The reliability of immunoassays to detect autoantibodies in patients with myositis is dependent on autoantibody specificity

OBJECTIVES: In order to address the reliability of commercial assays to identify myositis-specific and -associated autoantibodies, we aimed to compare the results of two commercial immunoassays with the results obtained by protein immunoprecipitation.METHODS: Autoantibody status was determined using radio-labelled protein immunoprecipitation for patients referred to our laboratory for myositis autoantibody characterization. For each autoantibody of interest, the sera from 25 different patients were analysed by line blot (Euroline Myositis Antigen Profile 4, EuroImmun, Lübeck, Germany) and dot blot (D-Tek BlueDiver, Diagnostic Technology, Belrose, NSW, Australia). Sera from 134 adult healthy controls were analysed.RESULTS: Overall commercial assays performed reasonably well, with high agreement (Cohen's κ &gt;0.8). Notable exceptions were the detection of rarer anti-synthetases with κ &lt; 0.2 and detection of anti-TIF1γ, where κ was 0.70 for the line blot and 0.31 for dot blot. Further analysis suggested that the proportion of patients with anti-TIF1γ may recognize a conformational epitope, limiting the ability of blotting-based assays that utilize denatured antigen to detect this clinically important autoantibody. A false-positive result occurred in 13.7% of samples analysed by line blot and 12.1% analysed by dot blot.CONCLUSION: The assays analysed do not perform well for all myositis-specific and -associated autoantibodies and overall false positives are relatively common. It is crucial that clinicians are aware of the limitations of the methods used by their local laboratory. Results must be interpreted within the clinical context and immunoprecipitation should still be considered in selected cases, such as apparently autoantibody-negative patients where anti-synthetase syndrome is suspected.</p

A Commercial Anti-TIF1γ ELISA Is Superior to Line and Dot Blot and Should Be Considered as Part of Routine Myositis-Specific Antibody Testing

OBJECTIVES: Anti-TIF1γ is an important autoantibody in the diagnosis of cancer-associated dermatomyositis and the most common autoantibody in juvenile onset dermatomyositis. Its reliable detection is important to instigate further investigations into underlying malignancy in adults. We previously showed that commercial assays using line and dot blots do not reliably detect anti-TIF1γ. We aimed to test a new commercial ELISA and compare with previously obtained protein immunoprecipitation. METHODS: Radio-labelled immunoprecipitation had previously been used to determine the autoantibody status of patients with immune-mediated inflammatory myopathies and several healthy controls. ELISA was undertaken on healthy control and anti-TIF1γ sera and compared to previous immunoprecipitation data. RESULTS: A total of 110 serum samples were analysed: 42 myositis patients with anti- TIF1γ and 68 autoantibody negative healthy control sera. Anti-TIF1γ was detected by ELISA in 41 out of 42 of the anti-TIF1γ-positive samples by immunoprecipitation, and in none of the healthy controls, giving a sensitivity of 97.6% and specificity of 100%. The false negative rate was 2%. CONCLUSION: ELISA is an affordable and time-efficient method which is accurate in detecting anti-TIF1γ

Switching spectroscopic measurement of surface potentials on ferroelectric surfaces via an open-loop Kelvin probe force microscopy method

We report a method for switching spectroscopy Kelvin probe force microscopy (SS-KPFM). The method is established as a counterpart to switching spectroscopy piezoresponse force microscopy (SS-PFM) in Kelvin probe force microscopy. SS-KPFM yields quantitative information about the surface charge state during a local bias-induced polarization switching process, complementary to the electromechanical coupling properties probed via SS-PFM. Typical ferroelectric samples of a Pb-based relaxor single crystal and a BiFeO3 thin film were investigated using both methods. We briefly discuss the observed surfacecharging phenomena and their influence on the associated piezoresponse hysteresis loops.Q.L., Y.L., D.W., and R.L.W. acknowledge the support of the Australian Research Council (ARC) in the form of ARC Discovery Grants. Y.L. also acknowledges support from the ARC Future Fellowships Program

Exploratory application of a cannulation model in recently weaned pigs to monitor longitudinal changes in the enteric microbiome across varied porcine reproductive and respiratory syndrome virus (PRRSV) infection status

The enteric microbiome and its possible modulation to improve feed conversion or vaccine efficacy is gaining more attention in pigs. Weaning pigs from their dam on top of many routine procedures is stressful. A better understanding of the impact of this process on the microbiome may be important to improve pig production. The objective of this study was to develop a weaner pig cannulation model allowing ileum content collection from the same pig over time for 16S rRNA sequencing under different porcine reproductive and respiratory syndrome virus (PRRSV) infection status. Methods: Fifteen 3-week-old pigs underwent abdominal surgery and were fitted with an ileum cannula and ileum contents were collected over time. In this pilot study, treatment groups included a NEG-CONTROL group (no vaccination, no PRRSV challenge), a POS-CONTROL group (no vaccination, PRRSV challenge), a VAC-PRRSV group (vaccinated, PRRSV challenged), a VAC-PRO-PRRSV group (supplemented with probiotics, vaccinated, challenged with PRRSV) and a VAC-ANTI-PRRSV group (antibiotic administration, vaccinated, PRRSV challenged). We assessed the microbiome over time, measured anti-PRRSV serum antibodies, PRRSV load in serum and nasal samples, and severity of lung lesions. Results: Vaccination was protective against PRRSV challenge, irrespective of other treatments. All vaccinated pigs mounted an immune response to PRRSV within 1 week after vaccination. A discernible impact of treatment on the diversity, structure, and taxonomic abundance of enteric microbiome among the groups was not observed. Instead, significant influences on ileum microbiome were observed in relation to time and treatment. Discussion: The cannulation model described in this pilot study has the potential to be useful to study the impact of weaning, vaccination, disease challenge, and antimicrobial administration on the enteric microbiome and its impact on pig health and production. Remarkably, despite the cannulation procedures, all vaccinated pigs exhibited robust immune responses and remained protected against PRRSV challenge, as evidenced by development of anti-PRRSV serum antibodies and viral shedding data

Tolerogenic effects of 1,25-dihydroxyvitamin D on dendritic cells involve induction of fatty acid synthesis

The active form of vitamin D, 1,25-dihydroxyvitamin D (1,25D) is a potent regulator of immune function, promoting anti-inflammatory, tolerogenic T cell responses by modulating antigen presentation by dendritic cells (DC). Transcriptomic analyses indicate that DC responses to 1,25D involve changes in glycolysis, oxidative phosphorylation, electron transport and the TCA cycle. To determine the functional impact of 1,25D-mediated metabolic remodelling, human monocyte-derived DC were differentiated to immature (+vehicle, iDC), mature (+LPS, mDC), and immature tolerogenic DC (+1,25D, itolDC) and characterised for metabolic function. In contrast to mDC which showed no change in respiration, itolDC showed increased basal and ATP-linked respiration relative to iDC. Tracer metabolite analyses using (13)C -labeled glucose showed increased lactate and TCA cycle metabolites. Analysis of lipophilic metabolites of (13)C-glucose revealed significant incorporation of label in palmitate and palmitoleate, indicating that 1,25D promotes metabolic fatty acid synthesis in itolDC. Inhibition of fatty acid synthesis in itolDC altered itolDC morphology and suppressed expression of CD14 and IL-10 by these cells. These data indicate that the ability of 1,25D to induce tolerogenic DC involves metabolic remodelling leading to synthesis of fatty acids

Determination of the Solubilizing Character of 1-(2-Hydroxyethyl)-1-Methylimidazolium Tris(Pentafluoroethyl)Trifluorophosphate Based on the Abraham Solvation Parameter Model

Article discussing the solubilizing character of 1-(2-hydroxyethyl)-1-methylimidazolium tris(pentafluoroethyl)trifluorophosphate based on the Abraham solvation parameter model

Spectroscopy of the unbound nucleus 18Na

Expérience GANIL, SPIRALInternational audienceThe unbound nucleus 18Na, the intermediate nucleus in the two-proton radioactivity of 19Mg, is studied through the resonant elastic scattering 17Ne(p,17Ne)p. The spectroscopic information obtained in this experiment is discussed and put in perspective with previous measurements and the structure of the mirror nucleus 18N

A Commercial Anti-TIF1γ ELISA Is Superior to Line and Dot Blot and Should Be Considered as Part of Routine Myositis-Specific Antibody Testing

Objectives: Anti-TIF1γ is an important autoantibody in the diagnosis of cancer-associated dermatomyositis and the most common autoantibody in juvenile onset dermatomyositis. Its reliable detection is important to instigate further investigations into underlying malignancy in adults. We previously showed that commercial assays using line and dot blots do not reliably detect anti-TIF1γ. We aimed to test a new commercial ELISA and compare with previously obtained protein immunoprecipitation. Methods: Radio-labelled immunoprecipitation had previously been used to determine the autoantibody status of patients with immune-mediated inflammatory myopathies and several healthy controls. ELISA was undertaken on healthy control and anti-TIF1γ sera and compared to previous immunoprecipitation data. Results: A total of 110 serum samples were analysed: 42 myositis patients with anti- TIF1γ and 68 autoantibody negative healthy control sera. Anti-TIF1γ was detected by ELISA in 41 out of 42 of the anti-TIF1γ-positive samples by immunoprecipitation, and in none of the healthy controls, giving a sensitivity of 97.6% and specificity of 100%. The false negative rate was 2%. Conclusion: ELISA is an affordable and time-efficient method which is accurate in detecting anti-TIF1γ.</p