Analytical performance of the AtheNA MultiLyte® ANA II assay in sera from lupus patients with multiple positive ANAs

The purpose of this study was to evaluate the precision and accuracy of a commercial multiplexed kit for the measurement of 9 anti-nuclear antibodies (ANAs; anti-SS/A, anti-SS/B, anti-Sm, anti-RNP, anti-Jo-1, anti-Scl-70, anti-dsDNA, anti-Centromere B, and anti-Histone), and to compare these results to a subset of ANAs measured by enzyme-linked immunosorbent assays (ELISA) and immunodiffusion (ID). Sera were obtained from 22 systemic lupus erythematosus (SLE) patients, twelve controls and five others (commercial source) with various autoimmune diseases. ANA results from the AtheNA MultiLyte ANA II Assay (AtheNA) were compared to ELISA results (controls) and patients (ID). The AtheNA interassay coefficients of variation (CVs, N = 39, performed in duplicate; replicated 3x) ranged from 6.2% to 16.7% (mean = 9.8%), while the intra-assay CVs ranged from 5.8% to 14.3% (mean = 10.8%). Compared to results for SLE cases and controls, the sensitivity of AtheNA ranged from 85.7% to 100% (mean = 97.1%), while diagnostic specificity ranged from 16.7% to 100% (mean = 71.6%). There was significant agreement (P values ranging from 0.0001 to 0.03) when analytes coanalyzed by AtheNA and ELISA/ID were evaluated using Cohen's kappa (kappa values ranging from 0.376 to 1.000). No false positive ANA results were observed for either the control or commercial source autoimmune disease sera. These results indicate that the AtheNA assay is a precise and accurate alternative for performing multiple ELISAs or IDs in the diagnosis of autoimmune diseases, especially when the number of sera to be tested is large, such as in clinical screening or epidemiologic studies. It also appears that the AtheNA assay identifies positive ANA specificities which are missed by ID techniques, suggesting that it may have greater analytical sensitivity for some ANAs.     

Macrophage Migration Inhibitory Factor (MIF) and Its Homologue D-Dopachrome Tautomerase (DDT) Inversely Correlate with Inflammation in Discoid Lupus Erythematosus

Discoid Lupus Erythematosus (DLE) is a chronic cutaneous disease of unknown etiology and of immunoinflammatory origin that is characterized by inflammatory plaques and may lead to disfiguring scarring and skin atrophy. Current treatments are limited, with a large proportion of patients either poorly or not responsive, which makes DLE an unmet medical need. Macrophage migration inhibitory factor (MIF) is the prototype of a pleiotropic family of cytokine that also includes the recently discovered homologue D-dopachrome tautomerase (DDT) or MIF2. MIF and DDT/MIF-2 exert several biological properties, primarily, but not exclusively of a proinflammatory nature. MIF and DDT have been suggested to play a key role in the pathogenesis of several autoimmune diseases, such as multiple sclerosis and type 1 diabetes, as well as in the development and progression of certain forms of cancers. In the present study, we have performed an immunohistochemistry analysis for the evaluation of MIF in DLE lesions and normal skin. We found high levels of MIF in the basal layer of the epidermis as well as in the cutaneous appendage (eccrine glands and sebocytes) of normal skin. In DLE lesions, we observed a significant negative correlation between the expression of MIF and the severity of inflammation. In addition, we performed an analysis of MIF and DDT expression levels in the skin of DLE patients in a publicly available microarray dataset. Interestingly, while these in silico data only evidenced a trend toward reduced levels of MIF, they demonstrated a significant pattern of expression and correlation of DDT with inflammatory infiltrates in DLE skins. Overall, our data support a protective role for endogenous MIF and possibly DDT in the regulation of homeostasis and inflammation in the skin and open up novel avenues for the treatment of DLE.     

IA免疫吸附剂血液灌流治疗系统性红斑狼疮病例研究

本文介绍用IA免疫吸附剂血液灌流临床治疗系统性红斑狼疮患者的研究结果。     

A new autosomal STR nineplex for canine identification and parentage testing

A single multiplex PCR assay capable of simultaneously amplifying nine canine‐specific autosomal STR markers (FH3210, FH3241, FH2004, FH2658, FH4012, REN214L11, FH2010, FH2361 and the newly described C38) was developed for individual identification and parentage testing in domestic dogs. In order to increase genotyping efficiency, amplicon sizes were optimized for a 90–350 bp range, with fluorescently labelled primers for use in Applied Biosystems, Inc., platforms. The performance of this new multiplex system was tested in 113 individuals from a case‐study population and 12 random dogs from mixed‐breed origin. Co‐dominant inheritance of STR alleles was investigated in 101 father, mother and son trios. Expected heterozygosity values vary between 0.5648 for REN214L11 and 0.9050 for C38. The high level of genetic diversity observed for most markers provides this multiplex with a very high discriminating power (matching probability=1.63/10¹⁰ and matching probability among siblings=4.9/10³). Allele sequences and a proposal for standardized nomenclature are also herein presented, aiming at implementing the use of this system in forensic DNA typing and population genetic studies. This approach resulted in an optimized and well‐characterized canine DNA genotyping system that is highly performing and straightforward to integrate and employ routinely. Although this STR multiplex was developed for use and tested in a case‐study population, the Portuguese breed Cão de Gado Transmontano, it proved to be useful for general identification purposes or parentage testing.     

Biosensor analyses of serum autoantibodies: application to antiphospholipid syndrome and systemic lupus erythematosus

Autoimmune disorders are rare human diseases characterized by the presence of circulating autoantibodies that bind the body’s own structural compounds as target antigens. The detection of autoantibodies is important for the diagnostic process. Immunofluorescence and immunoassay methods do not allow a reliable characterization of binding characteristics. Therefore, novel analytical techniques should be considered. This review describes the application of surface plasmon resonance biosensor systems for the diagnosis of autoimmune disorders. The covalent attachment of native antigens to the sensor chip is a suitable method for obtaining highly reproducible analyses of autoantibodies, allowing the evaluation of kinetic rate and affinity constants, and it may enable the identification of disease-relevant autoantibodies linked to disease progression. The autoantibody microarray is another future-oriented technique. Patterns of differential antigen recognition should allow early diagnosis. This is due to the fact that a broad range of autoreactive B cell responses in autoimmune disorders can only be mirrored by including a sufficient number of antigens in a microarray format.     

A framework for the development of STR genotyping in domestic animal species: Characterization and population study of 12 canine X‐chromosome loci

This study reports the methodology used to search, select and characterize STR loci on the canine X chromosome using publicly available genome resources and following the current guidelines for human and non‐human forensic testing. After several rounds of selection, 12 X‐STR markers were optimized for simultaneous co‐amplification in a single PCR, and genetic profiles were determined in a sample of 103 unrelated dogs. Mendelian inheritance was verified and mutation rates were assessed using family groups. Alleles that varied in size were sequenced to create a standardized nomenclature proposal based on the number of repeats. All loci conformed to Hardy–Weinberg expectations. The resulting panel showed high forensic efficiency, presenting high values of power of discrimination (in males and females) and mean exclusion chance, both in trios involving female offspring and in duos composed of dam and male offspring. Its use may complement the information obtained by autosomal STR analysis and contribute to the resolution of complex cases of kinship in dogs. The presented methodology for the de novo construction of an STR multiplex may also provide a helpful framework for analogous work in other animal species. As an increasing number of reference genomes become available, convenient tools for individual identification and parentage testing based on STR loci selected from autosomes or sex chromosomes' sequences may be created following this strategy.     

Urinary metabolomic study of systemic lupus erythematosus based on gas chromatography/mass spectrometry

Systemic lupus erythematosus (SLE) is an autoimmune disease with heterogeneous organ and system manifestations. In this study, urinary metabolic alterations related to SLE were investigated by performing gas chromatography/mass spectrometry (GC/MS) based metabolomics and multivariate statistical analysis. Patients with SLE and healthy controls could be clearly differentiated in view of the metabolic abnormity in urine. Among 70 identified endogenous metabolites, 23 metabolites were dramatically increased in SLE patients, which involved in several key metabolic pathways including energy metabolism, nucleotide metabolism, oxidative stress and gut‐microbiome‐derived metabolism. This noninvasive and GC/MS‐based metabolomic technique is a promising and potent strategy for identifying novel biomarkers and understanding pathogenesis of SLE. Copyright © 2016 John Wiley & Sons, Ltd.     

Mass spectrometric and peptide chip characterization of an assembled epitope: analysis of a polyclonal antibody model serum directed against the Sjøgren/systemic lupus erythematosus autoantigen TRIM21

We demonstrate the development of a mass spectrometry‐based epitope‐mapping procedure in combination with Western blot analysis that works also with antigens that are insoluble in nondenaturing buffers consuming minute amounts of antigen (approximately 200 pmol) and antibody (approximately 15 pmol), respectively. A polyclonal anti‐TRIM21 rabbit antibody serum is applied as a model serum for future patient analyses to set up the system. The major epitope that is recognized by the anti‐TRIM21 serum spans the central TRIM21 region LQ‐EE REL GE‐KE, showing that immunization with a 139‐amino acid residue long peptide resulted in a ‘monospecific’ polyclonal antibody repertoire. Protein structure investigations, secondary structure predictions, and surface area calculations revealed that the best matching partial sequence to fulfill all primary and secondary structure requirements was the four amino acid spanning motif ‘L–E–Q–L’, which is present in both the sequential and the α‐helical peptide conformation. Peptide chip analyses confirmed the mass spectrometric results and showed that the peptide chip platform is an appropriate method for displaying secondary structure‐relying epitope conformations. As the same secondary structures are present in vivo, patient antibody screening, e.g., to identify subgroups of patients according to distinct epitope antibody reactivities, is feasible. Copyright © 2013 John Wiley & Sons, Ltd.     

Evaluation of Ethanolic Extracts of Mullaca (Physalis Angulata L.) Herbs for Treatment of Lupus Disease in Mice Induced Pristane

Systemic lupus erythematosus is a systemic autoimmune disease characterized by multisystem inflammation due to immune complex deposits in the organs like kidneys, joints, pleura, skin, and so on. Physalisangulata known as mullaca, has been widely studied for its pharmacological activities such as antiinflammatory, immunosuppressive, cytotoxic, and also inhibition of organ rejection in transplantation. This study was directed to investigate the activities of P. angulata extract as an immunomodulatory agent. P. angulata powder was extracted by maceration with 70% of ethanol. An animal model of lupus was obtained by an injection 0.7 mL of pristane, i.p. Successful induction obtained in two weeks after injection which can be monitored by measuring total leukocyte count. For ensuring successful induction, another test was done four weeks and eight weeks after injection by detecting the presence of specific antinuclear antibodies using SDS PAGE method. Other measured parameters were including nonspecific immune response (measurement of total leukocyte count and differential leukocyte count), specific humoral immune response (hemagglutination test), specific cellular immune responses (delayed type hypersensitivity test), organ index and histology of kidney and spleen. As the results shown, ethanolic extract of Physalisangulata at dose of 1000 mg/kg BW orally, gave immunomodulatory effect in Lupus rat model. The extract worked primarily on specific immune response by lowering immune response near to the normal value, it was not suppressing immune response as prednisone.