Myosin 1g and 1f: A Prospective Analysis in NK Cell Functions.

NK cells are contained in the ILC1 group; they are recognized for their antiviral and antitumor cytotoxic capacity; NK cells also participate in other immune response processes through cytokines secretion. However, the mechanisms that regulate these functions are poorly understood since NK cells are not as abundant as other lymphocytes, which has made them difficult to study. Using public databases, we identified that NK cells express mRNA encoding class I myosins, among which Myosin 1g and Myosin 1f are prominent. Therefore, this mini-review aims to generate a model of the probable participation of Myosin 1g and 1f in NK cells, based on information reported about the function of these myosins in other leukocytes.

Myo1g is required for efficient adhesion and migration of activated B lymphocytes to inguinal lymph nodes.

Cell migration is a dynamic process that involves adhesion molecules and the deformation of the moving cell that depends on cytoskeletal remodeling and actin-modulating proteins such as myosins. In this work, we analyzed the role of the class I Myosin-1 g (Myo1g) in migratory processes of LPS + IL-4 activated B lymphocytes in vivo and in vitro. In vivo, the absence of Myo1g reduced homing of activated B lymphocytes into the inguinal lymph node. Using microchannel chambers and morphology analysis, we found that the lack of Myo1g caused adhesion and chemotaxis defects. Additionally, deficiency in Myo1g causes flaws in adopting a migratory morphology. Our results highlight the importance of Myo1g during B cell migration.

An antibody against an Anopheles albimanus midgut myosin reduces Plasmodium berghei oocyst development.

BACKGROUND: Malaria parasites are transmitted by Anopheles mosquitoes. Although several studies have identified mosquito midgut surface proteins that are putatively important for Plasmodium ookinete invasion, only a few have characterized these protein targets and demonstrated transmission-blocking activity. Molecular information about these proteins is essential for the development of transmission-blocking vaccines (TBV). The aim of the present study was to test three monoclonal antibodies (mAbs), A-140, A-78 and A-10, for their ability to recognize antigens and block oocyst infection of the midgut of Anopheles albimanus, a major malaria vector in Latin America. METHOD: Western-blot of mAbs on antigens from midgut brush border membrane vesicles was used to select antibodies. Three mAbs were tested by membrane feeding assays to evaluate their potential transmission-blocking activity against Plasmodium berghei. The cognate antigens recognized by mAbs with oocyst-reducing activity were determined by immunoprecipitation followed by liquid chromatography tandem mass spectrometry. RESULTS: Only one mAb, A-140, significantly reduced oocyst infection intensity. Hence, its probable protein target in the Anopheles albimanus midgut was identified and characterized. It recognized three high-molecular mass proteins from a midgut brush border microvilli vesicle preparation. Chemical deglycosylation assays confirmed the peptide nature of the epitope recognized by mAb A-140. Immunoprecipitation followed by proteomic identification with tandem mass spectrometry revealed five proteins, presumably extracted together as a complex. Of these, AALB007909 had the highest mascot score and corresponds to a protein with a myosin head motor domain, indicating that the target of mAb A-140 is probably myosin located on the microvilli of the mosquito midgut. CONCLUSION: These results provide support for the participation of myosin in mosquito midgut invasion by Plasmodium ookinetes. The potential inclusion of this protein in the design of new multivalent vaccine strategies for blocking Plasmodium transmission is discussed.

Endothelial actin-binding proteins and actin dynamics in leukocyte transendothelial migration.

The endothelium is the first barrier that leukocytes have to overcome during recruitment to sites of inflamed tissues. The leukocyte extravasation cascade is a complex multistep process that requires the activation of various adhesion molecules and signaling pathways, as well as actin remodeling, in both leukocytes and endothelial cells. Endothelial adhesion molecules, such as E-selectin or ICAM-1, are connected to the actin cytoskeleton via actin-binding proteins (ABPs). Although the contribution of receptor-ligand interactions to leukocyte extravasation has been studied extensively, the contribution of endothelial ABPs to the regulation of leukocyte adhesion and transendothelial migration remains poorly understood. This review focuses on recently published evidence that endothelial ABPs, such as cortactin, myosin, or α-actinin, regulate leukocyte extravasation by controlling actin dynamics, biomechanical properties of endothelia, and signaling pathways, such as GTPase activation, during inflammation. Thus, ABPs may serve as targets for novel treatment strategies for disorders characterized by excessive leukocyte recruitment.

Troponin T autoantibodies correlate with chronic cardiomyopathy in human Chagas disease.

OBJECTIVE: To evaluate the potential involvement of anti-Trypanosoma cruzi and cardiac protein antibody (IgG total and isotypes) production and their possible association with different clinical forms of human chronic Chagas disease. METHODS: IgG total and isotypes were measured by ELISA, using epimastigote and trypomastigote forms of T. cruzi as antigens and human cardiac proteins (myosin and troponin T) in sera of patients with indeterminate (IND, n = 72), cardiac (CARD, n = 47) and digestive/cardiodigestive (DIG/CARD-DIG, n = 12) clinical forms of the disease. Samples from uninfected health individuals (CONT, n = 30) and patients with ischaemic cardiomyopathy (ISCH, n = 15) were used as controls. Autoantibody levels were correlated with parameters of cardiac function obtained by electrocardiographic, radiographic and echocardiographic examinations. RESULTS: Fifty five per cent of patients were classified as IND, 35.9% as CARD and 9.1% as DIG/CARD-DIG. Greater total IgG production was observed in IND, CARD and DIG/CARD-DIG chagasic patients than in CONT and ISCH, using trypomastigote, epimastigote and cardiac antigens. Moreover, patients with CARD and DIG/CARD-DIG presented greater total IgG production (trypomastigote and epimastigote antigen) than IND, and a negative correlation was determined between total IgG and left ventricular ejection fraction (LVEF). Patients with IND and CARD presented similar higher levels of total IgG specific to troponin T and myosin than CONT and ISCH individuals. Patients with chronic Chagas disease presented a negative correlation between left ventricular ejection fraction (LVEF) and the production of anti-myosin and troponin T autoantibodies. When grouped as low and high antibody producers and compared with LVEF, we observed that high anti-troponin T (P = 0.042) and myosin (P = 0.013) producers presented lower LVEF than low producers. Moreover, there was a positive correlation (r = 0.9508, P = 0.0001) between the production of troponin T and myosin autoantibodies. CONCLUSION: These findings indicate that increased production of anti-cardiac troponin T and myosin autoantibodies probably influences the left ventricular ejection fraction and could be related to chagasic cardiomyopathy.

A striking common O-linked N-acetylglucosaminyl moiety between cruzipain and myosin.

Single units of O-linked N-acetylglucosamine (GlcNAc), usually components of nuclear and cytoplasmatic proteins, are present at the C-terminal domain of cruzipain (Cz), a lysosomal major antigen from Trypanosoma cruzi. On the other hand, antibodies directed against some self-antigens like myosin are associated with Chagas heart disease. The participation of O-GlcNAc moieties in the molecular antigenicity of Cz was determined using GlcNAc linked to aprotinin by ELISA. The immune cross-reactivity between Cz and myosin is mainly focused in the C-T domain. ELISA inhibition assays using rabbit sera specific for Cz and C-T in conjunction with immune-gold electron microscopy analysis of heart tissues from mice immunized with C-T confronted with polyclonal rabbit sera specific for Cz and C-T prior and after myosin adsorption provided evidence which indicates that O-GlcNAc moieties constitute a common epitope between Cz and either myosin or other cardiac O-GlcNAc-containing proteins, showing a new insight into the molecular immune pathogenesis of Chagas heart disease.

The distribution of motor proteins in the muscles and flame cells of the Schistosoma mansoni miracidium and primary sporocyst.

Schistosoma mansoni eggs, miracidia and primary sporocysts were labelled with phalloidin-rhodamine to visualize filamentous actin structures. Analysis of these forms by confocal fluorescence microscopy revealed the presence of previously well-defined circular and longitudinal muscle layers. Besides these muscular layers that sustain and provide motility to these parasite forms, we found in these 3 consecutive developmental stages of the parasite previously unidentified actin-rich tubular structures. In the 3 forms, 4 actin-rich tubules could be observed by optical sectioning underneath the well-developed muscle layers. The tubules appear in pairs, transversal to the length of the parasite, and located towards the extremities. By using an anti-flame cell specific antibody we confirmed that the tubules co-localize with flame cells and also determined that the tubule core is filled with microtubules. The additional presence of myosin in these tubules strongly suggests that they are contractile structures.

Immune response to a major Trypanosoma cruzi antigen, cruzipain, is differentially modulated in C57BL/6 and BALB/c mice.

BALB/c mice immunized with cruzipain, a major Trypanosoma cruzi antigen, produce specific and autoreactive immune responses against heart myosin, associated with cardiac functional and structural abnormalities. Preferential activation of the Th2 phenotype and an increase in cell populations expressing CD19+, Mac-1+ and Gr-1+ markers were found in the spleens of these mice. The aim of the present study was to investigate whether cardiac autoimmunity could be induced by cruzipain immunization of C57BL/6 mice and to compare the immune response elicited with that of BALB/c mice. We demonstrate that immune C57BL/6 splenocytes, re-stimulated in vitro with cruzipain, produced high levels of IFNgamma and low levels of IL-4 compatible with a Th1 profile. In contrast to BALB/c mice, spleens from cruzipain immune C57BL/6 mice revealed no significant changes in the number of cells presenting CD19+, Mac-1+ and Gr-1+ markers. An increased secretion of TGFbeta and a greater number of CD4+ TGFbeta+ cells were found in immune C57BL/6 but not in BALB/c mice. These findings were associated with the lack of autoreactive response against heart myosin and a myosin- or cruzipain-derived peptide. Thus, the differential immune response elicited in C57BL/6 and BALB/c mice upon cruzipain immunization is implicated in the resistance or pathogenesis of experimental Chagas' disease.

Yersinia enterocolitica O:3 isolated from patients with or without reactive arthritis induces polyclonal activation of B cells and autoantibody production in vivo.

The mechanisms by which arthritis-provoking pathogens such as Yersinia enterocolitica interact with the human immune system to produce inflammatory synovitis are not well known. One of the immunomodulating mechanisms used against these pathogens is the polyclonal activation of lymphocytes. In this study, we investigated the extent of the B-lymphocyte activation induced in mice by a strain of Y. enterocolitica O:3 (FCF 526) isolated from a patient with arthritis, and compared it with two other strains, a virulent one (FCF 397[+]) isolated from a patient without arthritis and its plasmidless isogenic pair (FCF397[-]). Also we investigated the production of autoantibodies in mice infected with these different strains. SPF Swiss mice were infected intravenously with a suspension of Y. enterocolitica. Spleen cells were taken on days 7, 14, 21 and 28 after infection and the number of cells secreting nonspecific and specific antibodies of IgG1, IgG2a, IgG2b, IgG3, IgM and IgA isotypes were determined by the ELISPOT technique. The presence of autoantibodies in mouse serum was investigated by the dot-blot assay. The pattern of infection of the three bacterial strains were almost the same. We observed a general increase in the number of nonspecific Ig-secreting cells with all three strains, and the greatest increases observed were in the IgG2a and IgG3 isotypes. Only a small fraction of the immunoglobulins detected were antibacterial, suggesting that the rest resulted from polyclonal B cell activation. The strain isolated from the patient with arthritis (FCF526) induced the greatest production of autoantibodies, coinciding with the period in which the greatest activation of nonspecific B lymphocytes was seen. There were no signs of arthritis or inflammation in the joints of the infected animals. Based on our results, we were unable to determine whether there is an association between the arthritogenic capability of Y. enterocolitica and polyclonal activation of B cells.

Differential expression of adhesion moleculesshaping the T-cell subset prevalence during the early phase of autoimmune and Trypanosoma cruzi-elicited myocarditis.

The participation of cell adhesion molecules (CAMs) in the establishment of autoimmune and infectious myocarditis is an important matter of investigation and may have therapeutic implication. Trypanosoma cruzi infection induces a CD8-mediated myocarditis in patients with severe cardiomyopathy and experimental animals. Previously, we have proposed that this predominance of CD8+ T-cells is, at least in part, consequence of the differential expression of CAMs on circulating CD8+ lymphocytes. In the present study we investigated the participation of CAMs in shaping the phenotypic nature of the autoimmune CD4-mediated myosin-induced and the CD8-mediated T. cruzi-elicited myocarditis. We provide evidence that the prevalence of a certain T-cell subset inside the inflamed heart reflects the differential profile of the adhesion molecules VLA-4, LFA-1, and ICAM-1 displayed on a large proportion of this particular T-cell population in peripheral blood during the early phase of inflammation. Further, the expression of VCAM-1, ligand for VLA-4, and ICAM-1, counter-receptor for LFA-1, was up-regulated on vascular endothelium and paralleled the entrance of inflammatory cells into the cardiac tissue. Thus, this up-regulated expression of receptors-counter-receptors that regulate T-cell transmigration through the vascular endothelium may have an important role in the pathogenesis of the early phase of both autoimmune and infectious myocarditis.

Isolation of lipoxygenase isoforms from Glycine max embryo axes based on apparent cross-reactivity with anti-myosin antibodies.

Three lipoxygenase isoforms were isolated from Glycine max embryo axes. A number of proteins around 97 kDa cross-reacted with several anti-actin and anti-myosin antibodies and these were used to follow their purification through gel filtration, hydroxyapatite and anion exchange columns. The 97-kDa cross-reactive material eluted in the unbound fractions of the last anion exchange column, and displayed two components of pI's 6.2 and 6.3. Further phase partition of this fraction in TX-114 yielded a hydrophobic 97 kDa protein. Additionally, a 95-kDa protein was retained and eluted from this last column. Partial peptide sequences indicated that the 95 kDa protein was soybean lipoxygenase-1, the first 97 kDa protein was lypoxygenase-3, and the hydrophobic 97 kDa protein was lipoxygenase-2. Several possible reasons for the cross-reactivity with the antibodies are discussed. To our knowledge, this is the first example of individual lipoxygenase isoforms isolated from soybean embryo axes.

Effects of Yersinia enterocolitica O:3 derivatives on B lymphocyte activation in vivo.

The potential sequelae of intestinal infection with Yersinia enterocolitica include reactive arthritis, erythema nodosum, Reiter's syndrome and other autoimmune diseases. The role of the immune response in the pathogenesis of these diseases has not been fully defined, but autoimmune manifestations may be a consequence of the increase in autoantibodies as a result of polyclonal B-cell activation induced by Yersinia. We investigated the effects of Y. enterocolitica O:3 derivatives on B lymphocyte activation in vivo. Groups of five specific pathogen free (SPF) Swiss mice were inoculated with bacterial cell extract, Yersinia outermembrane proteins (Yops) or lipopolysaccharide (LPS) obtained from Y. enterocolitica O:3 and their immunoglobulin-secreting spleen cells were detected by isotype-specific protein A plaque assay. The presence of specific anti-Yersinia antibodies and autoantibodies was determined in mouse sera by ELISA. In all experiments a marked increase in the number of secretory cells of different isotypes was observed as early as the third day after inoculation. IgG and IgM anti-Yersinia antibodies were detected in the sera of all inoculated mice, and autoantibodies against myosin in the sera of those inoculated with bacterial cell extract. The sera from animals stimulated with LPS reacted with myelin, actin and laminin, while the sera from mice inoculated with Yops reacted with myelin, thyroglobulin and cardiolipin. These results suggest that SPF Swiss mice inoculated with any one of the Y. enterocolitica derivatives tested exhibited polyclonal activation of B lymphocytes as a result of stimulation by various bacterial components and not only LPS stimulation.

Experimental chronic Chagas' disease myocarditis is an autoimmune disease preventable by induction of immunological tolerance to myocardial antigens.

The protozoan Trypanosoma cruzi causes chronic Chagas' disease myocarditis (CCDM) in infected mammals. The pathogenesis of CCDM, however, is still unclear. Indirect evidence for either parasite- or heart-specific immune responses playing a pathogenic role is available. In this work, the participation of autoimmunity in the development of CCDM is demonstrated in mice in which immunological tolerance to heart antigens was induced or strengthened prior to their infection by T. cruzi. Tolerance was induced by heart antigen administration in the presence of complete Freund's adjuvant and anti-CD4 antibodies. Tolerized mice developed less intense CCDM than control non-tolerized animals that had received only anti-CD4 and adjuvant. This result confirms the important notion that tolerance to self, and in particular to heart antigens, may be reinforced/induced in normal animals, and raises the possibility that analogous interventions may prevent the development of CCDM in millions of T. cruzi -infected human beings.

T-cell reactivity against streptococcal antigens in the periphery mirrors reactivity of heart-infiltrating T lymphocytes in rheumatic heart disease patients.

T-cell molecular mimicry between streptococcal and heart proteins has been proposed as the triggering factor leading to autoimmunity in rheumatic heart disease (RHD). We searched for immunodominant T-cell M5 epitopes among RHD patients with defined clinical outcomes and compared the T-cell reactivities of peripheral blood and intralesional T cells from patients with severe RHD. The role of HLA class II molecules in the presentation of M5 peptides was also evaluated. We studied the T-cell reactivity against M5 peptides and heart proteins on peripheral blood mononuclear cells (PBMC) from 74 RHD patients grouped according to the severity of disease, along with intralesional and peripheral T-cell clones from RHD patients. Peptides encompassing residues 1 to 25, 81 to 103, 125 to 139, and 163 to 177 were more frequently recognized by PBMC from RHD patients than by those from controls. The M5 peptide encompassing residues 81 to 96 [M5(81-96) peptide] was most frequently recognized by PBMC from HLA-DR7+ DR53+ patients with severe RHD, and 46.9% (15 of 32) and 43% (3 of 7) of heart-infiltrating and PBMC-derived peptide-reactive T-cell clones, respectively, recognized the M5(81-103) region. Heart proteins were recognized more frequently by PBMC from patients with severe RHD than by those from patients with mild RHD. The similar pattern of T-cell reactivity found with both peripheral blood and heart-infiltrating T cells is consistent with the migration of M-protein-sensitized T cells to the heart tissue. Conversely, the presence of heart-reactive T cells in the PBMC of patients with severe RHD also suggests a spillover of sensitized T cells from the heart lesion.

Induction of antibodies reactive to cardiac myosin and development of heart alterations in cruzipain-immunized mice and their offspring.

Human and murine infection with Trypanosoma cruzi parasite is usually accompanied by strong humoral and cellular immune response to cruzipain, a parasite immunodominant antigen. In the present study we report that the immunization of mice with cruzipain devoid of enzymatic activity, was able to induce antibodies which bind to a 223-kDa antigen from a mouse heart extract. We identified this protein as the mouse cardiac myosin heavy chain by sequencing analysis. The study of IgG isotype profile revealed the occurrence of all IgG isotypes against cruzipain and myosin. IgG1 showed the strongest reactivity against cruzipain, whereas IgG2a was the main isotype against myosin. Anti-cruzipain antibodies purified by immunoabsorption recognized the cardiac myosin heavy chain, suggesting cross-reactive epitopes between cruzipain and myosin. Autoimmune response in mice immunized with cruzipain was associated to heart conduction disturbances. In addition, ultrastructural findings revealed severe alterations of cardiomyocytes and IgG deposit on heart tissue of immunized mice. We investigated whether antibodies induced by cruzipain transferred from immunized mothers to their offsprings could alter the heart function in the pups. All IgG isotypes against cruzipain derived from transplacental crossing were detected in pups' sera. Electrocardiographic studies performed in the offsprings born to immunized mothers revealed conduction abnormalities. These results provide strong evidence for a pathogenic role of autoimmune response induced by a purified T. cruzi antigen in the development of experimental Chagas' disease.

Cruzipain induces autoimmune response against skeletal muscle and tissue damage in mice.

The goal of the current study was to investigate whether cruzipain, a major Trypanosoma cruzi antigen, is able to induce in mice an autoimmune response and skeletal muscle damage. We demonstrate that immunization with cruzipain triggers immunoglobulin G antibody binding to a 210-kDa antigen from a syngeneic skeletal muscle extract. The absorption of immune sera with purified myosin completely eliminated this reactivity, confirming that the protein identified is really myosin. We also found that spleen cells from immunized mice proliferated in response to a skeletal muscle extract rich in myosin and to purified myosin. Cells from control mice did not proliferate against any of the antigens tested. In addition, we observed an increase in plasma creatine kinase activity, a biochemical marker of muscle damage. Histological studies showed inflammatory infiltrates and myopathic changes in skeletal muscle of immunized animals. Electromyographic studies of these mice revealed changes such as are found in inflammatory or necrotic myopathy. Altogether, our results suggest that this experimental model provides strong evidence for a pathogenic role of anticruzipain immune response in the development of muscle tissue damage.

Molecular cloning, purification and immunological responses of recombinants GroEL and DnaK from Streptococcus pyogenes.

To better understand the roles of heat shock proteins in streptococcal diseases, the groEL and dnaK genes from Streptococcus pyogenes were cloned and their products (GroEL and DnaK) and derivatives (F2GroEL, F3GroEL and C1DnaK) purified as His-tagged fusion proteins. Western blot analysis of the purified proteins with sera from individuals with streptococcal diseases demonstrated that 29 out of 36 sera tested were reactive with GroEL and eight recognized DnaK. Rabbit antiserum against myosin recognized both GroEL and DnaK. Antibodies raised against purified F2GroEL and DnaK reacted with myosin in the ELISA but not in a Western immunoblot. These data indicate that the S. pyogenes GroEL and DnaK may be important immunogens during streptococcal infections. Furthermore, we provide evidence of an immunogenic relatedness of the GroEL and DnaK proteins with myosin that could play a role in the pathogenesis of streptococcal non-suppurative sequelae.

Isolation and identification of actin-binding proteins in Plasmodium falciparum by affinity chromatography.

The invasion of the erythrocyte by Plasmodium falciparum depends on the ability of the merozoite to move through the membrane invagination. This ability is probably mediated by actin dependent motors. Using affinity columns with G-actin and F-actin we isolated actin binding proteins from the parasite. By immunoblotting and immunoprecipitation with specific antibodies we identified the presence of tropomyosin, myosin, a-actinin, and two different actins in the eluate corresponding to F-actin binding proteins. In addition to these, a 240-260 kDa doublet, different in size from the erythrocyte spectrin, reacted with an antibody against human spectrin. All the above mentioned proteins were metabolically radiolabeled when the parasite was cultured with 35S-methionine. The presence of these proteins in P. falciparum is indicative of a complex cytoskeleton and supports the proposed role for an actin-myosin motor during invasion.

Isolation and identification of actin-binding proteins in Plasmodium falciparum by affinity chromatography

The invasion of the erythrocyte by Plasmodium falciparum depends on the ability of the merozoite to move through the membrane invagination. This ability is probably mediated by actin dependent motors. Using affinity columns with G-actin and F-actin we isolated actin binding proteins from the parasite. By immunoblotting and immunoprecipitation with specific antibodies we identified the presence of tropomyosin, myosin, a-actinin, and two different actins in the eluate corresponding to F-actin binding proteins. In addition to these, a 240-260 kDa doublet, different in size from the erythrocyte spectrin, reacted with an antibody against human spectrin. All the above mentioned proteins were metabolically radiolabeled when the parasite was cultured with 35S-methionine. The presence of these proteins in P. falciparum is indicative of a complex cytoskeleton and supports the proposed role for an actin-myosin motor during invasion.