Gene Expression Changes Reflect Clinical Response in a Placebo-Controlled Randomized Trial of Abatacept in Patients with Diffuse Cutaneous Systemic Sclerosis

Systemic sclerosis is an autoimmune disease characterized by inflammation and fibrosis of the skin and internal organs. We sought to assess the clinical and molecular effects associated with response to intravenous abatacept in patients with diffuse cutaneous systemic

An integrated peptide-antigen microarray on plasmonic gold films for sensitive human antibody profiling.

High-throughput screening for interactions of peptides with a variety of antibody targets could greatly facilitate proteomic analysis for epitope mapping, enzyme profiling, drug discovery and biomarker identification. Peptide microarrays are suited for such undertaking because of their high-throughput capability. However, existing peptide microarrays lack the sensitivity needed for detecting low abundance proteins or low affinity peptide-protein interactions. This work presents a new peptide microarray platform constructed on nanostructured plasmonic gold substrates capable of metal enhanced NIR fluorescence enhancement (NIR-FE) by hundreds of folds for screening peptide-antibody interactions with ultrahigh sensitivity. Further, an integrated histone peptide and whole antigen array is developed on the same plasmonic gold chip for profiling human antibodies in the sera of systemic lupus erythematosus (SLE) patients, revealing that collectively a panel of biomarkers against unmodified and post-translationally modified histone peptides and several whole antigens allow more accurate differentiation of SLE patients from healthy individuals than profiling biomarkers against peptides or whole antigens alone

Interspecies somatic cell nuclear transfer is dependent on compatible mitochondrial DNA and reprogramming factors

Interspecies somatic cell nuclear transfer (iSCNT) involves the transfer of a nucleus or cell from one species into the cytoplasm of an enucleated oocyte from another. Once activated, reconstructed oocytes can be cultured in vitro to blastocyst, the final stage of preimplantation development. However, they often arrest during the early stages of preimplantation development; fail to reprogramme the somatic nucleus; and eliminate the accompanying donor cell’s mitochondrial DNA (mtDNA) in favour of the recipient oocyte’s genetically more divergent population. This last point has consequences for the production of ATP by the electron transfer chain, which is encoded by nuclear and mtDNA. Using a murine-porcine interspecies model, we investigated the importance of nuclear-cytoplasmic compatibility on successful development. Initially, we transferred murine fetal fibroblasts into enucleated porcine oocytes, which resulted in extremely low blastocyst rates (0.48%); and failure to replicate nuclear DNA and express Oct-4, the key marker of reprogramming. Using allele specific-PCR, we detected peak levels of murine mtDNA at 0.1460.055% of total mtDNA at the 2-cell embryo stage and then at ever-decreasing levels to the blastocyst stage (,0.001%). Furthermore, these embryos had an overall mtDNA profile similar to porcine embryos. We then depleted porcine oocytes of their mtDNA using 10 mM 29,39- dideoxycytidine and transferred murine somatic cells along with murine embryonic stem cell extract, which expressed key pluripotent genes associated with reprogramming and contained mitochondria, into these oocytes. Blastocyst rates increased significantly (3.38%) compared to embryos generated from non-supplemented oocytes (P,0.01). They also had significantly more murine mtDNA at the 2-cell stage than the non-supplemented embryos, which was maintained throughout early preimplantation development. At later stages, these embryos possessed 49.9962.97% murine mtDNA. They also exhibited an mtDNA profile similar to murine preimplantation embryos. Overall, these data demonstrate that the addition of species compatible mtDNA and reprogramming factors improves developmental outcomes for iSCNT embryos

Characterization of Influenza Vaccine Immunogenicity Using Influenza Antigen Microarrays

<div><p>Background</p><p>Existing methods to measure influenza vaccine immunogenicity prohibit detailed analysis of epitope determinants recognized by immunoglobulins. The development of highly multiplex proteomics platforms capable of capturing a high level of antibody binding information will enable researchers and clinicians to generate rapid and meaningful readouts of influenza-specific antibody reactivity.</p><p>Methods</p><p>We developed influenza hemagglutinin (HA) whole-protein and peptide microarrays and validated that the arrays allow detection of specific antibody reactivity across a broad dynamic range using commercially available antibodies targeted to linear and conformational HA epitopes. We derived serum from blood draws taken from 76 young and elderly subjects immediately before and 28±7 days post-vaccination with the 2008/2009 trivalent influenza vaccine and determined the antibody reactivity of these sera to influenza array antigens.</p><p>Results</p><p>Using linear regression and correcting for multiple hypothesis testing by the Benjamini and Hochberg method of permutations over 1000 resamplings, we identified antibody reactivity to influenza whole-protein and peptide array features that correlated significantly with age, H1N1, and B-strain post-vaccine titer as assessed through a standard microneutralization assay (p<0.05, <i>q</i> <0.2). Notably, we identified several peptide epitopes that were inversely correlated with regard to age and seasonal H1N1 and B-strain neutralization titer (p<0.05, <i>q</i> <0.2), implicating reactivity to these epitopes in age-related defects in response to H1N1 influenza. We also employed multivariate linear regression with cross-validation to build models based on age and pre-vaccine peptide reactivity that predicted vaccine-induced neutralization of seasonal H1N1 and H3N2 influenza strains with a high level of accuracy (84.7% and 74.0%, respectively).</p><p>Conclusion</p><p>Our methods provide powerful tools for rapid and accurate measurement of broad antibody-based immune responses to influenza, and may be useful in measuring response to other vaccines and infectious agents.</p></div

Peptide-antigen microarrays on plasmonic gold substrate for profiling antibodies in serum samples of SLE patients and healthy individuals.

<p>A) Box plot of serum IgG antibody reactivity against several peptides and a whole antigen for 20 SLE patients and 20 healthy controls. Acetylated histone H2B peptides were found to be able to differentiate SLE patients and healthy controls (top box plots) together with whole H2B protein (bottom left box plot). While the H3 peptide with the 18th lysine methylated were found not capable of telling SLE patient from healthy control (bottom right box plot). B–D) Heatmaps displaying antibody reactivity to (B) histone peptides only, (C) whole antigens only, and (D) a combination of histone peptides and whole antigens that are identified capable of differentiating SLE patients and healthy controls with false discovery rate (q value) <0.001. The dashed lines are drawn to highlight the separation of SLE and healthy groups identified by using the average linkage Euclidean distance hierarchical clustering method. Color intensity of each grid in the heatmap reflected mean fluorescence intensity of corresponding peptide or antigen spot on the microarray for each SLE patient or healthy individual. In (B) and (C), several SLE patients (labeled in blue and purple color) are misplaced in the healthy group. These patients are grouped in the SLE side in (D) that profiles antibodies against both peptides and whole antigens. However, one healthy individual is mis-placed in the SLE group by this approach, reducing the specificity of this analysis.</p

Plasmonic gold films for peptide microarrays.

<p>A) Scanning electron microscopy (SEM) of an as-made plasmonic gold film containing nano-islands formed on a glass substrate. Inset: an X-ray photoelectron spectrum (XPS) of the film. B) SEM of a plasmonic gold film coated with a layer of avidin proteins (avidin coating caused blurred features of the underlying gold nano-islands). Inset, an XPS spectrum taken on the avidin coated gold substrate showing the presence of nitrogen (in the avidin) over the film. C) A schematic drawing of a multiplexed peptide array (two peptides are shown, red and blue; peptide spots are surrounded by PEG-star coating) on gold for antibody screening based on a 3-layer assay with a NIR fluorophore reporter. D) Intensity of IRDye800 fluorescence (emission ∼ 800 nm) measured on a layer of IRDye800 labeled avidin coated on glass vs. on a plamonic gold film. A ∼100 fold fluorescence enhancement was observed on gold.</p

An integrated peptide-antigen microarray for profiling serum antibodies of 20 patients with systemic lupus erythematosus (SLE).

<p>A) Peptide-antigen microarray layout including modified and unmodified histone H2A, H2B, H3, H4 peptides as well as whole antigens. Ac: acetylated; aa: amino acid; Me1: methylated; Me2: dimethylated; Me3: trimethylated; Ph: phosphorylated; K: Lysine; S: Serine. Number indicates amino acid position from the N-terminus of its corresponding histone proteins. Peptide sequences are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071043#pone.0071043.s008" target="_blank">Table S1</a>. B) Microarray imaging results of a SLE patient serum probed on a plasmonic gold substrate (left) vs. on a commercial streptavidin-glass substrate (right) shown in the same IRDye800 fluorescence intensity scale. C) Dynamic range of fluorescence signal reading among 20 SLE patients measured on each peptide and antigen spots on avidin/gold slide (upper panel) and commercial streptavidin/glass slide (lower panel). Each dot represents a fluorescence intensity measured on a peptide or antigen spot shown along the x-axis in the serum of each of the 20 SLE patients. A dot is drawn only when the fluorescence intensity is above the background by 2 standard deviation of the background signal.</p