Trans-ancestral fine-mapping of MHC reveals key amino acids associated with spontaneous clearance of hepatitis C in HLA-DQß1.

Spontaneous clearance of acute hepatitis C virus (HCV) infection is associated with single nucleotide polymorphisms (SNPs) on the MHC class II. We fine-mapped the MHC region in European (n = 1,600; 594 HCV clearance/1,006 HCV persistence) and African (n = 1,869; 340 HCV clearance/1,529 HCV persistence) ancestry individuals and evaluated HCV peptide binding affinity of classical alleles. In both populations, HLA-DQß1Leu26 (p valueMeta = 1.24 × 10-14) located in pocket 4 was negatively associated with HCV spontaneous clearance and HLA-DQß1Pro55 (p valueMeta = 8.23 × 10-11) located in the peptide binding region was positively associated, independently of HLA-DQß1Leu26. These two amino acids are not in linkage disequilibrium (r2 < 0.1) and explain the SNPs and classical allele associations represented by rs2647011, rs9274711, HLA-DQB1∗03:01, and HLA-DRB1∗01:01. Additionally, HCV persistence classical alleles tagged by HLA-DQß1Leu26 had fewer HCV binding epitopes and lower predicted binding affinities compared to clearance alleles (geometric mean of combined IC50 nM of persistence versus clearance; 2,321 nM versus 761.7 nM, p value = 1.35 × 10-38). In summary, MHC class II fine-mapping revealed key amino acids in HLA-DQß1 explaining allelic and SNP associations with HCV outcomes. This mechanistic advance in understanding of natural recovery and immunogenetics of HCV might set the stage for much needed enhancement and design of vaccine to promote spontaneous clearance of HCV infection.

TREM-1 isoforms in bacterial infections: to immune modulation and beyond.

The triggering receptor expressed on myeloid cells 1 (TREM-1) is an innate immunity receptor associated with the amplification of inflammation in sterile and non-sterile inflammatory disorders. Since its first description, the two isoforms of the receptor, membrane and soluble (mTREM-1 and sTREM-1, respectively) have been largely explored in the immunopathogenesis of several bacterial diseases and sepsis. The role of the receptor in these scenarios seems to be at least partly dependent on the source/type of bacteria, host and context. As uncontrolled inflammation is a result of several bacterial infections, the inhibition of the receptor has been considered as a promising approach to treat such conditions. Further, sTREM-1 has been explored as a biomarker for diagnosis and/or prognosis of several bacterial diseases. Therefore, this review aims to provide an updated insight into how the receptor influences and is influenced by bacterial infections, highlighting the advances regarding the use/manipulation of TREM-1 isoforms in biomedical research and clinical practice.

Participation of 14-3-3ε and 14-3-3ζ proteins in the phagocytosis, component of cellular immune response, in Aedes mosquito cell lines.

BACKGROUND: Better knowledge of the innate immune system of insects will improve our understanding of mosquitoes as potential vectors of diverse pathogens. The ubiquitously expressed 14-3-3 protein family is evolutionarily conserved from yeast to mammals, and at least two isoforms of 14-3-3, the ε and ζ, have been identified in insects. These proteins have been shown to participate in both humoral and cellular immune responses in Drosophila. As mosquitoes of the genus Aedes are the primary vectors for arboviruses, causing several diseases such as dengue fever, yellow fever, Zika and chikungunya fevers, cell lines derived from these mosquitoes, Aag-2 from Aedes aegypti and C6/36 HT from Aedes albopictus, are currently used to study the insect immune system. Here, we investigated the role of 14-3-3 proteins (ε and ζ isoform) in phagocytosis, the main cellular immune responses executed by the insects, using Aedes spp. cell lines. RESULTS: We evaluated the mRNA and protein expression of 14-3-3ε and 14-3-3ζ in C6/36 HT and Aag-2 cells, and demonstrated that both proteins were localised in the cytoplasm. Further, in C6/36 HT cells treated with a 14-3-3 specific inhibitor we observed a notable modification of cell morphology with filopodia-like structure caused through cytoskeleton reorganisation (co-localization of 14-3-3 proteins with F-actin), more importantly the decrease in Salmonella typhimurium, Staphylococcus aureus and E. coli phagocytosis and reduction in phagolysosome formation. Additionally, silencing of 14-3-3ε and 14-3-3ζ expression by mean of specific DsiRNA confirmed the decreased phagocytosis and phagolysosome formation of pHrodo labelled E. coli and S. aureus bacteria by Aag-2 cells. CONCLUSION: The 14-3-3ε and 14-3-3ζ proteins modulate cytoskeletal remodelling, and are essential for phagocytosis of Gram-positive and Gram-negative bacteria in Aedes spp. cell lines.

Resistance (R) Genes: Applications and Prospects for Plant Biotechnology and Breeding.

The discovery of novel plant resistance (R) genes (including their homologs and analogs) opened interesting possibilities for controlling plant diseases caused by several pathogens. However, due to environmental pressure and high selection operated by pathogens, several crop plants have lost specificity, broad-spectrum or durability of resistance. On the other hand, the advances in plant genome sequencing and biotechnological approaches, combined with the increasing knowledge on Rgenes have provided new insights on their applications for plant genetic breeding, allowing the identification and implementation of novel and efficient strategies that enhance or optimize their use for efficiently controlling plant diseases. The present review focuses on main perspectives of application of R-genes and its co-players for the acquisition of resistance to pathogens in cultivated plants, with emphasis on biotechnological inferences, including transgenesis, cisgenesis, directed mutagenesis and gene editing, with examples of success and challenges to be faced.

Plant Elite Squad: First Defense Line and Resistance Genes - Identification, Diversity and Functional Roles.

Plants exhibit sensitive mechanisms to respond to environmental stresses, presenting some specific and non-specific reactions when attacked by pathogens, including organisms from different classes and complexity, as viroids, viruses, bacteria, fungi and nematodes. A crucial step to define the fate of the plant facing an invading pathogen is the activation of a compatible Resistance (R) gene, the focus of the present review. Different aspects regarding R-genes and their products are discussed, including pathogen recognition mechanisms, signaling and effects on induced and constitutive defense processes, splicing and post transcriptional mechanisms involved. There are still countless challenges to the complete understanding of the mechanisms involving R-genes in plants, in particular those related to the interactions with other genes of the pathogen and of the host itself, their regulation, acting mechanisms at transcriptional and post-transcriptional levels, as well as the influence of other types of stress over their regulation. A magnification of knowledge is expected when considering the novel information from the omics and systems biology.

Transcription Factors Involved in Plant Resistance to Pathogens.

Phytopathogenic microorganisms have a significant influence on survival and productivity of several crop plants. Transcription factors (TFs) are important players in the response to biotic stresses, as insect attack and pathogen infection. In face of such adversities many TFs families have been previously reported as differentially expressed in plants as a reaction to bacterial, fungal and viral infection. This review highlights recent progresses in understanding the structure, function, signal regulation and interaction of transcription factors with other proteins in response to pathogens. Hence, we focus on three families of transcription factors: ERF, bZIP and WRKY, due to their abundance, importance and the availability of functionally well-characterized members in response to pathogen attack. Their roles and the possibilities related to the use of this knowledge for engineering pathogen resistance in crop plants are also discussed.

Preparation of human tau exon-2- and -10-specific monoclonal antibodies for the recognition of brain tau proteins in various mammals.

The aggregations of tau protein in brain tissue have been described in a large number of neurodegenerative diseases; however, due to the lack of tau isoform- or exon-specific antibodies, the exact situations under which various brain tau isoforms can be found and their exact contributions during disease progression remain unknown. Therefore, in this study, we prepared tau exon-specific monoclonal antibodies (mAbs) that recognize different mammalian tau isoforms. Briefly, 3 Balb/c mice were separately immunized (3 mice per antigen) with the recombinant GST-fusion proteins, GST-tE2 and GST-tE10. Two hybridoma cell lines, 4A8 and 3E12, secreting antibodies against human tau exon-2 and -10 were established using the hybridoma technique. The sensitivity and specificity of the prepared mAbs were evaluated using indirect ELISA and western blot analysis. The ability of the prepared mAbs, 4A8 and 3E12, to recognize endogenous tau protein in the brain tissues of various mammals was estimated by immunoprecipitation. Based on the results of various verification methods, we found that the prepared mAbs, 4A8 and 3E12, not only specifically reacted with the individual recombinant GST tau exon fusion proteins, but also correctly recognized the recombinant human tau isoforms containing respective exon sequences, as shown by western blot analysis. Furthermore, western blot analysis and immunoprecipitation assays verified that the mAbs, 4A8 and 3E12, recognized endogenous tau proteins in human brain tissue, as well as tau proteins in a series of mammalian tissues, including goat, bovine, rabbit, hamster and mouse. Thus, in the present study, using the hybridoma technique, we successfully prepared the mAbs, 4A8 against tau exon-2 and 3E12 against tau exon-10, which provide useful tools for determining potential alternations of tau isoforms in neurodegenerative diseases.

Transcriptional and posttranscriptional regulations of the HLA-G gene.

HLA-G has a relevant role in immune response regulation. The overall structure of the HLA-G coding region has been maintained during the evolution process, in which most of its variable sites are synonymous mutations or coincide with introns, preserving major functional HLA-G properties. The HLA-G promoter region is different from the classical class I promoters, mainly because (i) it lacks regulatory responsive elements for IFN-γ and NF-κB, (ii) the proximal promoter region (within 200 bases from the first translated ATG) does not mediate transactivation by the principal HLA class I transactivation mechanisms, and (iii) the presence of identified alternative regulatory elements (heat shock, progesterone and hypoxia-responsive elements) and unidentified responsive elements for IL-10, glucocorticoids, and other transcription factors is evident. At least three variable sites in the 3' untranslated region have been studied that may influence HLA-G expression by modifying mRNA stability or microRNA binding sites, including the 14-base pair insertion/deletion, +3142C/G and +3187A/G polymorphisms. Other polymorphic sites have been described, but there are no functional studies on them. The HLA-G coding region polymorphisms might influence isoform production and at least two null alleles with premature stop codons have been described. We reviewed the structure of the HLA-G promoter region and its implication in transcriptional gene control, the structure of the HLA-G 3'UTR and the major actors of the posttranscriptional gene control, and, finally, the presence of regulatory elements in the coding region.

The influence of chitin on the immune response to the house dust mite allergen Blo T 12.

BACKGROUND: Information about the biological properties of Blomia tropicalis allergens is scarce. It is predicted that Blo t 12, an allergen with two described isoforms, contains a chitin-binding domain, similar to that found in peritrophins. Th2 adjuvant properties have been described for chitin. Therefore, it is feasible that binding to this carbohydrate influences its allergenicity. We aimed to evaluate the chitin-binding activity of Blo t 12 isoallergens and its effect on airway inflammation and antibody responses in a murine model of allergen sensitization. METHODS: Chitin-binding assays were conducted with the recombinant isoallergens Blo t 12.0101 and Blo t 12.0102. BALB/c mice were sensitized via i.p. with any of the two isoforms (alone, with chitin or alum) and then challenged intranasally. Methacholine-induced bronchial hyperreactivity was tested by whole-body plethysmography and lung sections were stained with hematoxylin and eosin and periodic-acid Schiff. Total IgE and allergen-specific IgE, IgG1 and IgG2 levels were measured by ELISA. RESULTS: The two isoforms bound chitin, but Blo t 12.0101 showed a stronger binding capacity. Both isoforms induced total and allergen-specific IgE, airway hyperreactivity, bronchial inflammation and mucus secretion without any adjuvant; however, when administered with chitin, Blo t 12.0101 induced higher total IgE levels. The IgG1/IgG2a ratio was significantly higher in mice immunized with Blo t 12.0101 than those immunized with Blo t 12.0102. As peritrophins, Blo t 12 was detected in mite feces. CONCLUSIONS: Blo t 12 isoforms are chitin-binding proteins that induce airway inflammation and bronchial hyperreactivity. However, for Blo t 12.0101, chitin reinforces its effects on total IgE production.

Production and evaluation of chicken antibodies against a synthetic peptide from glial growth factor.

Neuregulins (NRG) are proteins that belong to the family of epidermal growth factors. It is well established that these factors are essential for the development and maintenance of the nervous system. Due to the difficulty of purifying enough quantities of these factors and the lack of specificity from commercially available antibodies, the aim of this work was to produce antibodies against a synthetic peptide capable to detect and identify neuregulin GGFbeta isoforms. To accomplish this goal, polyclonal antibodies were raised in hens against a synthetic peptide designed from the GGFbeta1 extracellular sequence. The sequence analysis was made using different epitope-predicting programs. Our results showed that the peptide sequence selected was immunogenic because it was capable of inducing a specific type B immune response in the experimental animal model. These antibodies were also capable of recognizing a recombinant GGF protein and GGF isoforms present in different samples. Our results suggest that the development of immunoglobulin Y (IgY) using synthetic peptides represents, a valuable tool for neuroscience research.

Production and evaluation of chicken antibodies against a synthetic peptide from glial growth factor / Producción y evaluación de anticuerpos de gallinas contra un péptido sintético del factor de crecimiento glial

Neuregulins (NRG) are proteins that belong to the family of epidermal growth factors. It is well established that these factors are essential for the development and maintenance of the nervous system. Due to the difficulty of purifying enough quantities of these factors and the lack of specificity from commercially available antibodies, the aim of this work was to produce antibodies against a synthetic peptide capable to detect and identify neuregulin GGFb isoforms. To accomplish this goal, polyclonal antibodies were raised in hens against a synthetic peptide designed from the GGFb1 extracellular sequence. The sequence analysis was made using different epitope-predicting programs. Our results showed that the peptide sequence selected was immunogenic because it was capable of inducing a specific type B immune response in the experimental animal model. These antibodies were also capable of recognizing a recombinant GGF protein and GGF isoforms present in different samples. Our results suggest that the development of immunoglobulin Y (IgY) using synthetic peptides represents, a valuable tool for neuroscience research.

Las Neuregulinas (NRG) son proteínas que pertenecen a la familia de los factores de crecimiento epidermal. Se ha demostrado que estos factores son esenciales para el desarrollo y mantenimiento de la funcionalidad del sistema nervioso. Debido a la dificultad para purificar estas proteínas y la falta de especificidad de los anticuerpos disponibles comercialmente, el objetivo de este trabajo fue producir anticuerpos contra un péptido sintético capaz de detectar e identificar una isoforma de la Neuregulina (GGFb). Para lograr este objetivo, se desarrollaron anticuerpos en gallinas (IgY) contra un péptido sintético diseñado a partir de la secuencia aminoacídica de la región extracelular de GGFb, utilizando programas de predicción de epítopes. Los resultados demuestran que el péptido seleccionado fue immunogénico debido a que estimuló una respuesta inmune específica tipo B en el modelo utilizado. Estos anticuerpos fueron también capaces de reconocer una proteína recombinante e isoformas de GGF presentes en diferentes muestras biológicas. Nuestros resultados demuestran el potencial valor de las inmunoglobulinas Y (IgY) contra péptidos sintéticos como una herramienta de aplicación para la investigación en neurociencia.

Identification of CD3ε, CD4, CD8ß splice variants of Atlantic salmon.

In vertebrates, CD3 complex and CD4 and CD8 co-receptors are essential for signal transduction during T cell activation. In the present study, we report the mRNA spliced variants of the Atlantic salmon CD3ε, CD4 and CD8ß and the effect of pathogen encounter on the expression of these variants. CD3ε is alternatively spliced in thymus, head kidney, spleen and gills to give rise to the complete mRNA sequence and to an alternative product that lacks the transmembrane exon. CD4 is also alternatively spliced in the thymus, head kidney, spleen and gills to form two variants, although the alternative product is barely detectable. The alternative product lacks the exon 1B encoding the D1 domain, which is essential for binding to MHC class II proteins. Two amplicons were also found for the CD8ß gene; sequencing analysis revealed that the main PCR product corresponds to the previously reported CD8ß sequence, whereas the variant sequence encodes a potential protein that lacks the Ig-like domain. The expression of CD3, CD4, CD8ß genes also analyzed in head kidney of LPS-treated and IPNV infected salmon and different patterns of expression were observed. The presence and balance of the different variants of T cell co-receptors could be related to the ability of fish to induce a particular type of immune response, as well as, the ability of the pathogen to modify the fish immune response.

The neural cell adhesion molecule is involved in the metastatic capacity in a murine model of lung cancer.

Neural cell adhesion molecule (NCAM) is involved in cell growth, migration, and differentiation. Its expression and/or polysialylation appear to be deregulated in many different cancer types. We employed the lung tumor cell line LP07, syngeneic in BALB/c mice to investigate the role of NCAM in malignant progression. LP07 cells express the three main NCAM isoforms, all of them polysialylated. This cells line, pretreated with an anti-NCAM antibody and inoculated intravenously (i.v.) into syngeneic mice, developed less and smaller lung metastases. In vitro studies showed that NCAM bound antibody inhibited cell growth, mainly due to an increase in apoptosis, associated with a decrease of cyclin D1 and enhanced expression of active caspase 3 and caspase 9. Anti-NCAM-treated LP07 cells showed impairment in their ability to migrate and adhere to several extracellular matrix components. Secreted uPA activity was also reduced. NCAM-140 knocked-down by siRNA in LP07 cells pretreated or not with anti-NCAM showed an impaired metastasizing ability upon i.v. inoculation into mice. These results suggest that anti-NCAM treatment could be mimicking homophilic trans-interactions and NCAM-140 knocked-down impairs heterophilic interactions, both leading to inhibition of metastatic dissemination. The involvement of NCAM in lung tumor progression was confirmed in human NSCLC tumors. Sixty percent of the cases expressed NCAM at tumor cell level. A multivariate analysis indicated that NCAM expression was associated with a shorter overall survival in this homogeneous series of Stages I and II NSCLC patients. NCAM may be able to modulate mechanisms involved in lung carcinoma progression and represents an attractive target to control metastatic progression.

[Fcgamma receptor polymorphisms and systemic lupus erythematosus]. / Polimorfismos de los receptores fcgamma y lupus eritematoso sistémico.

The G Immunoglobulin Fc Receptors (FcgammaR) belong to the TNFR5 receptors family, of the immunoglobulin superfamily and are widely expressed in the immune system; their function follows in importance after the complement receptors for immunocomplexes clearance. On the other hand, the systemic lupus erythematosus (SLE) is the prototype of the autoimmune diseases mediated by immunocomplexes and several studies have shown an impaired handle of these ones in part due to dysfunction of the FcgammaR. Among all types of Fcgamma receptors, the FcgammaRIIA, FcgammaRIIB, FcgammaRIIIA and FcgammaRIIIB have well characterized polymorphisms that produce an alteration in the receptor function. A number of studies have been done worldwide to probe an association between these polymorphism and SLE or some of its clinical features, among these the most important are two meta-analyses in which it is shown that the FcygammaRIIA-R131 polymorphism present a significant association with SLE susceptibility (OR: 1.3, 95% CI: 1.10-1.52), while the FcgammaRIIIA F176 polymorphism showed to be associated with lupic nephritis (OR: 1.47, 95% CI: 1.11-1.93, p = 0.006) but not with SLE susceptibility, the results in the rest of the polymorphisms studied are still contradictories.

Immunological characterization of a Blo t 12 isoallergen: identification of immunoglobulin E epitopes.

BACKGROUND: Differences in the IgE response to isoallergens could have clinical implications; therefore, its analysis will contribute to the design of better strategies for the diagnosis and treatment of allergic respiratory diseases. Several isoforms have been described from mites but there is no information about the clinical impact of Blomia tropicalis isoallergens. OBJECTIVE: To evaluate the differences in the IgE response against two Blo t 12 isoallergens. METHODS: The IgE-binding properties of Blo t 12 isoallergens were analysed by ELISA, a skin prick test and ELISA cross-inhibition. Epitope mapping was performed using synthetic overlapping peptides. Fold recognition methods were used to model the chitin-binding domain of the two isoallergens. RESULTS: The frequency and strength of the IgE response were greater for Blo t 12.0101 than for Blo t 12.0102. Three IgE-binding areas were identified in Blo t 12.0101; one of them included two residues that are different in Blo t 12.0102. Modelling of the chitin-binding domains of these allergens predicted that they have structural differences that could influence antibody recognition of one of these epitopes. CONCLUSION: In silico structural analysis and immunological characterization of Blo t 12 reveals that allergen polymorphism influences IgE reactivity. Blo t 12.0101 is the most IgE-reactive isoform in Cartagena. The identified IgE epitopes could be mutated to obtain hypoallergenic molecules of potential use for immunotherapy.

[Characterizing the CD3 epsilon chain from the New World primate Aotus nancymaae]. / Caracterización de la cadena CD3 épsilon en el primate del nuevo mundo Aotus nancymaae.

INTRODUCTION: The T-cell receptor (TCR)-associated complex, CD3 (d, g, e) and z-chains are essential transmembrane proteins for signal transduction during T cell activation and immune response, as well as during thymocyte development. OBJECTIVE: This work established the CD3epsilon-chain primary structure for the New World owl monkey Aotus nancymaae. MATERIALS AND METHODS: Total RNA was isolated from peripheral blood mononuclear cells; CD3epsilon molecule was amplified, cloned and sequenced. RESULTS: The CD3epsilon amino acid sequence was deduced for the owl monkey Aotus nancymaae.> It has an identity for nucleotide and amino acid sequences with the human counterpart of 84% and 76%, respectively. As described in other species, the Aotus CD3-e molecule is very variable in the extracellular region and greatly conserved in the intracellular domain. Even though high variability occurs in the CD3epsilon-extracellular domain, the subregions involved in ectodomain folding are conserved. CONCLUSIONS: The primary structure suggested that the Aotus protein has a functional role similar to that of humans, and that the initial T-cell activation steps are also similar. However, the great variation observed in CD3epsilon-extracellular region in humans in contrast to the Aotus (especially in areas that are surface-exposed) indicated that some monoclonal antibodies against the human CD3 complex will not recognize these Aotus determinants.

Methotrexate regulates the expression of glucocorticoid receptor alpha and beta isoforms in normal human peripheral mononuclear cells and human lymphocyte cell lines in vitro.

MTX is an effective therapy for autoimmune-inflammatory diseases. The mechanisms that mediate these actions are not completely clear. It is accepted that many of these effects are mediated through the release of adenosine with the activation of the adenosine receptor A2. MTX is used as a steroid sparing agent. An improved in vitro GC cell sensitivity in GC insensitive asthma patients has been demonstrated after MTX treatment. Most GC actions are mediated by the GCR. The effect of MTX on GCRs expression has not been previously evaluated. Therefore, we evaluate if MTX regulates the expression of glucocorticoid receptors, increasing the expression of the active receptor (GCR alpha) and/or decreasing the expression of the dominant negative receptor (GCR beta). We show that MTX increases the mRNA and protein levels of GCR alpha and decreases or leaves unchanged the protein expression of the GCR beta in CEM cells in culture. This effect was also observed in other lymphocytes (Jurkat and Raji) and in PBMNC from healthy volunteers. We also show that upon MTX treatment PBMC from normal volunteers exhibit a higher sensitivity to DEX inhibition on LPS-induced TNF alpha release. To explore if these actions are mediated by adenosine through the adenosine receptor A2 we evaluate the effect of adenosine on the GCRs expression and the effect of an A2 receptor blocker (DMPX) on MTX effects on GCRs expression. Our results show that adenosine does not mimic and DMPX can enhance MTX effects on these receptors. We conclude that MTX increases the GCR alpha/GCR beta ratio of expression in lymphocytes which could mediate its previously reported effects in improving cell glucocorticoid sensitivity. These actions are not mediated by the adenosine receptor A2.

Signaling capacity of FcgammaRII isoforms in B-CLL cells.

Two main isoforms of Fcgamma receptor II (CD 32) have been described in humans: activatory FcgammaRIIA and inhibitory FcgammaRIIB. We have previously reported that B cells from a subset of chronic lymphocytic leukemia (B-CLL) patients express not only FcgammaRIIB, as normal B lymphocytes, but also the myeloid FcgammaRIIA. The aim of this study was to evaluate the signaling capacity of both FcgammaRII isoforms in B-CLL cells. We found that FcgammaRIIA expressed by leukemic cells failed to induce Ca(2+) mobilization or protein tyrosine phosphorylation, suggesting that the receptor is not functional. By contrast, FcgammaRIIB effectively diminished BCR-triggered ERK 1 phosphorylation, which indicates that it is able to transduce inhibitory signals in B-CLL cells. Moreover, we found that FcgammaRIIB homoaggregation in either B-CLL or non-malignant tonsillar B cells did not result in apoptosis as was reported for murine B splenocytes. Together, these results show that FcgammaRIIB, but not FcgammaRIIA is biologically active in B-CLL cells and might influence leukemic cell physiology in vivo.

Muscular myosin isoforms of Taenia solium (Cestoda).

Type II myosin, the primary component of the thick filament of muscle fibers, is organized as a dimeric high molecular weight protein, and is composed of a pair of heavy chains (MHC) and two pairs of light chains. Myosin II transforms ATP energy into mechanical force. All type II myosins are conserved proteins but they have two variable regions that are located in different places of the molecule. Myosin molecules are encoded by a multigene family and many isoforms are generated. The expression of myosins depends on the developmental stage and on the type and degree of contractile activity and tissue, therefore several myosin isoforms are found in the same organism. Here we describe the use of different techniques that allowed demonstrating the presence of isoforms of the heavy chain type II myosin of Taenia solium cysticerci (larvae) and tapeworms (adults), a cestode parasite of importance in public health in many developing countries. Myosin was purified and used in comparative proteolytic fragmentation, ATPase activity, detection of antigenic differences and electrophoretic separation. The results obtained showed biochemical and immunochemical differences among cysticerci and tapeworms, and demonstrate the presence of myosin isoforms in T. solium that are probably associated to physiological requirements of each developmental stage.

Prolactin in eyes of patients with retinopathy of prematurity: implications for vascular regression.

PURPOSE: Disruption of the anti-angiogenic environment of the retina leads to neovascular eye diseases, including retinopathy of prematurity (ROP). Prolactin (PRL), the hormone originally associated with milk secretion, is proteolytically processed to 16K-PRL, a fragment with potent antiangiogenic, proapoptotic effects. Whether 16K-PRL is produced in eyes of patients with ROP and promotes the regression of intraocular blood vessels associated with this disease was investigated. METHODS: PRL was quantified in the aqueous humor, subretinal fluid, and serum from patients with stage 5 ROP and in patients with non-neovascular eye disorders. Intraocular expression of PRL was evaluated by RT-PCR, in situ hybridization, and Western blot analysis. AntiPRL antibodies were injected intravitreously in neonatal rats, and apoptosis of hyaloid vessels determined by TUNEL and ELISA. RESULTS: PRL was elevated in ocular fluids and serum from ROP patients. There was no correlation between PRL in ocular fluids and its level in serum, whereas PRL in aqueous humor and subretinal fluid were significantly correlated. PRL mRNA was expressed in blood vessels and leukocytes within retrolental fibrovascular membranes of ROP patients, and these membranes contained a 16 kDa immunoreactive PRL. The 16K-PRL isoform was more concentrated in subretinal fluid than in serum and was generated from PRL by subretinal fluid proteases. Intravitreous injection of neutralizing antiPRL antibodies inhibited the apoptosis of hyaloid vessels in neonatal rats. CONCLUSIONS: 16K-PRL derived from PRL internalized from the circulation or synthesized intraocularly can stimulate apoptosis-induced vascular regression and contribute to the development and progression of ROP.