for its activities. For example, tyrosine kinases, such as Lyn, were implicated in the generation of a Fas receptor-linked transmembrane death signal in eosinophil...

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.

Sera from patients with chronic Chagas heart disease recognize the carboxyl-terminal regions of the Trypanosoma cruzi ribosomal P proteins defined by B cell epitopes P013 (EDDDDDFGMGALF) and R13 (EEEDDDMGFGLFD) corresponding to the T. cruzi ribosomal P0 (TcP0) and P2beta (TcP2beta) proteins, respectively. It has been hypothesized that both epitopes may induce antibodies that cross-react and stimulate the beta1-adrenoreceptor. However, no proof as to their pathogenicity has been obtained. We investigated the consequences of immunizing mice with either TcP0 or TcP2beta proteins. Of 24 immunized animals, 16 generated antibodies against the carboxyl-terminal end of the corresponding protein, 13 of which showed an altered ECG (P<0.001, 81%). Immunization with TcP0 induced anti-P013 antibodies that bind to and stimulate cardiac G-protein-coupled receptors and are linked to the induction of supraventricular arrhythmia, repolarization, and conduction abnormalities as monitored by serial electrocardiographic analysis. In contrast, immunization with TcP2beta generated anti-R13 antibodies with an exclusive beta1-adrenergic-stimulating activity whose appearance strictly correlated with the recording of supraventricular tachycardia and death. These findings demonstrate that anti-P antibodies are arrhythmogenic in the setting of a normal heart, since no inflammatory lesions or fibrosis were evident to light microscopic examination.

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.

Molecular expression cloning techniques revealed that patients with severe chronic Chagas heart disease showed a strong humoral response against the cloned C-terminal portion of the Trypanosoma cruzi ribosomal P2beta protein, previously named JL5. The main linear epitope of this polypeptide was mapped to the 13 C-terminal amino acid sequence EEEDDDMGFGLFD (named R13), which is almost identical to the mammalian ribosomal P consensus sequence EESDDDMGFGLFD (named H13). Enzyme-linked immunosorbent assay measurements demonstrated that sera from patients with chronic Chagas heart disease presented a very specific anti-P humoral response with high anti-R13, but low H13 antibody levels. We attempted to develop an animal model that would reproduce, at least partially, two features of the human infection: (1) the serological pattern of the anti-P response, and (2) specific cardiac symptoms. To this effect, mice were immunized with T. cruzi P2beta recombinant protein. Immunization reproduced the typical anti-P antibody profile defined for chronic infections, but did not induce cardiac inflammatory lesions. However, it altered significantly the electrocardiograms of immunized mice. It is suggested that this assay represents a functional test for assessing the biological activity of antibodies against T. cruzi ribosomal P protein on cardiac muscle.

No abstract

Immunoglobulins undergo isotype switching in response to antigenic stimulation. The C(H) domains, in particular the hinge region, impose structural constraints on the interaction of antibody with antigen, especially multivalent antigens such as HIV. We previously showed that switching the IgG1 anti-HIV human monoclonal antibody (HMAb) F105 to an IgG3 resulted in significantly enhanced neutralization of HIV. To further investigate the influence of isotype, including the functional activity of HMAbs switched to IgA, which may be important in mucosal defenses, isotype switched antibodies have been generated for the anti-V3 loop base IgG2 HMAb F425B4e8. Reactivity of the IgG1 antibody was greater than the parental IgG2 antibody for SF2 infected cells but less for primary isolate virions. In contrast, there was less reactivity of the IgG3 with either infected cells or virions. IgA reacted significantly more with infected cells and virions as compared to the IgG subclasses. In contrast to previous studies whereby IgG3 enhanced neutralization, comparable neutralization of primary isolate virus was observed for IgG subclasses (IgG1, IgG2, IgG3) and IgA. This may reflect differences in the exposure of epitopes recognized by the HMAb with antibody flexibility being important to neutralization by antibodies reactive with obscured epitopes (e.g., CD4 binding site). Further analysis of the in vitro activity of isotype or subclass switched antibodies, IgA in particular, alone and in combination with other HMAbs, will provide important information on the role of IgG subclass and IgA antibodies on protective immunity to HIV.

Molecular expression cloning techniques revealed that patients with severe chronic Chagas heart disease showed a strong humoral response against the cloned C-terminal portion of the Trypanosoma cruzi ribosomal P2beta protein, previously named JL5. The main linear epitope of this polypeptide was mapped to the 13 C-terminal amino acid sequence EEEDDDMGFGLFD (named R13), which is almost identical to the mammalian ribosomal P consensus sequence EESDDDMGFGLFD (named H13). Enzyme-linked immunosorbent assay measurements demonstrated that sera from patients with chronic Chagas heart disease presented a very specific anti-P humoral response with high anti-R13, but low H13 antibody levels. We attempted to develop an animal model that would reproduce, at least partially, two features of the human infection: (1) the serological pattern of the anti-P response, and (2) specific cardiac symptoms. To this effect, mice were immunized with T. cruzi P2beta recombinant protein. Immunization reproduced the typical anti-P antibody profile defined for chronic infections, but did not induce cardiac inflammatory lesions. However, it altered significantly the electrocardiograms of immunized mice. It is suggested that this assay represents a functional test for assessing the biological activity of antibodies against T. cruzi ribosomal P protein on cardiac muscle.