Single-cell western blotting.

Cell heterogeneity is a variation in cellular processes in functionally similar cells. Cells from the same tissue which are considered genetically identical may have difference in size, structure, and level of protein expression which can lead to major impact on the functions of cell leading to difference in physiological consequences. Single-cell proteome-wide studies are used to detect cell heterogeneity.

SDS-PAGE to Immunoblot in One Hour.

An ultra-rapid method for electrophoresing proteins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transfer of proteins to nitrocellulose membranes, and immunoblotting is described here. Electrophoresis of the autoantigens La and Ro60, as well as molecular weight standards on a 4-20 % gradient gel, was performed in about 10 min using heated (70-75 °C) normal Laemmli running buffer. Electrophoretic transfer of these proteins was achieved in 7 min using a semidry transfer method.

T Cell ELISPOT: For the Identification of Specific Cytokine-Secreting T Cells.

The ELISPOT is a powerful functional assay used to detect biological activity and immunological secretions from immune cells. In this chapter, we specifically discuss T cell ELISPOT methods for the detection of secreted cytokines. A detailed protocol is given enabling the detection of interferon gamma-secreting CD8(+) T cells and/or peripheral blood mononuclear cells following their isolation and polyclonal activation.

B-Cell ELISPOT: For the Identification of Antigen-Specific Antibody-Secreting Cells.

The B-cell ELISPOT assay is a sensitive tool that can be utilized to measure total immunoglobulin (Ig) and antigen-specific antibody-secreting cells. Typically, membrane-bound antigen enables binding of antibody secreted by B-cells. Bound antibody is then detected by using an anti-Ig antibody and a colorimetric substrate, resulting in colored spots on the membrane that can be easily enumerated.

Grid-immunoblotting.

Grid-immunoblotting is a fast, simple, and efficient method for simultaneously testing multiple allergens utilizing small amount of antibody.

Strip immunoblotting of multiple antigenic peptides.

Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis can be employed to efficiently separate multiple antigenic peptides (MAPs). Moreover, the electrophoresed MAPs are amenable for transfer to nitrocellulose membrane for immunoblotting. MAPs involve a hepta lysine core with end groups for anchoring multiple copies of the same synthetic peptide.

Membrane strip affinity purification of autoantibodies.

A method for affinity purification of autoantibodies from a membrane strip using small volumes of human sera is described. The membrane strip is excised from a western blot containing a target antigen electrophoretically transferred from a sodium dodecyl sulfate (SDS) polyacrylamide gel. This method is a very useful alternative for affinity column chromatography, particularly when the antigen of interest is of low abundance in a HeLa cell extract.

Western blotting of high and low molecular weight proteins using heat.

A method for the electrophoretic transfer of high and low molecular weight proteins to nitrocellulose membranes following sodium dodecyl sulfate (SDS) polyacrylamide gel is described here. The transfer was performed with heated (70-75 °C) normal transfer buffer from which methanol had been omitted. Complete transfer of high and low molecular weight antigens (molecular weight protein standards, a purified protein, and proteins from a human tissue extract) could be carried out in 10 min for a 7 % (0.

Multiple Immunoblots by Passive Diffusion of Proteins from a Single SDS-PAGE Gel.

Western blotting enables the detection and characterization of proteins of low abundance. Sodium dodecyl sulfate (SDS) polyacrylamide gel-separated proteins are normally transferred electrophoretically to nitrocellulose or polyvinylidene difluoride membranes. Here we describe the transfer proteins [Ro 60 (or SSA) autoantigen, 220 and 240 kDa spectrin antigens, and prestained molecular weight standards] by diffusion from SDS polyacrylamide gels at 37 °C.

Concordance of increased B1 cell subset and lupus phenotypes in mice and humans is dependent on BLK expression levels.

Polymorphisms in the B lymphoid tyrosine kinase (BLK) gene have been associated with autoimmune diseases, including systemic lupus erythematosus, with risk correlating with reduced expression of BLK. How reduced expression of BLK causes autoimmunity is unknown. Using Blk(+/+) , Blk(+/-) , and Blk(-/-) mice, we show that aged female Blk(+/-) and Blk(-/-) mice produced higher anti-dsDNA IgG Abs and developed immune complex-mediated glomerulonephritis, compared with Blk(+/+) mice.

High Affinity Antibodies against Influenza Characterize the Plasmablast Response in SLE Patients After Vaccination.

Breakdown of B cell tolerance is a cardinal feature of systemic lupus erythematosus (SLE). Increased numbers of autoreactive mature naïve B cells have been described in SLE patients and autoantibodies have been shown to arise from autoreactive and non-autoreactive precursors. How these defects, in the regulation of B cell tolerance and selection, influence germinal center (GC) reactions that are directed towards foreign antigens has yet to be investigated.

Interaction between innate immunity and Ro52-induced antibody causes Sjögren's syndrome-like disorder in mice.

Objectives: Autoantibodies reactive with Ro52 are often found in sera of patients with Sjögren's syndrome (SS). This study was undertaken to investigate the role of Ro52-induced immune responses in pathogenesis of SS.

Methods: New Zealand Mixed (NZM) 2758 mice were immunised with Ro52 in alum adjuvant.

Lupus after kidney donation to an affected male relative.

We report a case of lupus occurring in a 52 year-old woman approximately 2 years after donating a kidney to her brother who had end-stage-renal-disease on the basis of lupus nephritis (LN). At the time of donation, the patient had been asymptomatic with a normal physical examination, laboratory and imaging studies. At the time of her diagnosis of lupus she was found to be ANA negative but positive for anti-double stranded DNA.

Ribosomal and immune transcripts associate with relapse in acquired ADAMTS13-deficient thrombotic thrombocytopenic purpura.

Approximately 40% of patients who survive acute episodes of thrombotic thrombocytopenic purpura (TTP) associated with severe acquired ADAMTS13 deficiency experience one or more relapses. Risk factors for relapse other than severe ADAMTS13 deficiency and ADAMTS13 autoantibodies are unknown. ADAMTS13 autoantibodies, TTP episodes following infection or type I interferon treatment and reported ensuing systemic lupus erythematosus in some patients suggest immune dysregulation.

Epigenome profiling reveals significant DNA demethylation of interferon signature genes in lupus neutrophils.

Recent evidence suggests that neutrophils play an important role in the pathogenesis of lupus. The goal of this study was to characterize the epigenetic architecture, by studying the DNA methylome, of neutrophils and low density granulocytes (LDGs) in lupus patients. We studied 15 lupus patients and 15 healthy age, sex, and ethnicity matched controls.

Defective selection of thymic regulatory T cells accompanies autoimmunity and pulmonary infiltrates in Tcra-deficient mice double transgenic for human La/Sjögren's syndrome-B and human La-specific TCR.

A human La/Sjögren's syndrome-B (hLa)-specific TCR/hLa neo-self-Ag double-transgenic (Tg) mouse model was developed and used to investigate cellular tolerance and autoimmunity to the ubiquitous RNA-binding La Ag often targeted in systemic lupus erythematosus and Sjögren's syndrome. Extensive thymic clonal deletion of CD4(+) T cells occurred in H-2(k/k) double-Tg mice presenting high levels of the I-E(k)-restricted hLa T cell epitope. In contrast, deletion was less extensive in H-2(k/b) double-Tg mice presenting lower levels of the epitope, and some surviving thymocytes were positively selected as thymic regulatory T cells (tTreg).

Genetic association of CD247 (CD3ζ) with SLE in a large-scale multiethnic study.

A classic T-cell phenotype in systemic lupus erythematosus (SLE) is the downregulation and replacement of the CD3ζ chain that alters T-cell receptor signaling. However, genetic associations with SLE in the human CD247 locus that encodes CD3ζ are not well established and require replication in independent cohorts. Our aim was therefore to examine, localize and validate CD247-SLE association in a large multiethnic population.

Differential expression of the transcription factor ARID3a in lupus patient hematopoietic progenitor cells.

Although hematopoietic stem/progenitor cells (HSPCs) are used for transplantation, characterization of the multiple subsets within this population in humans has lagged behind similar studies in mice. We found that expression of the DNA-binding protein, ARID3a, in mouse stem cells was important for normal development of hematopoietic lineages; however, progenitors expressing ARID3a in humans have not been defined. We previously showed increased numbers of ARID3a(+) B cells in nearly half of systemic lupus erythematosus (SLE) patients, and total numbers of ARID3a(+) B cells were associated with increased disease severity.

A coding IRAK2 protein variant compromises Toll-like receptor (TLR) signaling and is associated with colorectal cancer survival.

Within innate immune signaling pathways, interleukin-1 receptor-associated kinases (IRAKs) fulfill key roles downstream of multiple Toll-like receptors and the interleukin-1 receptor. Although human IRAK4 deficiency was shown to lead to severe immunodeficiency in response to pyogenic bacterial infection during childhood, little is known about the role of human IRAK2. We here identified a non-synonymous IRAK2 variant, rs35060588 (coding R214G), as hypofunctional in terms of NF-κB signaling and Toll-like receptor-mediated cytokine induction.

Early effects of Staphylococcus aureus biofilm secreted products on inflammatory responses of human epithelial keratinocytes.

Background: Chronic wounds such as diabetic foot ulcers, pressure ulcers, and venous leg ulcers contribute to a considerable amount of mortality in the U.S. annually.

GWAS identifies novel SLE susceptibility genes and explains the association of the HLA region.

In a genome-wide association study (GWAS) of individuals of European ancestry afflicted with systemic lupus erythematosus (SLE) the extensive utilization of imputation, step-wise multiple regression, lasso regularization and increasing study power by utilizing false discovery rate instead of a Bonferroni multiple test correction enabled us to identify 13 novel non-human leukocyte antigen (HLA) genes and confirmed the association of four genes previously reported to be associated. Novel genes associated with SLE susceptibility included two transcription factors (EHF and MED1), two components of the NF-κB pathway (RASSF2 and RNF114), one gene involved in adhesion and endothelial migration (CNTN6) and two genes involved in antigen presentation (BIN1 and SEC61G). In addition, the strongly significant association of multiple single-nucleotide polymorphisms (SNPs) in the HLA region was assigned to HLA alleles and serotypes and deconvoluted into four primary signals.

Alum, an aluminum-based adjuvant, induces Sjögren's syndrome-like disorder in mice.

Objectives: Adjuvant-induced innate immune responses have been suspected to play a role in the initiation of certain autoimmune disorders. This study investigates the role of alum, an aluminum-based adjuvant in the induction of Sjögren's syndrome-like disorder in mice.

Methods: Inbred, female New Zealand Mixed (NZM) 2758 strain of mice were injected with alum.

Two functional lupus-associated BLK promoter variants control cell-type- and developmental-stage-specific transcription.

Efforts to identify lupus-associated causal variants in the FAM167A/BLK locus on 8p21 are hampered by highly associated noncausal variants. In this report, we used a trans-population mapping and sequencing strategy to identify a common variant (rs922483) in the proximal BLK promoter and a tri-allelic variant (rs1382568) in the upstream alternative BLK promoter as putative causal variants for association with systemic lupus erythematosus. The risk allele (T) at rs922483 reduced proximal promoter activity and modulated alternative promoter usage.

MHC associations with clinical and autoantibody manifestations in European SLE.

Systemic lupus erythematosus (SLE) is a clinically heterogeneous disease affecting multiple organ systems and characterized by autoantibody formation to nuclear components. Although genetic variation within the major histocompatibility complex (MHC) is associated with SLE, its role in the development of clinical manifestations and autoantibody production is not well defined. We conducted a meta-analysis of four independent European SLE case collections for associations between SLE sub-phenotypes and MHC single-nucleotide polymorphism genotypes, human leukocyte antigen (HLA) alleles and variant HLA amino acids.

PXK locus in systemic lupus erythematosus: fine mapping and functional analysis reveals novel susceptibility gene ABHD6.

Objectives: To perform fine mapping of the PXK locus associated with systemic lupus erythematosus (SLE) and study functional effects that lead to susceptibility to the disease.

Methods: Linkage disequilibrium (LD) mapping was conducted by using 1251 SNPs (single nucleotide polymorphism) covering a 862 kb genomic region on 3p14.3 comprising the PXK locus in 1467 SLE patients and 2377 controls of European origin.