RESUMO
Aire is a transcriptional controller in medullary thymic epithelial cells (mTECs) modulating a set of peripheral tissue antigens (PTAs) and non-PTA mRNAs as well as miRNAs. Even miRNAs exerting posttranscriptional control of mRNAs in mTECs, the composition of miRNA-mRNA networks may differ. Under reduction in Aire expression, networks exhibited greater miRNA diversity controlling mRNAs. Variations in the number of 3'UTR binding sites of Aire-dependent mRNAs may represent a crucial factor that influence the miRNA interaction. To test this hypothesis, we analyzed through bioinformatics the length of 3'UTRs of a large set of Aire-dependent mRNAs. The data were obtained from existing RNA-seq of mTECs of wild type or Aire-knockout (KO) mice. We used computational algorithms as FASTQC, STAR and HTSEQ for sequence alignment and counting reads, DESEQ2 for the differential expression, 3USS for the alternative 3'UTRs and TAPAS for the alternative polyadenylation sites. We identified 152 differentially expressed mRNAs between these samples comprising those that encode PTAs as well as transcription regulators. In Aire KO mTECs, most of these mRNAs featured an increase in the length of their 3'UTRs originating additional miRNA binding sites and new miRNA controllers. Results from the in silico analysis were statistically significant and the predicted miRNA-mRNA interactions were thermodynamically stable. Even with no in vivo or in vitro experiments, they were adequate to show that lack of Aire in mTECs might favor the downregulation of PTA mRNAs and transcription regulators via miRNA control. This could unbalance the overall transcriptional activity in mTECs and thus the self-representation.
Assuntos
Regiões 3' não Traduzidas , RNA Mensageiro/genética , Timo/metabolismo , Fatores de Transcrição/genética , Algoritmos , Animais , Antígenos/genética , Sítios de Ligação/genética , Simulação por Computador , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Camundongos , Camundongos Knockout , MicroRNAs/genética , Poliadenilação/genética , Poliendocrinopatias Autoimunes/genética , RNA-Seq , Alinhamento de Sequência , Timo/citologia , Timo/imunologia , Fatores de Transcrição/deficiência , Proteína AIRERESUMO
Rhipicephalus microplus (formerly Boophilus microplus) ticks are potential vectors of several pathogens of livestock especially in tropical and subtropical regions where may have substantial effects on economic development. Among tick-borne pathogens, Anaplasma marginale is considered one of the most important in domestic and wild ruminants worldwide. Different molecular mechanisms have been employed by both ticks and these intracellular pathogens, in order to be able to adapt and survive. Subolesin, originally called 4D8, is an evolutionarily well-preserved protein among ixodid tick species. This new antigen was found to be protective against tick infestations when used as a vaccine, as it has an essential role in tick blood digestion, development and infection of host cells by A. marginale. Recent studies have demonstrated that infection of both tick and vertebrate host cells with this microorganism changed gene expression. Therefore, the main objective of this study was to investigate subolesin expression in uninfected and A. marginale-infected R. microplus salivary glands by real-time reverse transcriptase (RT)-PCR. To analyze the differential expression of the recombinant protein subolesin, the gene was previously expressed from ticks infected with A. marginale. Results from this study revealed that, the expression of subolesin was significantly higher in salivary glands of infected R. microplus in comparison to uninfected ones.
Assuntos
Anaplasma marginale/fisiologia , Antígenos/genética , Proteínas de Artrópodes/genética , Expressão Gênica , Rhipicephalus/genética , Rhipicephalus/microbiologia , Anaplasmose/imunologia , Anaplasmose/microbiologia , Animais , Antígenos/metabolismo , Proteínas de Artrópodes/metabolismo , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/microbiologia , Feminino , Reação em Cadeia da Polimerase/veterinária , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rhipicephalus/metabolismo , Glândulas Salivares/metabolismo , Glândulas Salivares/microbiologiaRESUMO
The 2016-2017 epidemic of influenza A (H7N9) virus in China prompted concern that a genetic change may underlie increased virulence. Based on an evolutionary analysis of H7N9 viruses from all five outbreak waves, we find that additional subclades of the H7 and N9 genes have emerged. Our analysis indicates that H7N9 viruses inherited NP genes from co-circulating H7N9 instead of H9N2 viruses. Genotypic diversity among H7N9 viruses increased following wave I, peaked during wave III, and rapidly deceased thereafter with minimal diversity in wave V, suggesting that the viruses entered a relatively stable evolutionary stage. The ZJ11 genotype caused the majority of human infections in wave V. We suggest that the largest outbreak of wave V may be due to a constellation of genes rather than a single mutation. Therefore, continuous surveillance is necessary to minimize the threat of H7N9 viruses.
Assuntos
Subtipo H7N9 do Vírus da Influenza A/genética , Influenza Humana/patologia , Substituição de Aminoácidos , Antígenos/genética , Antígenos/imunologia , Antígenos/metabolismo , China/epidemiologia , Surtos de Doenças , Evolução Molecular , Genótipo , Humanos , Subtipo H7N9 do Vírus da Influenza A/isolamento & purificação , Subtipo H7N9 do Vírus da Influenza A/patogenicidade , Influenza Humana/epidemiologia , Influenza Humana/virologia , Proteínas do Nucleocapsídeo , Filogenia , Proteínas de Ligação a RNA/classificação , Proteínas de Ligação a RNA/genética , RNA Polimerase Dependente de RNA/classificação , RNA Polimerase Dependente de RNA/genética , Proteínas do Core Viral/classificação , Proteínas do Core Viral/genética , Proteínas Virais/classificação , Proteínas Virais/genéticaRESUMO
Twenty years ago, the autoimmune regulator (Aire) gene was associated with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy, and was cloned and sequenced. Its importance goes beyond its abstract link with human autoimmune disease. Aire identification opened new perspectives to better understand the molecular basis of central tolerance and self-non-self distinction, the main properties of the immune system. Since 1997, a growing number of immunologists and molecular geneticists have made important discoveries about the function of Aire, which is essentially a pleiotropic gene. Aire is one of the functional markers in medullary thymic epithelial cells (mTECs), controlling their differentiation and expression of peripheral tissue antigens (PTAs), mTEC-thymocyte adhesion and the expression of microRNAs, among other functions. With Aire, the immunological tolerance became even more apparent from the molecular genetics point of view. Currently, mTECs represent the most unusual cells because they express almost the entire functional genome but still maintain their identity. Due to the enormous diversity of PTAs, this uncommon gene expression pattern was termed promiscuous gene expression, the interpretation of which is essentially immunological - i.e. it is related to self-representation in the thymus. Therefore, this knowledge is strongly linked to the negative selection of autoreactive thymocytes. In this update, we focus on the most relevant results of Aire as a transcriptional and post-transcriptional controller of PTAs in mTECs, its mechanism of action, and its influence on the negative selection of autoreactive thymocytes as the bases of the induction of central tolerance and prevention of autoimmune diseases.
Assuntos
Seleção Clonal Mediada por Antígeno/genética , Seleção Clonal Mediada por Antígeno/imunologia , Timócitos/citologia , Timócitos/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Antígenos/genética , Antígenos/imunologia , Antígenos/metabolismo , Apoptose , Autoimunidade , Biomarcadores , Adesão Celular/genética , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Humanos , Tolerância Imunológica/genética , Mutação , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Proteína AIRERESUMO
BACKGROUND: In the present work we described the recombinant production and characterization of heterodimeric construction ZnT8-Arg-Trp325 fused to thioredoxin using a high-performance expression system such as Escherichia coli. In addition, we apply this novel recombinant antigen in a non-radiometric method, with high sensitivity, low operational complexity and lower costs. RESULTS: ZnT8 was expressed in E. coli as a fusion protein with thioredoxin (TrxZnT8). After 3 h for induction, recombinant protein was obtained from the intracellular soluble fraction and from inclusion bodies and purified by affinity chromatography. The expression and purification steps, analyzed by SDS-PAGE and western blot, revealed a band compatible with TrxZnT8 expected theoretical molecular weight (≈ 36.8 kDa). The immunochemical ability of TrxZnT8 to compete with [35S]ZnT8 (synthesized with rabbit reticulocyte lysate system) was assessed qualitatively by incubating ZnT8A positive patient sera in the presence of 0.2-0.3 µM TrxZnT8. Results were expressed as standard deviation scores (SDs). All sera became virtually negative under antigen excess (19.26-1.29 for TrxZnT8). Also, radiometric quantitative competition assays with ZnT8A positive patient sera were performed by adding TrxZnT8 (37.0 pM-2.2 µM), using [35S]ZnT8. All dose-response curves showed similar protein concentration that caused 50% inhibition (14.9-0.15 nM for TrxZnT8). On the other hand, preincubated bridge ELISA for ZnT8A detection was developed. This assay showed 51.7% of sensitivity and 97.1% of specificity. CONCLUSIONS: It was possible to obtain with high-yield purified heterodimeric construction of ZnT8 in E. coli and it was applied in cost-effective immunoassay for ZnT8A detection.
Assuntos
Autoanticorpos/sangue , Diabetes Mellitus Tipo 1/imunologia , Escherichia coli/genética , Transportador 8 de Zinco/genética , Transportador 8 de Zinco/imunologia , Animais , Antígenos/genética , Antígenos/imunologia , Autoanticorpos/imunologia , Ensaio de Imunoadsorção Enzimática/economia , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Coelhos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Tiorredoxinas/química , Tiorredoxinas/genéticaRESUMO
Computational methods have traditionally struggled to predict the effect of mutations in antibody-antigen complexes on binding affinity. This has limited their usefulness during antibody engineering and development, and their ability to predict biologically relevant escape mutations. Here we present mCSM-AB, a user-friendly web server for accurately predicting antibody-antigen affinity changes upon mutation which relies on graph-based signatures. We show that mCSM-AB performs better than comparable methods that have been previously used for antibody engineering. mCSM-AB web server is available at http://structure.bioc.cam.ac.uk/mcsm_ab.
Assuntos
Anticorpos/genética , Anticorpos/imunologia , Afinidade de Anticorpos/genética , Antígenos/imunologia , Internet , Mutação , Software , Anticorpos/química , Afinidade de Anticorpos/imunologia , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/genética , Complexo Antígeno-Anticorpo/imunologia , Antígenos/química , Antígenos/genética , Benchmarking , Conjuntos de Dados como Assunto , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/genética , Anticorpos Anti-HIV/imunologia , Engenharia de Proteínas , Vacinas/química , Vacinas/genética , Vacinas/imunologiaRESUMO
The objective was to investigate a family from Argentina. The proposita was a 51-year-old woman who had a moderate bleeding tendency. Some of her children showed a mild bleeding tendency. Her mother and the husband were asymptomatic. Clotting, immunological and molecular biology techniques were used. Partial thromboplastin, prothrombin, Russell Viper venom-clotting times were moderately prolonged in the proposita, whereas they were slightly prolonged in the children and in her mother. Factor X (FX) activity was about 2-3% of normal in all assay systems. FX antigen was less than 5%. Other clotting factors and platelet were normal. Genetic analysis showed a compound heterozygosis: combination of a 'new' mutation (Gln138Arg) with an already known mutation (Glu350Lys). The children had intermediate FX levels (35-63% of normal) and were carriers of one of the two mutations present in the proposita. This is the first observation of a FX deficiency in Argentina.
Assuntos
Antígenos/genética , Deficiência do Fator X/genética , Fator X/genética , Hemorragia/genética , Heterozigoto , Mutação , Adolescente , Adulto , Antígenos/sangue , Argentina , Sequência de Bases , Testes de Coagulação Sanguínea , Análise Mutacional de DNA , Deficiência do Fator X/sangue , Deficiência do Fator X/diagnóstico , Feminino , Expressão Gênica , Hemorragia/sangue , Hemorragia/diagnóstico , Humanos , Masculino , Pessoa de Meia-Idade , LinhagemRESUMO
Human platelet antigens (HPA) are immunogenic structures that result from single nucleotide polymorphisms (SNPs) leading to single amino acid substitutions. This study sought to determine the allele and genotype frequencies of HPA-1, HPA-2, HPA-3, HPA-4, HPA-5 and HPA-15 in platelet donors from the state of Rio Grande do Sul (RS), Brazil, and compare their allele frequencies to those observed in other populations. HPA genotyping was performed by PCR-SSP method. The study sample comprised 201 platelet donors (167 Caucasians and 34 non-Caucasians). Allele 'a' was that most commonly found for HPA-1 to 5 in both groups. The HPA-15ab genotype predominated over homozygous genotypes of this system. Fisher's exact test revealed statistically significant differences for the HPA-5 system, with a greater prevalence of the HPA-5b allele in non-Caucasians. The neighbour-joining method and principal components analysis revealed genetic proximity between our Caucasian group and European populations. We conclude that the allele frequencies of HPA-1 to 5 and HPA-15 found in our Caucasian sample are similar to those reported for European populations. These findings corroborate the ethnic makeup of the population of RS. The higher frequency of the HPA-5b allele found in the non-Caucasian group of our sample suggests the possibility of allosensitization in patients who receive platelet transfusions from genetically incompatible donors.
Assuntos
Antígenos/genética , Doadores de Sangue , Plaquetas/metabolismo , Técnicas de Genotipagem/métodos , Adulto , Brasil , Feminino , Frequência do Gene/genética , Loci Gênicos , Genótipo , Humanos , Masculino , Filogenia , Análise de Componente PrincipalRESUMO
Taenia solium cysticercosis is a major parasitic disease that affects the human health and the economy in underdeveloped countries. Porcine cysticercosis, an obligatory stage in the parasite life cycle, is a suitable target for vaccination. While several recombinant and synthetic antigens proved to be effective as vaccines, the cost and logistic difficulties have prevented their massive use. Taking this into account, a novel strategy for developing a multi-epitope low-cost vaccine is herein explored. The S3Pvac vaccine components (KETc1, KETc12, KETc7, and GK1 [KETc7]) and the protective HP6/TSOL18 antigen were expressed in a Helios2A polyprotein system, based on the 'ribosomal skip' mechanism mediated by the 2A sequence (LLNFDLLKLAGDVESNPG-P) derived from the Foot-and-mouth disease virus, which induces self-cleavage events at a translational level. This protein arrangement was expressed in transgenic tobacco cells. The inserted sequence and its transcript were detected in several Helios2A lines, with some lines showing recombinant protein accumulation levels up to 1.3 µg/g of fresh weight in leaf tissues. The plant-derived Helios2A vaccine was recognized by antibodies in the cerebral spinal fluid from neurocysticercosis patients and elicited specific antibodies in BALB/c immunized mice. These evidences point to the Helios2A polyprotein as a promising system for expressing multiple antigens of interest for vaccination and diagnosis in one single construction.
Assuntos
Antígenos/genética , Cisticercose/imunologia , Epitopos/genética , Vacinas/imunologia , Animais , Antígenos/biossíntese , Cisticercose/parasitologia , Cisticercose/prevenção & controle , Epitopos/imunologia , Vírus da Febre Aftosa/genética , Humanos , Imunização , Camundongos , Células Vegetais , Proteínas Recombinantes/genética , Ribossomos/genética , Suínos , Taenia solium/genética , Taenia solium/patogenicidade , Vacinas/genéticaRESUMO
The downregulation of PTA genes in mTECs is associated with the loss of self-tolerance, and the role of miRNAs in this process is not fully understood. Therefore, we studied the expression of mRNAs and miRNAs in mTECs from autoimmune NOD mice during the period when loss of self-tolerance occurs in parallel with non-autoimmune BALB/c mice. Although the expression of the transcriptional regulator Aire was unchanged, we observed downregulation of a set of PTA mRNAs. A set of miRNAs was also differentially expressed in these mice. The reconstruction of miRNA-mRNA interaction networks identified the controller miRNAs and predicted the PTA mRNA targets. Interestingly, the known Aire-dependent PTAs exhibited pronounced refractoriness in the networking interaction with miRNAs. This study reveals the existence of a new mechanism in mTECs, and this mechanism may have importance in the control of self-tolerance.