Cytoplasmic filamentous rods and rings (RR) structures were identified using human autoantibodies as probes. In the present study, the formation of these conserved structures in mammalian cells and ...functions linked to these structures were examined.
Distinct cytoplasmic rods (∼3-10 µm in length) and rings (∼2-5 µm in diameter) in HEp-2 cells were initially observed in immunofluorescence using human autoantibodies. Co-localization studies revealed that, although RR had filament-like features, they were not enriched in actin, tubulin, or vimentin, and not associated with centrosomes or other known cytoplasmic structures. Further independent studies revealed that two key enzymes in the nucleotide synthetic pathway cytidine triphosphate synthase 1 (CTPS1) and inosine monophosphate dehydrogenase 2 (IMPDH2) were highly enriched in RR. CTPS1 enzyme inhibitors 6-diazo-5-oxo-L-norleucine and Acivicin as well as the IMPDH2 inhibitor Ribavirin exhibited dose-dependent induction of RR in >95% of cells in all cancer cell lines tested as well as mouse primary cells. RR formation by lower concentration of Ribavirin was enhanced in IMPDH2-knockdown HeLa cells whereas it was inhibited in GFP-IMPDH2 overexpressed HeLa cells. Interestingly, RR were detected readily in untreated mouse embryonic stem cells (>95%); upon retinoic acid differentiation, RR disassembled in these cells but reformed when treated with Acivicin.
RR formation represented response to disturbances in the CTP or GTP synthetic pathways in cancer cell lines and mouse primary cells and RR are the convergence physical structures in these pathways. The availability of specific markers for these conserved structures and the ability to induce formation in vitro will allow further investigations in structure and function of RR in many biological systems in health and diseases.
During the 12th International Workshop on Autoantibodies and Autoimmunity held in Sao Paulo, Brazil, on August 28, 2014, a full day session was devoted to establishing a consensus on the nomenclature ...of staining patterns observed in the antinuclear antibody (ANA) indirect immunofluorescence test on HEp-2 cells. The current report summarizes the collective agreements with input from the host Brazilian and international communities that represented research, clinical, and diagnostic service laboratories. Patterns are categorized in three major groups (nuclear, cytoplasmic, and mitotic patterns) and each pattern has been defined and described in detail. The consensus nomenclature and representative patterns are made available online at the international consensus on antinuclear antibody pattern (ICAP) website (www.ANApatterns.org). To facilitate continuous improvement and input, specific comments on ICAP are encouraged and these will be discussed in subsequent ICAP meetings. The ultimate goal with the establishment of the ICAP is to promote harmonization and understanding of autoantibody test nomenclature, as well as interpretation guidelines for ANA testing, thereby optimizing usage in patient care.
Dermatomyositis (DM) is a rare idiopathic inflammatory myopathy characterized by muscle weakness and cutaneous manifestations in adults and children. Calcinosis, a complication of DM, is the abnormal ...deposition of insoluble calcium salts in tissues, including skin, subcutaneous tissue, tendons, fascia, and muscle. Calcinosis is more commonly seen in juvenile DM (JDM), but also develops in adult DM. Although the mechanism of calcinosis remains unclear, several pathogenic hypotheses have been proposed, including intracellular accumulation of calcium secondary to an alteration of the cellular membrane by trauma and inflammation, local vascular ischemia, dysregulation of mechanisms controlling the deposition and solubility of calcium and phosphate, and mitochondrial damage of muscle cells. Identifying calcinosis biomarkers is important for early disease detection and risk assessment, and may lead to novel therapeutic targets for the prevention and treatment of DM-associated calcinosis. In this review, we summarize myositis autoantibodies associated with calcinosis in DM, histopathology and chemical composition of calcinosis, genetic and inflammatory markers that have been studied in adult DM and JDM-associated calcinosis, as well as potential novel biomarkers.
•Anti-NXP2 autoantibodies have been associated with calcinosis in both adult and juvenile dermatomyositis (DM).•The structure and composition of calcinosis differ from normal trabecular bone, with a mineral content of carbonate apatite.•Inflammation plays a central role in the calcinosis process in DM, with calcium as a chemoattractant for inflammatory cells.•Further research is needed to identify serum biomarkers for calcinosis in DM.
The International Consensus on ANA Patterns (ICAP) was initiated as a workshop aiming to thoroughly discuss and achieve consensus regarding the morphological patterns observed in the indirect ...immunofluorescence assay on HEp-2 cells. One of the topics discussed at the second ICAP workshop, and addressed in this paper, was the harmonization of reporting ANA test results. This discussion centered on the issue if cytoplasmic and mitotic patterns should be reported as positive or negative. This report outlines the issues that impact on two major different reporting methods. Although it was appreciated by all participants that cytoplasmic and mitotic patterns are clinically relevant, implications for existing diagnostic/classification criteria for ANA-associated diseases in particular hampered a final consensus on this topic. Evidently, a more concerted action of all relevant stakeholders is required. Future ICAP workshops may help to facilitate this action.
Nucleotide biosynthesis is a highly regulated process necessary for cell growth and replication. Cytoplasmic structures in mammalian cells, provisionally described as rods and rings (RR), were ...identified by human autoantibodies and recently shown to include two key enzymes of the CTP/GTP biosynthetic pathways, cytidine triphosphate synthetase (CTPS) and inosine monophosphate dehydrogenase (IMPDH). Several studies have described CTPS filaments in mammalian cells, Drosophila, yeast, and bacteria. Other studies have identified IMPDH filaments in mammalian cells. Similarities among these studies point to a common evolutionarily conserved cytoplasmic structure composed of a subset of nucleotide biosynthetic enzymes. These structures appear to be a conserved metabolic response to decreased intracellular GTP and/or CTP pools. Antibodies to RR were found to develop in some hepatitis C patients treated with interferon-α and ribavirin. Additionally, the presence of anti-RR antibodies was correlated with poor treatment outcome.
Effective estimation of wildlife population abundance is an important component of population monitoring, and ultimately essential for the development of conservation actions. Diurnal line‐transect ...surveys are one of the most applied methods for abundance estimations. Local ecological knowledge (LEK) is empirically acquired through the observation of ecological processes by local people. LEK‐based methods have only been recognized as valid scientific methods for surveying fauna abundance in the last three decades. However, the agreement between both methods has not been extensively analysed.
We compared concomitant abundance data for 91 wild species (mammals, birds and tortoises) from diurnal line transects (9,221 km of trails) and a LEK‐based method (291 structured interviews) at 18 sites in Central and Western Amazonia. We used biological and socioecological factors to assess the agreements and divergences between abundance indices obtained from both methods.
We found a significant agreement of population abundance indices for diurnal and game species. This relationship was also positive regardless of species sociality (solitary or social), body size and locomotion mode (terrestrial and arboreal); and of sampled forest type (upland and flooded forests). Conversely, we did not find significant abundance covariances for nocturnal and non‐game species. Despite the general agreement between methods, line transects were not effective at surveying many species occurring in the area, with 40.2% and 39.8% of all species being rarely and never detected in at least one of the survey sites. On the other hand, these species were widely reported by local informants to occur at intermediate to high abundances.
Although LEK‐based methods have been long neglected by ecologists, our comparative study demonstrated their effectiveness for estimating vertebrate abundance of a wide diversity of taxa and forest environments. This can be used simultaneously with line‐transect surveys to calibrate abundance estimates and record species that are rarely sighted during surveys on foot, but that are often observed by local people during their daily extractive activities. Thus, the combination of local and scientific knowledge is a potential tool to improve our knowledge of tropical forest species and foster the development of effective strategies to meet biodiversity conservation goals.
Resumen
La estimación eficaz de la abundancia de las poblaciones de fauna silvestre es un importante componente del monitoreo poblacional, y, en último término, es fundamental para el desarrollo de actividades dirigidas a la conservación. Los transectos lineales diurnos son uno de los métodos más frecuentemente utilizados para estimar la abundancia. El Conocimiento Ecológico Local (LEK) se adquiere de forma empírica a través de una aproximación más holística y ecocéntrica de los procesos ecológicos. Los métodos basados en LEK sólo han sido reconocidos como método científico de monitoreo de fauna silvestre en las últimas tres décadas. Sin embargo, aún no se ha evaluado en profundidad la concordancia entre ambos métodos.
En este estudio comparamos la abundancia relativa de 91 especies silvestres (mamíferos, aves y tortugas) obtenida a partir de transectos lineales diurnos (9,221 km de recorrido) y de un método basado en LEK (291 entrevistas estructuradas) en 18 sitios de la Amazonía Central y Oeste. Usamos factores socio‐ecológicos y biológicos para analizar las concordancias y divergencias entre la índices de abundancia estimada mediante ambos métodos.
Se observó concordancia en los índices de abundancia poblacional de especies diurnas y especies cinegéticas estimada mediante ambos métodos. Esta concordancia fue independiente de la sociabilidad de la especie (solitaria o social), tamaño corporal, modo de locomoción (terrestre o arbórea), y tipo de bosque (bosque de altura e inundable). A la inversa, no se observó concordancia en el índice abundancia de especies nocturnas y no‐cinegéticas. A pesar de la concordancia general entre ambos métodos, los transectos lineales diurnos no fueron efectivos para monitorear muchas de las especies, con un 40.2% y 39.8% de todas las especies poco frecuentes (<0.1 individuos/km) o no detectadas, respectivamente, en al menos uno de los sitos muestreados. Algo que llama la atención, ya que la mayor parte de los informantes locales reportaron que las abundancias de esas especies eran intermedias o elevadas.
Aunque los ecólogos han tendido a infravalorar los métodos basados en LEK, nuestro estudio comparativo muestra la eficacia de estos métodos para estimar el índice de abundancia de vertebrados en una amplia variedad de taxones y de ambientes. Los métodos basados en LEK son rentables y pueden ayudar a superar las restricciones financieras y logísticas propias del monitoreo de fauna continuado y de largo término de proyectos de conservación. Así, la combinación del conocimiento local y científico podría ser una herramienta importante para mejorar el conocimiento sobre especies tropicales y para promover el desarrollo de estrategias efectivas que permitan cumplir con los objetivos de conservación de la biodiversidad.
Serum autoantibodies are frequently detected in patients with chronic HCV infection, reflecting the wide spectrum of immune reactions related to this virus. In the present study, a novel autoantibody ...to cytoplasmic rods and rings (RR) in chronic HCV patients was characterized.
Sera from 75 previously untreated HCV patients were investigated by indirect immunofluorescence using HEp-2 cell substrate before and during pegylated interferon (PEG-IFN)/ribavirin (RBV) therapy. HEp-2 cells were cultured and fixed either following standard protocols or with the addition of RBV in culture medium.
In 15 out of 75 (20%) patients, analysis revealed the presence of antibodies to rod-like cytoplasmic structures ranging approximately 3-10 μm in length and rings approximately 2-5 μm in diameter. These RR structures became detectable in >95% of cells after addition of RBV in culture medium, whereas they were absent in untreated cells. Anti-RR antibodies were found in sera collected during PEG-IFN/RBV treatment only, but never detected before antiviral therapy nor in control groups. More importantly, these anti-RR antibodies were more often detected in non-responder/relapsers than in responder patients (33% versus 11%; P-value =0.037).
An RBV-induced autoantibody was identified to a new cytoplasmic autoantigenic structure developed in HCV patients after PEG-IFN/RBV and this same structure can be induced by RBV in in vitro culture. Owing to the onset of anti-RR antibodies in PEG-IFN/RBV-treated patients and their association with a treatment failure, studies are deemed necessary to clarify whether anti-RR plays a role in the response to PEG-IFN/RBV therapy.
Antibodies to extractable nuclear antigens (ENA) are found in a variety of collagen vascular diseases. Determining the individual specificities of these antibodies is extremely useful in establishing ...the disease diagnosis and in some cases the prognosis. With a multiplexed fluorescent microsphere immunoassay, reactivity to five of the most diagnostically useful ENA was measured in 249 serum samples, including samples from 56 patients previously documented to have systemic lupus erythematosus (SLE). Results of the multiplexed assay were compared to results from established ENA enzyme-linked immunosorbent assays (ELISAs), and the agreement, sensitivity, and specificity, respectively, for the five ENA evaluated were as follows: SSA, 99.1, 100.0, and 98.8%; SSB, 98.6, 88.9, and 99.5%; Sm, 97.6, 95.8, and 97.9%; RNP, 97.2, 92.7, and 98.8%; Scl-70, 93.6, 50.0, and 99.0%. In the 56 confirmed SLE patients, the frequency of significant concentrations of autoantibodies with the multiplexed assay was 21.4% for SSA, 7.1% for SSB, 10.7% for Sm, 32.1% for RNP, and 0% for Scl-70. The new flow cytometric bead-based multiplexed assay showed excellent correlation with the well-established single-analyte ELISA methods for four of five the ENA markers investigated in this study. The most notable discrepancies between the two assays were for the Scl-70 antigen, which was most often resolved in favor of the multiplexed assay. Our studies show that the multiplexed microsphere-based immunoassay is a sensitive and specific method for the detection and semiquantitation of ENA antibodies in human sera.
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