Antibodies to the endoplasmic reticulum-resident chaperones calnexin, BiP and Grp94 in patients with rheumatoid arthritis and systemic lupus erythematosus

Objectives. To investigate the presence of autoantibodies against mammalian chaperones of the endoplasmic reticulum (ER) in patients with RA and other immune-mediated diseases. Methods. Sera from healthy donors, from early RA patients with two follow-up samples, patients with SLE, SSc and IBD were collected and analysed for anti-ER chaperone antibodies. Detection of serum IgG antibodies against immunoglobulin heavy chain binding protein (BiP), glucose-regulated protein 94 (Grp94) and calnexin was carried out using ELISA. The specificity of sera positive for individual ER chaperones was confirmed by immunoblotting. Statistical analysis was performed using Welch's t-test, Mann-Whitney U-test, partial correlation and Pearson's correlation. Results. In patients with RA and SLE, autoantibody titres against BiP, Grp94 and calnexin were significantly higher than those in healthy controls. These autoantibodies were detectable in patients with early RA and titres remained stable for at least 6-12 months. Also several SSc and IBD patients exhibited autoantibodies against these ER chaperones; however, titres and frequencies were lower than in RA or SLE patients. Furthermore, anti-calnexin antibodies correlated significantly with the presence of BiP and Grp94 autoantibodies in patients with RA and SLE. Conclusion. Calnexin and Grp94 were identified as novel autoantigens in RA and calnexin in SLE. Since calnexin, Grp94 and BiP are ER-resident proteins of eukaryotic cells, our data suggest that autoantibody generation against ER chaperones is independent of initial exposure to the corresponding bacterial chaperones; rather, ER chaperones may represent genuine autoantigen

Mutation in utp15 Disrupts Vascular Patterning in a p53-Dependent Manner in Zebrafish Embryos

Angiogenesis is the process by which the highly branched and functional vasculature arises from the major vessels, providing developing tissues with nutrients, oxygen, and removing metabolic waste. During embryogenesis, vascular patterning is dependent on a tightly regulated balance between pro- and anti-angiogenic signals, and failure of angiogenesis leads to embryonic lethality. Using the zebrafish as a model organism, we sought to identify genes that influence normal vascular patterning.In a forward genetic screen, we identified mutant LA1908, which manifests massive apoptosis during early embryogenesis, abnormal expression of several markers of arterial-venous specification, delayed angiogenic sprouting of the intersegmental vessels (ISV), and malformation of the caudal vein plexus (CVP), indicating a critical role for LA1908 in cell survival and angiogenesis. Genetic mapping and sequencing identified a G to A transition in the splice site preceding exon 11 of utp15 in LA1908 mutant embryos. Overexpression of wild type utp15 mRNA suppresses all observed mutant phenotypes, demonstrating a causative relationship between utp15 and LA1908. Furthermore, we found that injecting morpholino oligonucleotides inhibiting p53 translation prevents cell death and rescues the vascular abnormalities, indicating that p53 is downstream of Utp15 deficiency in mediating the LA1908 phenotypes.Taken together, our data demonstrate an early embryonic effect of Utp15 deficiency on cell survival and the normal patterning of the vasculature and highlight an anti-angiogenic role of p53 in developing embryos

Utilization of Benchtop Next Generation Sequencing Platforms Ion Torrent PGM and MiSeq in Noninvasive Prenatal Testing for Chromosome 21 Trisomy and Testing of Impact of In Silico and Physical Size Selection on Its Analytical Performance

OBJECTIVES: The aims of this study were to test the utility of benchtop NGS platforms for NIPT for trisomy 21 using previously published z score calculation methods and to optimize the sample preparation and data analysis with use of in silico and physical size selection methods. METHODS: Samples from 130 pregnant women were analyzed by whole genome sequencing on benchtop NGS systems Ion Torrent PGM and MiSeq. The targeted yield of 3 million raw reads on each platform was used for z score calculation. The impact of in silico and physical size selection on analytical performance of the test was studied. RESULTS: Using a z score value of 3 as the cut-off, 98.11% - 100% (104-106/106) specificity and 100% (24/24) sensitivity and 99.06% - 100% (105-106/106) specificity and 100% (24/24) sensitivity were observed for Ion Torrent PGM and MiSeq, respectively. After in silico based size selection both platforms reached 100% specificity and sensitivity. Following the physical size selection z scores of tested trisomic samples increased significantly-p = 0.0141 and p = 0.025 for Ion Torrent PGM and MiSeq, respectively. CONCLUSIONS: Noninvasive prenatal testing for chromosome 21 trisomy with the utilization of benchtop NGS systems led to results equivalent to previously published studies performed on high-to-ultrahigh throughput NGS systems. The in silico size selection led to higher specificity of the test. Physical size selection performed on isolated DNA led to significant increase in z scores. The observed results could represent a basis for increasing of cost effectiveness of the test and thus help with its penetration worldwide