Search

Systemic Lupus Erythematosus (SLE) is a complex autoimmune disease characterized by overproduction of autoantibodies by B cells and the loss of tolerance to nucleic acids. Our previous studies show that AT-rich interacting domain 3a (ARID3a), a DNA-binding protein, is more abundant in SLE B cells than in healthy control B cells. Increased numbers of ARID3a+ B cells in SLE correlated with increased disease activity in patients. ARID3a expression in SLE patient blood occurred in naïve B cell subsets which do not express ARID3a in healthy controls. Studies by the Sanz laboratory revealed that activated naïve B cells and may be precursors for the double negative (DN) B cells linked to pathogenic responses in SLE. We hypothesize that ARID3a expression in naïve B cells also defines precursors of pathogenic B cells. In individual patients, ARID3a is bimodally expressed, such that only a fraction of the naïve B cells express ARID3a at any given time. We took advantage of this bimodal expression using single cell technology to perform RNA-seq analyses of ARID3a+ and ARID3a− SLE B cells to identify differentially expressed genes associated with ARID3a. PCA analyses of scRNA seq data show that ARID3a+ naïve B cells cluster together and away from ARID3a− cells. This allows for analyses within and among individual samples by binning cells based on ARID3a expression. Preliminary data indicate that ARID3a expression is correlated with innate immune response genes, transcription factors, and epigenetic regulators. These studies will be informative regarding how ARID3a expression contributes to disease pathogenesis in patients with SLE and will provide novel information defining a new subset of naive B cells that may be directly linked to breaches in tolerance.

Our previous studies demonstrated that AT-rich interacting domain 3a (ARID3a), a DNA-binding protein associated with increased immunoglobulin heavy chain transcription and chromatin accessibility, is required for normal hematopoiesis in mice and in human cells. ARID3a knockout mice exhibited low numbers of erythrocytes and died due to failed erythropoiesis between days 9 and 12 of gestation. The role of ARID3a in human erythropoiesis has not been studied. Stimulation of the human K562 cell line with hemin induces fetal globin synthesis and erythrocyte differentiation. K562 cells constitutively express ARID3a, so we used this cell line to determine if ARID3a is important for human erythrocyte differentiation. Knockdown of ARID3a with shRNA in hemin-stimulated K562 cells resulted in >75% inhibition of globin. To identify genes affected by ARID3a loss, we performed RNA-seq experiments at various times after hemin stimulation with and without ARID3a. ARID3a shRNA-inhibited cells confirmed inhibition of globin gene products, including HBA1, HBA2, and HBZ. Erythroid specific transcription factors were also inhibited in samples treated with ARID3a shRNA while myeloid surface markers were induced. Several critical upstream regulators of ARID3a were also identified in these analyses. These data suggest that ARID3a is important for cell fate decisions in erythropoiesis in human cells and identifies potential important targets of ARID3a in this process.

Population studies estimate that 40% of European and US populations will be over the age of 60 by 2050. Aged individuals exhibit impaired immune responses consisting of poor responses to vaccines and increases in myeloid lineage cells. The reasons for decreased immunity in the elderly haven’t been completely elucidated, making studies of the mechanisms leading to these poor responses important. One possibility is that defects in early hematopoiesis may contribute to poor immune responses in the elderly. Previous studies in our lab indicated that inhibition of the DNA-binding protein ARID3a in hematopoietic cultures of human cord blood led to hematopoietic developmental skewing toward myeloid lineage rather than lymphoid lineage progenitors. Furthermore, we previously reported that numbers of ARID3a-expressing hematopoietic stem cells (HSCs) can be highly variable in some patient subsets as well as in healthy individuals. ARID3a expression levels in hematopoietic progenitor cells have not previously been analyzed in relationship to age. We hypothesized that alterations in ARID3a expression levels in early hematopoietic progenitors could contribute to poor immune responses observed in elderly people. Our data suggest that hematopoietic progenitors obtained from peripheral blood of aged healthy persons show decreased frequencies of ARID3a-expressing HSCs in comparison to HSCs from young individuals. Furthermore, lineage committed progenitor numbers were also altered in aged persons compared young people. Ongoing analyses to investigate the gene expression data in young and aged HSCs in relation to ARID3a expression will assess potential genes regulated by ARID3a that contributes to lineage commitment and development.

Chlorine is a public health concern and potential threat due to its high reactivity, ease and scale of production, widespread industrial use, bulk transportation, massive stockpiles, and history as a chemical weapon. This work describes a new, sensitive, and rapid stable isotope dilution method for the retrospective detection and quantitation of two chlorine adducts. The biomarkers 3-chlorotyrosine (Cl-Tyr) and 3,5-dichlorotyrosine (Cl2-Tyr) were isolated from the pronase digest of chlorine exposed whole blood, serum, or plasma by solid phase extraction (SPE), separated by reversed-phase HPLC, and detected by tandem mass spectrometry (MS/MS). The calibration range is 2.50–1000 ng/ml (R2 ≥ 0.998) with a lowest reportable limit (LRL) of 2.50 ng/mL for both analytes, an accuracy of ≥ 93%, and an LOD of 0.443 ng/mL for Cl-Tyr and 0.396 ng/mL for Cl2-Tyr. Inter- and intra-day precision of quality control samples had coefficients of variation of ≤ 10% and ≤ 7.0%, respectively. Blood and serum samples from 200 healthy individuals and 175 individuals with chronic inflammatory disease were analyzed using this method to assess background levels of chlorinated tyrosine adducts. Results from patients with no known inflammatory disease history (healthy) showed baseline levels of

Systemic Lupus Erythematosus (SLE) is a complex autoimmune disease characterized by overproduction of autoantibodies by B cells and the loss of tolerance to nucleic acids. Our previous studies demonstrated that AT-rich interacting domain 3a (ARID3a), a DNA-binding protein associated with immunoglobulin transcription, is more abundant in SLE B lymphocytes than in healthy control B cells. ARID3a expression in SLE patient blood occurred in naïve B cell subsets which do not express ARID3a in healthy controls. Importantly, increased numbers of ARID3a+ B cells in SLE correlated with increased disease activity in patients. Our recent data indicated that ARID3a is critical for the expression of the key inflammatory cytokine, interferon-alpha (IFNa), in B cells and other blood cells. ARID3a+ B cells appear to denote a new kind of B effector cell that can induce IFNa expression in other cell types. We hypothesize that ARID3a is an important contributor to increased disease activity in SLE patients. Therefore, it is important to better define the characteristics of these cells. ARID3a is bimodally expressed, such that only a fraction of the cells within any B cell subset express ARID3a at a given time. Our current studies took advantage of this bimodal expression using single-cell technology to perform RNA-seq analyses of ARID3a+ and ARID3a− SLE B cells to identify differentially expressed genes associated with ARID3a. To date, ARID3a expression is correlated with innate immune response genes and epigenetic regulators. We predict these studies will be informative regarding how ARID3a expression contributes to disease pathogenesis in patients with SLE, and will provide novel information defining a new subset of effector B cells.

Sulfur mustard binds to reactive cysteine residues, forming a stable sulfur-hydroxyethylthioethyl [S-HETE] adduct that can be used as a long-term biomarker of sulfur mustard exposure in humans. The digestion of sulfur mustard-exposed blood samples with proteinase K following total protein precipitation with acetone produces the tripeptide biomarker [S-HETE]-Cys-Pro-Phe. The adducted tripeptide is purified by solid phase extraction, separated by ultra-high pressure liquid chromatography, and detected by isotope dilution tandem mass spectrometry. This approach was thoroughly validated and characterized in our laboratory. The average interday relative standard deviation was ≤ 9.49%, and the range of accuracy was between 96.1-109% over a concentration range of 3.00 to 250. ng/mL with a calculated limit of detection of 1.74 ng/mL. A full 96-well plate can be processed and analyzed in 8 h which is five times faster than our previous 96-well plate method and only requires 50 µL of serum, plasma, or whole blood. Extensive ruggedness and stability studies and matrix comparisons were conducted to create a robust, easily transferrable method. As a result, a simple and high-throughput method has been developed and validated for the quantitation of sulfur mustard blood protein adducts in low volume blood specimens which should be readily adaptable for quantifying human exposures to other alkylating agents.

This work describes a new specific, sensitive, and rapid stable isotope dilution method for the simultaneous detection of the organophosphorus nerve agents (OPNAs) tabun (GA), sarin (GB), soman (GD), cyclosarin (GF), VR, VX, and VM adducts to tyrosine (Tyr). Serum, plasma, and lysed whole blood samples (50 μL) were prepared by protein precipitation followed by digestion with Pronase. Specific Tyr adducts were isolated from the digest by a single solid phase extraction (SPE) step, and the analytes were separated by reversed-phase ultra high performance liquid chromatography (UHPLC) gradient elution in less than 2 min. Detection was performed on a triple quadrupole tandem mass spectrometer using time-triggered selected reaction monitoring (SRM) in positive electrospray ionization (ESI) mode. The calibration range was characterized from 0.100-50.0 ng/mL for GB- and VR-Tyr and 0.250-50.0 ng/mL for GA-, GD-, GF-, and VX/VM-Tyr (R(2) ≥ 0.995). Inter- and intra-assay precision had coefficients of variation of ≤17 and ≤10%, respectively, and the measured concentration accuracies of spiked samples were within 15% of the targeted value for multiple spiking levels. The limit of detection was calculated to be 0.097, 0.027, 0.018, 0.074, 0.023, and 0.083 ng/mL for GA-, GB-, GD-, GF-, VR-, and VX/VM-Tyr, respectively. A convenience set of 96 serum samples with no known nerve agent exposure was screened and revealed no baseline values or potential interferences. This method provides a simple and highly specific diagnostic tool that may extend the time postevent that a confirmation of nerve agent exposure can be made with confidence.