Karina A. Gómez

Anti-β1-adrenergic receptor autoantibodies in patients with chronic Chagas heart disease

Chronic Chagas heart disease (cChHD), a chronic manifestation of the Trypanosoma cruzi infection, is characterized by high antibody levels against the C‐terminal region of the ribosomal P proteins (i.e. peptide R13, EEEDDDMGFGLFD) which bears similarity with the second extracellular loop of β1‐adrenergic receptor (β1‐AR, peptide H26R HWWRAESDEARRCYNDPKCCDFVTNR). Because it has not been demonstrated clearly that IgGs from cChHD patients bind to native human β1‐AR, the aim of this study was to investigate further the physical interaction between cChHD IgGs and the human β1‐AR. Immunofluorescence assays demonstrated the binding of these antibodies to the receptor expressed on stably transfected cells, together with a β1‐AR agonist‐like effect. In addition, immunoadsorption of the serum samples from cChHD patients with a commercially available matrix, containing peptides representing the first and the second extracellular loop of the β1‐AR, completely abolished reactivity against the H26R peptide and the physiological response to the receptor. The follow‐up of this specificity after in vitro immunoadsorption procedures suggests that this treatment might be used to diminish significantly the serum levels of anti‐β1‐AR antibodies in patients with Chagas heart disease.

Galectin-1 Controls Cardiac Inflammation and Ventricular Remodeling during Acute Myocardial Infarction

Galectin-1 (Gal-1), an evolutionarily conserved β-galactoside-binding lectin, plays essential roles in the control of inflammation and neovascularization. Although identified as a major component of the contractile apparatus of cardiomyocytes, the potential role of Gal-1 in modulating heart pathophysiology is uncertain. Here, we aimed to characterize Gal-1 expression and function in the infarcted heart. Expression of Gal-1 was substantially increased in the mouse heart 7 days after acute myocardial infarction (AMI) and in hearts from patients with end-stage chronic heart failure. This lectin was localized mainly in cardiomyocytes and inflammatory infiltrates in peri-infarct areas, but not in remote areas. Both simulated hypoxia and proinflammatory cytokines selectively up-regulated Gal-1 expression in mouse cardiomyocytes, whereas anti-inflammatory cytokines inhibited expression of this lectin or had no considerable effect. Compared with their wild-type counterpart, Gal-1-deficient (Lgals1(-/-)) mice showed enhanced cardiac inflammation, characterized by increased numbers of macrophages, natural killer cells, and total T cells, but reduced frequency of regulatory T cells, leading to impaired cardiac function at baseline and impaired ventricular remodeling 7 days after nonreperfused AMI. Treatment of mice with recombinant Gal-1 attenuated cardiac damage in reperfused AMI. Taken together, our results indicate a protective role for Gal-1 in normal cardiac homeostasis and postinfarction remodeling by preventing cardiac inflammation. Thus, Gal-1 treatment represents a potential novel strategy to attenuate heart failure in AMI.

Identification of two liver proteins recognized by autoantibodies elicited in mice infected with mouse hepatitis virus A59

Western blot experiments showed that sera from mice infected with the mouse hepatitis virus strain A59 (MHV‐A59) contained autoantibodies (autoAb) that bound to a 40‐kDa protein present in liver and kidney extracts. No reaction was observed with extracts of the heart, muscles, spleen, brain and lung. The Ab cross‐reacted with a 40‐kDa protein from human, rat and sheep liver, but not withliver extracts from the silver side fish (Odontesthes bonariensis). No correlation was found between the development of the hypergammaglobulinemia that followed the viral infection and theoccurrence of the autoAb. Reactive immunoglobulins pertained to the IgG1, IgG2a and IgG2b subclasses, recognized cryptic epitopes and were detected from 10 days up to 8 weeks after MHV‐infection. The 40‐kDa protein was purified from mouse liver extracts by ion‐exchange chromatography, gel filtration and SDS‐PAGE. Because the N‐terminal was blocked, we digested the protein in‐gel with trypsin and sequenced various peptides. Results indicated a 100% homology of sequence between the protein recognized by the autoAb and liver fumarylacetoacetate hydrolase (FAH), the enzyme that mediates the last step of tyrosine catabolism. Additionally, a second protein recognized by the autoAb was detected during FAH purification steps and was identified as liver alcohol dehydrogenase.

Antibodies against the Trypanosoma cruzi ribosomal P proteins induce apoptosis in HL-1 cardiac cells.

High levels of antibodies (Abs) against the C-terminal end of the Trypanosoma cruzi ribosomal P2β protein, defined by the R13 peptide, are detected in sera from patients with chronic Chagas heart disease (cChHD). These Abs can cross-react with the β1-adrenergic receptor (β1-AR), inducing a functional response in cardiomyocytes. In this study, we report that a monoclonal Ab against the R13 peptide, called mAb 17.2, and its single-chain Fv fragment (scFv), C5, caused apoptosis of murine adult cardiac HL-1 cells, and this effect was inhibited by pre-incubation with the β-blocker, propranolol. In addition, apoptosis induced by mAb 17.2 might involve the mitochondrial pathway evidenced by an increase in pro-apoptotic molecule, Bax/anti-apoptotic molecule, Bcl(XL), mRNA levels. HL-1 cells also underwent apoptosis after incubation with nine of 23 IgGs from cChHD patients (39.1%) that presented reactivity against R13 peptide and β1-AR. The apoptotic effect caused by these IgGs was partially abolished by pre-incubation with R13 peptide or propranolol, suggesting the involvement of the C-terminal end of ribosomal P proteins and the β-adrenergic pathway. Moreover, we observed high rates of cardiomyocyte apoptosis in two tissue samples from cChHD patients by using a TUNEL assay and staining of active caspase-3. Our data demonstrate that Abs developed during T. cruzi infection have a strong cardiomyocyte apoptosis inducing ability, which could contribute to the heart disease developed in patients with cChHD.

Changes in the specificity of antibodies in mice infected with lactate dehydrogenase-elevating virus

Infection with lactate dehydrogenase‐elevating virus (LDV) modifies the isotypic distribution of antibodies (Ab) directed to several antigenic proteins with a preferential production of IgG2a. Because it was not known whether the virus could also affect the Ab specificity, the authors addressed this point using human growth hormone (hGH) as a model antigen. Anti‐hGH monoclonal antibodies (MoAb) were used as probes to study the occurrence of Ab to three native hGH epitopes (3C11, F11 and 10D6) in sera from LDV‐infected CBA/Ht and BALB/c mice immunized with hGH. Competition ELISA was used to determine the extent of Ab directed to cryptic hGH epitopes, i.e. antigenic determinants hidden in the native hormone. Results indicated that in LDV‐infected CBA/Ht mice the titres of anti‐hGH Ab were lower than in controls, although a consistent isotypic shift to IgG2a subclass was observed. Concurrently, the presence of Ab to epitopes 3C11, F11 and/or 10D6 were markedly reduced in infected animals and most anti‐hGH Ab were directed to hGH cryptic epitopes. By contrast, LDV infection increased the amount of anti‐KLH Ab elicited by CBA/Ht mice and did not affect Ab specificity, whilst control and LDV‐infected BALB/c mice showed similar concentrations of anti‐hGH Ab. Furthermore, the proportion of Ab to cryptic hGH epitopes did not change in infected animals even though an important shift to IgG2a was detected. Thus, data presented herein suggest that LDV infection modifies Ab specificity depending on the mice genetic background and on the antigenic characteristics of the immunogen.

Synergistic monoclonal antibodies‘ interactions and their use for determination of antibody specificities

Three monoclonal antibodies (MAb 3C11, F11 and 10D6) to human growth hormone (hGH) recognize independent epitopes and show mutually enhancing properties. Thus, 125I-hGH binding to each of these MAb augmented significantly in the presence of each one of the other two MAb. Moreover, preincubation of the hormone with paired MAb gave rise to ternary complexes (Ag:Ab1:Ab2) which bound better than the free tracer to the third MAb previously captured on a solid-phase. Highly stable quaternary complexes (Ag:Ab1:Ab2:Ab3) were thus formed. Since Fab fragments from the three MAb displayed the same behavior as the whole Ab molecule, neither the formation of multimolecular cyclic complexes nor the occurrence of interactions through Fc fragments could explain the reciprocal MAb binding enhancement. Therefore, the results obtained suggest that MAb 3C11, F11 and 10D6 produce some modification in the Ag, each one improving the binding of the two other MAb. Additionally, the inhibition of the formation of quaternary complexes between the MAb and hGH was used to evaluate specific Ab populations in polyclonal antisera, avoiding the masking effect of enhancing Ab. The results obtained indicate that Ab directed to the hGH antigenic domains defined by MAb 3C11, F11 and 10D6 could be detected in spite of the presence of enhancing Ab to all three MAb.

Sequence similarity and structural homologies are involved in the autoimmune response elicited by mouse hepatitis virus A59

Abstract The features of autoantibodies (autoAb) to liver fumarylacetoacetate hydrolase (FAH) elicited in mice infected with mouse hepatitis virus (MHV) were studied by ELISA and western-blot competition assays. All sera tested contained Ab to cryptic FAH epitopes according with results from western-blot tests, whereas ELISA data indicated that some of these same sera did recognize native epitopes of the autoantigen (autoAg). Such differences were detected in individual sera from various mouse strains, and were ascribed to the fact that proteins insolubilized on solid supports expose a variety of conformational and cryptic antigenic determinants. On the other hand, whereas results from both experimental protocols showed that anti-MHV Ab did not cross-react with the soluble autoAg, the opposite situation did not show analogous results. Thus, binding of autoAb to insolubilized FAH could be inhibited by MHV depending on the mouse serum or the experimental protocol used. Additionally, a set of synthetic homologous peptides from mouse FAH and various viral proteins was employed to analyze the Ab repertoire of MHV-infected mice. Results indicated that two homologous peptides were recognized by most Ab: the N-terminal sequences (1–10) from FAH and the nucleocapside, both sharing 50% of identity, and sequence 2317–2326 of the RNA polymerase, a peptide showing 30% of identity with FAH 11–20. Results indicated that MHV-infection triggers at least three distinct Ab populations: anti-MHV, anti-FAH and cross-reacting Ab. This cross-reaction implies either sequential or conformational epitopes from both the viral proteins and the autoAg and may differ between individuals.

Autoantibodies to Cryptic Epitopes Elicited by Infection with Lactate Dehydrogenase‐Elevating Virus

Lactate dehydrogenase‐elevating virus (LDV) produces a permanent infection in mice with a B‐lymphocyte polyclonal activation leading to hypergammaglobulinaemia. Since LDV specifically suppressed antibodies to native epitopes in CBA/Ht, but not BALB/c, mice immunized against a protein antigen, we explored the relationship between such a change in antibody specificity and the expression of autoantibodies under the influence of LDV. Again in CBA/Ht, but not BALB/c, mice we observed another effect of LDV: the sera from infected CBA/Ht mice were found by enzyme‐linked immunosorbant assay to contain antibodies to various mouse tissue extracts. Immunoblots revealed a large spectrum of autoantigens that differed markedly between animals. Western‐blot competition experiments showed that the protein autoantigens had to be denatured to react with most of the autoantibodies. Despite the presence of these autoantibodies directed to cryptic epitopes, no specific tissue lesions could be ascribed to the autoimmune response elicited by LDV infection, since both mouse strains showed mild inflammatory reactions in liver and kidney.

Galectin-1 Prevents Infection and Damage Induced by Trypanosoma cruzi on Cardiac Cells

Background Chronic Chagas cardiomyopathy caused by Trypanosoma cruzi is the result of a pathologic process starting during the acute phase of parasite infection. Among different factors, the specific recognition of glycan structures by glycan-binding proteins from the parasite or from the mammalian host cells may play a critical role in the evolution of the infection. Methodology and Principal Findings Here we investigated the contribution of galectin–1 (Gal–1), an endogenous glycan-binding protein abundantly expressed in human and mouse heart, to the pathophysiology of T. cruzi infection, particularly in the context of cardiac pathology. We found that exposure of HL–1 cardiac cells to Gal–1 reduced the percentage of infection by two different T. cruzi strains, Tulahuén (TcVI) and Brazil (TcI). In addition, Gal–1 prevented exposure of phosphatidylserine and early events in the apoptotic program by parasite infection on HL–1 cells. These effects were not mediated by direct interaction with the parasite surface, suggesting that Gal–1 may act through binding to host cells. Moreover, we also observed that T. cruzi infection altered the glycophenotype of cardiac cells, reducing binding of exogenous Gal–1 to the cell surface. Consistent with these data, Gal–1 deficient (Lgals1 -/-) mice showed increased parasitemia, reduced signs of inflammation in heart and skeletal muscle tissues, and lower survival rates as compared to wild-type (WT) mice in response to intraperitoneal infection with T. cruzi Tulahuén strain. Conclusion/Significance Our results indicate that Gal–1 modulates T. cruzi infection of cardiac cells, highlighting the relevance of galectins and their ligands as regulators of host-parasite interactions.

Effects of various adjuvants and a viral infection on the antibody specificity toward native or cryptic epitopes of a protein antigen.

An immunization protocol that induces antibodies (Abs) directed to cryptic epitopes of a protein antigen (Ag) reduces the efficacy of vaccines that ideally should induce Abs against native epitopes. We have shown earlier that viral infections concomitant with immunization against a protein tend to shift the Ab specificity toward cryptic epitopes and tend to induce the production of autoantibodies (autoAbs). Here, we show the effects of three adjuvants on the Ab specificity in the absence or presence of a viral infection (lactate dehydrogenase‐elevating virus or LDV), with human growth hormone (hGH) being, as before, the protein Ag.