RESUMO
PURPOSE: Indirect immunofluorescence (IIF) on the human epithelial cell-line HEp-2 (or derivatives) serves as the gold standard in antinuclear antibody (ANA) screening. IIF, and its evaluation, is a labor-intensive method, making ANA testing a major challenge for present clinical laboratories. Nowadays, several automated ANA pattern recognition systems are on the market. In the current study, the EUROPattern Suite is evaluated for its use in daily practice in a routine setting. METHODS: A total of 1033 consecutive routine samples was used to screen for ANA. Results (positive/negative ANA screening, pattern identification and titer) were compared between software-generated results (EUROPattern) and visual interpretation (observer) of automatically acquired digital images. RESULTS: Considering the visual interpretation as reference, a relative sensitivity of 99.3% and a relative specificity of 88.9% were obtained for negative and positive discrimination by the software (EPa). A good agreement between visual and software-based interpretation was observed with respect to pattern recognition (mean kappa: for 7 patterns: 0.7). Interestingly, EPa software distinguished more patterns per positive sample than the observer (on average 1.5 and 1.2, respectively). Finally, a concordance of 99.3% was observed within the range of 1 titer step difference between EPa and observer. CONCLUSIONS: The ANA IIF results reported by the EPa software are in very good agreement with the results reported by the observer with respect to being negative/positive, pattern recognition and titer, making automated ANA IIF evaluation an objective and time-efficient tool for routine testing.
RESUMO
Autoantibodies against neuronal cell surface antigens are tightly associated with immunotherapy-responsive autoimmune encephalitis, and a considerable number of corresponding autoantigens has been identified in recent years. Most patients initially present with overlapping symptoms, and a broad range of autoantibodies has to be considered to establish the correct diagnosis and initiate treatment as soon as possible to prevent irreversible and sometimes even life-threatening damage to the brain. Recombinant cell-based immunofluorescence allows to authentically present fragile membrane-associated surface antigens and, in combination with multiparametric analysis in the form of biochip mosaics, has turned out to be highly beneficial for parallel and prompt determination of anti-neuronal autoantibodies and comprehensive differential diagnostics. For the evaluation of recombinant cell-based IIFT, a semi-automated novel function was introduced into an established platform for computer-aided immunofluorescence microscopy. The system facilitates the microscopic analysis of the tests and supports the laboratory personnel in the rapid issuance of diagnostic findings, which is of major importance for autoimmune encephalitis patients since timely initiation of treatment may lead to their full recovery.
Assuntos
Encefalite/sangue , Doença de Hashimoto/sangue , Autoanticorpos/sangue , Encéfalo/patologia , Encefalite/diagnóstico , Doença de Hashimoto/diagnóstico , Humanos , Interpretação de Imagem Assistida por Computador , Microscopia de FluorescênciaRESUMO
The synaptonemal complex (SC), a key structure of meiosis that assembles during prophase I, has been initially described 60 years ago. Since then, the structure has been described in many sexually reproducing organisms. However, the SC protein components were characterized in only few model organisms. Surprisingly, they lacked an apparent evolutionary relationship despite the conserved structural organization of the SC. For better understanding of this obvious discrepancy, the evolutionary history of the SC and its individual components has been investigated in Metazoa in detail. The results are consistent with the notion of a single origin of the metazoan SC and provide evidence for a dynamic evolutionary history of the SC components. In this mini review, we recapitulate and discuss new insights into metazoan SC evolution.
Assuntos
Evolução Molecular , Complexo Sinaptonêmico/genética , Complexo Sinaptonêmico/metabolismo , Animais , HumanosRESUMO
Systemic lupus erythematosus (SLE) is a severe rheumatic autoimmune disease with various clinical manifestations. Anti-dsDNA antibodies are an important immunological hallmark of SLE and their occurrence represents a major criterion for the diagnosis. Among the commonly applied test systems for determination of anti-dsDNA antibodies, the indirect immunofluorescence test (IIFT) using the flagellated kinetoplastida Crithidia luciliae is considered to be highly disease specific at moderate sensitivity. Since IIFT, however, is claimed to be affected by subjective interpretation and a lack of standardization, there has been an increasing demand for automated pattern interpretation of immunofluorescence reactions in recent years. Corresponding platforms are already available for evaluation of anti-nuclear antibody (ANA) IIFT on HEp-2 cells, the recommended "gold standard" for ANA screening in the diagnosis of various systemic rheumatic autoimmune diseases. For one of these systems, the "EUROPattern-Suite" computer-aided immunofluorescence microscopy (CAIFM), automated interpretation of microscopic fluorescence patterns was extended to the Crithidia luciliae based anti-dsDNA IIFT.
Assuntos
Anticorpos Antinucleares/imunologia , Crithidia/imunologia , Técnica Indireta de Fluorescência para Anticorpo , Automação Laboratorial , Humanos , Lúpus Eritematoso Sistêmico/diagnóstico , Lúpus Eritematoso Sistêmico/imunologia , Microscopia de Fluorescência , Reprodutibilidade dos Testes , Doenças Reumáticas/diagnóstico , Doenças Reumáticas/imunologia , Sensibilidade e Especificidade , Fluxo de TrabalhoRESUMO
Indirect immunofluorescence assay is the recommended gold standard to test for antinuclear antibodies (ANA), which are important biomarkers for systemic rheumatic autoimmune diseases. It is internationally accepted that indirect immunofluorescence assay ANA screening is most sensitive on human epithelial (HEp-2) cells. The cells present a multitude of antigens that display distinguishable localization patterns in interphase and mitotic cells in indirect immunofluorescence analysis. Here, we present the IFA 40: HEp-20-10 test kit (Euroimmun AG, Lübeck, Germany), which is cleared for sale on the US market by the FDA. The test has been designed for qualitative and semiquantitative screening of ANA in human sera. It uses the commonly applied 1:40 cutoff dilution and the enhanced HEp-20-10 cell line for more efficient pattern recognition and has been validated in various studies and by method comparison. The IFA 40: HEp-20-10 test fulfills the essential criteria for reliable application in autoimmune diagnostics.
Assuntos
Anticorpos Antinucleares/imunologia , Técnica Indireta de Fluorescência para Anticorpo/métodos , Anticorpos Antinucleares/sangue , Doenças Autoimunes/sangue , Doenças Autoimunes/diagnóstico , Técnica Indireta de Fluorescência para Anticorpo/normas , Humanos , Kit de Reagentes para Diagnóstico/normas , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
The synaptonemal complex (SC) is an evolutionarily well-conserved structure that mediates chromosome synapsis during prophase of the first meiotic division. Although its structure is conserved, the characterized protein components in the current metazoan meiosis model systems (Drosophila melanogaster, Caenorhabditis elegans, and Mus musculus) show no sequence homology, challenging the question of a single evolutionary origin of the SC. However, our recent studies revealed the monophyletic origin of the mammalian SC protein components. Many of them being ancient in Metazoa and already present in the cnidarian Hydra. Remarkably, a comparison between different model systems disclosed a great similarity between the SC components of Hydra and mammals while the proteins of the ecdysozoan systems (D. melanogaster and C. elegans) differ significantly. In this review, we introduce the basal-branching metazoan species Hydra as a potential novel invertebrate model system for meiosis research and particularly for the investigation of SC evolution, function and assembly. Also, available methods for SC research in Hydra are summarized.
Assuntos
Hydra/metabolismo , Meiose/fisiologia , Complexo Sinaptonêmico/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Linhagem da Célula , Drosophila melanogaster/metabolismo , Evolução Molecular , Hydra/citologia , Hydra/genética , Modelos Animais , Especificidade da Espécie , VertebradosRESUMO
During the first meiotic prophase, chromosome synapsis is mediated by the synaptonemal complex (SC), an evolutionarily conserved meiosis-specific structure. In mammals, 7 SC protein components have been identified so far. Despite some controversy in the past, we have shown that SC proteins are ancient in metazoans and very likely formed an ancestral SC structure in the ancestor of metazoans. Protein components SYCP1, SYCP3, SYCE2, and TEX12 were identified in basal-branching metazoans, while other components (SYCE1 and SYCE3) are more recent elements. However, the evolutionary history of mammalian SYCP2 is not known. Here, we investigated this aspect with the aid of bioinformatic tools as well as with RNA and protein expression analysis. We conclude that SYCP2 belongs to the group of ancient SC proteins that was already present in the common ancestor of metazoans more than 500 million years ago.
Assuntos
Hydra/metabolismo , Mamíferos/metabolismo , Complexo Sinaptonêmico/genética , Complexo Sinaptonêmico/metabolismo , Testículo/metabolismo , Animais , Biologia Computacional/métodos , Evolução Molecular , Hydra/anatomia & histologia , Hydra/classificação , Hydra/genética , Masculino , Mamíferos/anatomia & histologia , Mamíferos/genética , Especificidade de Órgãos , Filogenia , Homologia de Sequência de AminoácidosRESUMO
During meiosis, the stable pairing of the homologous chromosomes is mediated by the assembly of the synaptonemal complex (SC). Its tripartite structure is well conserved in Metazoa and consists of two lateral elements (LEs) and a central region (CR) that in turn is formed by several transverse filaments (TFs) and a central element (CE). In a previous article, we have shown that not only the structure, but also the major structural proteins SYCP1 (TFs) and SYCP3 (LEs) of the mammalian SC are conserved in metazoan evolution. In continuation of this work, we now investigated the evolution of the mammalian CE-specific proteins using phylogenetic and biochemical/cytological approaches. In analogy to the observations made for SYCP1 and SYCP3, we did not detect homologs of the mammalian CE proteins in insects or nematodes, but in several other metazoan clades. We were able to identify homologs of three mammalian CE proteins in several vertebrate and invertebrate species, for two of these proteins down to the basal-branching phylum of Cnidaria. Our approaches indicate that the SC arose only once, but evolved dynamically during diversification of Metazoa. Certain proteins appear to be ancient in animals, but successive addition of further components as well as protein loss and/or replacements have also taken place in some lineages.
Assuntos
Evolução Molecular , Mamíferos/genética , Complexo Sinaptonêmico/genética , Sequência de Aminoácidos , Animais , Proteínas Cromossômicas não Histona/genética , Feminino , Humanos , Hydra/genética , Masculino , Meiose/genética , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , Proteínas Nucleares/genética , Filogenia , Homologia de Sequência de AminoácidosRESUMO
The synaptonemal complex (SC) is a key structure of meiosis, mediating the stable pairing (synapsis) of homologous chromosomes during prophase I. Its remarkable tripartite structure is evolutionarily well conserved and can be found in almost all sexually reproducing organisms. However, comparison of the different SC protein components in the common meiosis model organisms Saccharomyces cerevisiae, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, and Mus musculus revealed no sequence homology. This discrepancy challenged the hypothesis that the SC arose only once in evolution. To pursue this matter we focused on the evolution of SYCP1 and SYCP3, the two major structural SC proteins of mammals. Remarkably, our comparative bioinformatic and expression studies revealed that SYCP1 and SYCP3 are also components of the SC in the basal metazoan Hydra. In contrast to previous assumptions, we therefore conclude that SYCP1 and SYCP3 form monophyletic groups of orthologous proteins across metazoans.
Assuntos
Hydra/genética , Meiose/genética , Proteínas Nucleares/genética , Complexo Sinaptonêmico/genética , Sequência de Aminoácidos , Animais , Western Blotting , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Proteínas de Ligação a DNA , Expressão Gênica , Hydra/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Microscopia Eletrônica , Dados de Sequência Molecular , Proteínas Nucleares/classificação , Proteínas Nucleares/metabolismo , Filogenia , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Complexo Sinaptonêmico/metabolismo , Complexo Sinaptonêmico/ultraestruturaRESUMO
The synaptonemal complex (SC) is a proteinaceous structure of chromosome bivalents whose assembly is indispensable for the successful progression of the first meiotic division of sexually reproducing organisms. In this mini-review we will focus on recent progress dealing with the composition and assembly of the mammalian SC. These advances mainly resulted from the systematic use of knockout mice for all known mammalian SC proteins as well as from protein polymerization studies performed in heterologous systems.
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Mamíferos/fisiologia , Meiose/fisiologia , Multimerização Proteica/fisiologia , Recombinação Genética/fisiologia , Complexo Sinaptonêmico/fisiologia , Animais , Humanos , Mamíferos/genética , Mamíferos/metabolismo , Meiose/genética , Camundongos , Camundongos Knockout , Modelos Biológicos , Multimerização Proteica/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Subunidades Proteicas/fisiologia , Recombinação Genética/genética , Complexo Sinaptonêmico/genética , Complexo Sinaptonêmico/metabolismoRESUMO
The synaptonemal complex (SC) is a proteinaceous, meiosis-specific structure that is highly conserved in evolution. During meiosis, the SC mediates synapsis of homologous chromosomes. It is essential for proper recombination and segregation of homologous chromosomes, and therefore for genome haploidization. Mutations in human SC genes can cause infertility. In order to gain a better understanding of the process of SC assembly in a model system that would be relevant for humans, we are investigating meiosis in mice. Here, we report on a newly identified component of the murine SC, which we named SYCE3. SYCE3 is strongly conserved among mammals and localizes to the central element (CE) of the SC. By generating a Syce3 knockout mouse, we found that SYCE3 is required for fertility in both sexes. Loss of SYCE3 blocks synapsis initiation and results in meiotic arrest. In the absence of SYCE3, initiation of meiotic recombination appears to be normal, but its progression is severely impaired resulting in complete absence of MLH1 foci, which are presumed markers of crossovers in wild-type meiocytes. In the process of SC assembly, SYCE3 is required downstream of transverse filament protein SYCP1, but upstream of the other previously described CE-specific proteins. We conclude that SYCE3 enables chromosome loading of the other CE-specific proteins, which in turn would promote synapsis between homologous chromosomes.