Using PROMIS-29 to determine symptom burdens in the context of the Type 1 and 2 systemic lupus erythematosus (SLE) model: a cross sectional study.

OBJECTIVE: To account for heterogeneity in systemic lupus erythematosus (SLE) and bridge discrepancies between patient- and physician-perceived SLE activity, we developed the Type 1 and 2 SLE model. We examined PROMIS-29 scores, a composite patient-reported outcome (PRO) measure, through the lens of the model. METHODS: Patients completed PROMIS-29 and the polysymptomatic distress scale (PSD). Rheumatologists completed the SLE disease activity index (SLEDAI), and physician's global assessments (PGAs) for Type 1 and 2 SLE. We defined Type 1 SLE using SLEDAI, Type 1 PGA, and active nephritis, and Type 2 SLE using PSD and Type 2 PGA. We compared PROMIS-29 T-scores among Type 1 and 2 SLE groups and explored whether PROMIS-29 can predict Type 1 and 2 SLE activity. RESULTS: Compared to the general population, patients with isolated Type 1 SLE reported greater pain and physical dysfunction but less depression and improved social functions; patients with high Type 2 SLE (irrespective of Type 1 activity) reported high levels of pain, fatigue, and social and physical limitations. Patients with minimal Type 1 and 2 SLE had less depression and greater physical functioning with other domains similar to national norms. PROMIS-29 predicted Type 2 but not Type 1 SLE activity. CONCLUSION: PROMIS-29 similarities in patients with high Type 2 SLE, with and without active Type 1 SLE, demonstrate the challenges of using PROs to assess SLE inflammation. In conjunction with the Type 1 and 2 SLE model, however, PROMIS-29 identified distinct symptom patterns, suggesting that the model may help clinicians interpret PROs.

Pregnancy Outcomes in Undifferentiated Connective Tissue Disease Compared to Systemic Lupus Erythematosus: A Single Academic Center's Experience.

OBJECTIVE: Systemic lupus erythematosus (SLE) patients have more pregnancy complications than healthy patients. Data regarding pregnancy outcomes in women with undifferentiated connective tissue disease (UCTD) are more limited, and existing studies are concentrated in Italy and predominantly in patients with a new diagnosis. Our objective was to compare pregnancy outcomes for UCTD and SLE patients with established disease. METHODS: Between 2008 and 2017, patients with UCTD and SLE at an academic medical center were recruited to a prospective pregnancy registry. UCTD was defined as a positive autoantibody plus connective tissue disease symptoms not meeting criteria for another rheumatic diagnosis. SLE was defined by American College of Rheumatology or Systemic Lupus International Collaborating Clinics classification criteria or by physician diagnosis. Data were collected throughout pregnancy and postpartum. Comparator groups included UCTD, low-activity SLE, and high-activity SLE. RESULTS: A total of 150 SLE and 51 UCTD pregnancies were analyzed. Disease activity was low in most patients, although more patients with SLE had severe activity during pregnancy (12% versus 2%; P = 0.05). The frequencies of prematurity and preeclampsia were significantly lower in UCTD than in high-activity SLE patients (preterm 17% versus 45% [P = 0.004] and preeclampsia 6% versus 34% [P = 0.0008]), although similar to low-activity SLE patients. More infants who were small for gestational age were born to SLE than UCTD patients (33% versus 7% [P = 0.0005]), regardless of disease activity level. CONCLUSION: Pregnancies in women with UCTD managed by a rheumatologist have a high rate of pregnancy success and fewer risks than those in women with active SLE.

Interactions of E. coli with cylindrical micro-pillars of different geometric modifications.

Understanding the behavior of bacteria at the proximity of different surfaces is of great importance and interest. Despite recent exciting progress in geometric control of bacterial behavior around surfaces, a detailed comparison on the interaction of bacteria with cylindrical surfaces of different geometric modifications is still missing. Here, we investigated how bacteria interacted with cylindrical micro-pillars and modified cylindrical micro-pillars with sprocket, gear, and flower-like wall surface features. Using phase-contrast microscopy, we examined the motion of bacteria around the micro-pillars, and observed different responses of bacteria to each geometric modification. In addition, we extracted the trajectories of the bacteria and characterized several parameters (instantaneous velocity v, change of direction δ, approaching angle ϕ) to quantitatively compare the effects of the geometric modifications on the micro-pillars. We found that sharp spikes showed the largest effect, compared to smooth surface, convex and concave ripples. Lastly, we carried out numerical simulations, which explained the experimental observations and showed that the observed effects were due to the geometric modifications.

Photothermal Response Induced by Nanocage-Coated Artificial Extracellular Matrix Promotes Neural Stem Cell Differentiation.

Strategies to increase the proportion of neural stem cells that differentiate into neurons are vital for therapy of neurodegenerative disorders. In vitro, the extracellular matrix composition and topography have been found to be important factors in stem cell differentiation. We have developed a novel artificial extracellular matrix (aECM) formed by attaching gold nanocages (AuNCs) to glass coverslips. After culturing rat neural stem cells (rNSCs) on these gold nanocage-coated surfaces (AuNC-aECMs), we observed that 44.6% of rNSCs differentiated into neurons compared to only 27.9% for cells grown on laminin-coated glass coverslips. We applied laser irradiation to the AuNC-aECMs to introduce precise amounts of photothermally induced heat shock in cells. Our results showed that laser-induced thermal stimulation of AuNC-aECMs further enhanced neuronal differentiation (56%) depending on the laser intensity used. Response to these photothermal effects increased the expression of heat shock protein 27, 70, and 90α in rNSCs. Analysis of dendritic complexity showed that this thermal stimulation promoted neuronal maturation by increasing dendrite length as thermal dose was increased. In addition, we found that cells growing on AuNC-aECMs post laser irradiation exhibited action potentials and increased the expression of voltage-gated Na+ channels compared to laminin-coated glass coverslips. These results indicate that the photothermal response induced in cells growing on AuNC-aECMs can be used to produce large quantities of functional neurons, with improved electrochemical properties, that can potentially be transplanted into a damaged central nervous system to provide replacement neurons and restore lost function.

Therapeutic Opportunities for Intestinal Angioectasia- Targeting PPARγ and Oxidative Stress.

Recurrent and acute bleeding from intestinal tract angioectasia (AEC) presents a major challenge for clinical intervention. Current treatments are empiric, with frequent poor clinical outcomes. Improvements in understanding the pathophysiology of these lesions will help guide treatment. Using data from the US Food and Drug Administration (FDA)'s Adverse Event Reporting System (FAERS), we analyzed 12 million patient reports to identify drugs inversely correlated with gastrointestinal bleeding and potentially limiting AEC severity. FAERS analysis revealed that drugs used in patients with diabetes and those targeting PPARγ-related mechanisms were associated with decreased AEC phenotypes (P < 0.0001). Electronic health records (EHRs) at University of Cincinnati Hospital were analyzed to validate FAERS analysis. EHR data showed a 5.6% decrease in risk of AEC and associated phenotypes in patients on PPARγ agonists. Murine knockout models of AEC phenotypes were used to construct a gene-regulatory network of candidate drug targets and pathways, which revealed that wound healing, vasculature development and regulation of oxidative stress were impacted in AEC pathophysiology. Human colonic tissue was examined for expression differences across key pathway proteins, PPARγ, HIF1α, VEGF, and TGFß1. In vitro analysis of human AEC tissues showed lower expression of PPARγ and TGFß1 compared with controls (0.55 ± 0.07 and 0.49 ± 0.05). National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) RNA-Seq data was analyzed to substantiate human tissue findings. This integrative discovery approach showing altered expression of key genes involved in oxidative stress and injury repair mechanisms presents novel insight into AEC etiology, which will improve targeted mechanistic studies and more optimal medical therapy for AEC.

Patient-Reported Disease Activity and Adverse Pregnancy Outcomes in Systemic Lupus Erythematosus and Rheumatoid Arthritis.

OBJECTIVE: While increased rheumatic disease activity during pregnancy has been associated with adverse pregnancy outcomes, this disease activity is typically assessed by physicians. Little is known, however, about the association between patient-reported measures of disease activity and pregnancy outcomes. The aim of our study was to evaluate this association. METHODS: Univariate and multivariable regression models were used to assess the relationship between patient- and physician-reported measures of disease activity and adverse pregnancy outcomes in 225 patients with systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA). The patients were enrolled from 2008-2016 in a prospective registry at a single academic center. RESULTS: In women with RA, patient-reported disease activity was associated with preterm birth (odds ratio [OR] 5.9 [95% confidence interval (95% CI) 1.5, 23.9]) and gestational age in weeks (ß = -1.5 [95% CI -2.6, -0.4]). The physician assessment of disease activity also predicted preterm (OR 2.1 [95% CI 1.2, 3.5]), small for gestational age births (OR 1.8 [95% CI 1.03, 3.1]), and gestational age in weeks (ß = -0.6 [95% CI -0.9, -0.02]). Alternatively, in women with SLE, patient-reported disease activity measures, including the Health Assessment Questionnaire, pain, or global health measures, were not associated with adverse pregnancy outcomes. However, physician measures of SLE disease activity are associated with preterm birth (OR 2.9 [95% CI 1.3, 6.3]), cesarean delivery (OR 2.3 [95% CI 1.0, 5.3]), and preeclampsia (OR 2.8 [95% CI 1.3, 6.3]). The results did not appear to be driven by lupus nephritis or antiphospholipid syndrome. CONCLUSION: For women with RA, patient-reported measures of disease activity were associated with adverse pregnancy outcomes, and thus may be useful adjuncts to physician-reported measures in identifying pregnancies at greater risk. In contrast, in SLE, while physician measures of disease activity helped predict several adverse pregnancy outcomes, no patient-reported measures were associated with adverse outcomes.

UV light absorption parameters of the pathobiologically implicated bilirubin oxidation products, MVM, BOX A, and BOX B.

The formation of the bilirubin oxidation products (BOXes), BOX A ([4-methyl-5-oxo-3-vinyl-(1,5-dihydropyrrol-2-ylidene)acetamide]) and BOX B (3-methyl-5-oxo-4-vinyl-(1,5-dihydropyrrol-2-ylidene)acetamide), as well as MVM (4-methyl-3-vinylmaleimide) were synthesized by oxidation of bilirubin with H2O2 without and with FeCl3, respectively. Compound identity was confirmed with NMR and mass spectrometry (MS; less than 1 ppm, tandem MS up to MS4). UV absorption profiles, including λmax, and extinction coefficient (ε; estimated using NMR) for BOX A, BOX B, and MVM in H2O, 15% CH3CN plus 10 mM CF3CO2H, CH3CN, CHCl3, CH2Cl2, and 0.9% NaCl were determined. At longer wavelengths, λmax's for 1) BOX A were little affected by the solvent, ranging from 295-297 nm; 2) BOX B, less polar solvent yielded λmax's of lower wavelength, with values ranging from 308-313 nm, and 3) MVM, less polar solvent yielded λmax's of higher wavelength, with values ranging from 318-327 nm. Estimated ε's for BOX A and BOX B were approximately 5- to 10-fold greater than for MVM.

Anti-inflammatory activity of IgG1 mediated by Fc galactosylation and association of FcγRIIB and dectin-1.

Complement is an ancient danger-sensing system that contributes to host defense, immune surveillance and homeostasis. C5a and its G protein­coupled receptor mediate many of the proinflammatory properties of complement. Despite the key role of C5a in allergic asthma, autoimmune arthritis, sepsis and cancer, knowledge about its regulation is limited. Here we demonstrate that IgG1 immune complexes (ICs), the inhibitory IgG receptor FcγRIIB and the C-type lectin­like receptor dectin-1 suppress C5a receptor (C5aR) functions. IgG1 ICs promote the association of FcγRIIB with dectin-1, resulting in phosphorylation of Src homology 2 domain­containing inositol phosphatase (SHIP) downstream of FcγRIIB and spleen tyrosine kinase downstream of dectin-1. This pathway blocks C5aR-mediated ERK1/2 phosphorylation, C5a effector functions in vitro and C5a-dependent inflammatory responses in vivo, including peritonitis and skin blisters in experimental epidermolysis bullosa acquisita. Notably, high galactosylation of IgG N-glycans is crucial for this inhibitory property of IgG1 ICs, as it promotes the association between FcγRIIB and dectin-1. Thus, galactosylated IgG1 and FcγRIIB exert anti-inflammatory properties beyond their impact on activating FcγRs.

C5a receptor-deficient dendritic cells promote induction of Treg and Th17 cells.

C5a is a proinflammatory mediator that has recently been shown to regulate adaptive immune responses. Here we demonstrate that C5a receptor (C5aR) signaling in DC affects the development of Treg and Th17 cells. Genetic ablation or pharmacological targeting of the C5aR in spleen-derived DC results in increased production of TGF-beta leading to de novo differentiation of Foxp3(+) Treg within 12 h after co-incubation with CD4(+) T cells from DO11.10/RAG2(-/-) mice. Stimulation of C5aR(-/-) DC with OVA and TLR2 ligand Pam(3)CSK(4) increased TGF-beta production and induced high levels of IL-6 and IL-23 but only minor amounts of IL-12 leading to differentiation of Th cells producing IL-17A and IL-21. Th17 differentiation was also found in vivo after adoptive transfer of CD4(+) Th cell into C5aR(-/-) mice immunized with OVA and Pam(3)CSK(4). The altered cytokine production of C5aR(-/-) DC was associated with low steady state MHC class II expression and an impaired ability to upregulate CD86 and CD40 in response to TLR2. Our data suggest critical roles for C5aR in Treg and Th17-cell differentiation through regulation of DC function.

Commitment to the regulatory T cell lineage requires CARMA1 in the thymus but not in the periphery.

Regulatory T (T(reg)) cells expressing forkhead box P3 (Foxp3) arise during thymic selection among thymocytes with modestly self-reactive T cell receptors. In vitro studies suggest Foxp3 can also be induced among peripheral CD4(+) T cells in a cytokine dependent manner. T(reg) cells of thymic or peripheral origin may serve different functions in vivo, but both populations are phenotypically indistinguishable in wild-type mice. Here we show that mice with a Carma1 point mutation lack thymic CD4(+)Foxp3(+) T(reg) cells and demonstrate a cell-intrinsic requirement for CARMA1 in thymic Foxp3 induction. However, peripheral Carma1-deficient T(reg) cells could be generated and expanded in vitro in response to the cytokines transforming growth factor beta (TGFbeta) and interleukin-2 (IL-2). In vivo, a small peripheral T(reg) pool existed that was enriched at mucosal sites and could expand systemically after infection with mouse cytomegalovirus (MCMV). Our data provide genetic evidence for two distinct mechanisms controlling regulatory T cell lineage commitment. Furthermore, we show that peripheral T(reg) cells are a dynamic population that may expand to limit immunopathology or promote chronic infection.

Sustained CTL activation by murine pulmonary epithelial cells promotes the development of COPD-like disease.

Chronic obstructive pulmonary disease (COPD) is a lethal progressive lung disease culminating in permanent airway obstruction and alveolar enlargement. Previous studies suggest CTL involvement in COPD progression; however, their precise role remains unknown. Here, we investigated whether the CTL activation receptor NK cell group 2D (NKG2D) contributes to the development of COPD. Using primary murine lung epithelium isolated from mice chronically exposed to cigarette smoke and cultured epithelial cells exposed to cigarette smoke extract in vitro, we demonstrated induced expression of the NKG2D ligand retinoic acid early transcript 1 (RAET1) as well as NKG2D-mediated cytotoxicity. Furthermore, a genetic model of inducible RAET1 expression on mouse pulmonary epithelial cells yielded a severe emphysematous phenotype characterized by epithelial apoptosis and increased CTL activation, which was reversed by blocking NKG2D activation. We also assessed whether NKG2D ligand expression corresponded with pulmonary disease in human patients by staining airway and peripheral lung tissues from never smokers, smokers with normal lung function, and current and former smokers with COPD. NKG2D ligand expression was independent of NKG2D receptor expression in COPD patients, demonstrating that ligand expression is the limiting factor in CTL activation. These results demonstrate that aberrant, persistent NKG2D ligand expression in the pulmonary epithelium contributes to the development of COPD pathologies.

CD8+ T cells contribute to macrophage accumulation and airspace enlargement following repeated irritant exposure.

BACKGROUND: Persistent macrophage accumulation and alveolar enlargement are hallmark features of chronic obstructive pulmonary disease (COPD). A role for CD8(+) lymphocytes in the development of COPD is suggested based on observations that this T cell subset is increased in the airways and parenchyma of smokers that develop COPD with airflow limitation. In this study, we utilize a mouse model of COPD to examine the contributions of CD8(+) T cells in the persistent macrophage accumulation and airspace enlargement resulting from chronic irritant exposure. METHODS: We analyzed pulmonary inflammation and alveolar destruction in wild-type and Cd8-deficient mice chronically exposed to acrolein, a potent respiratory tract irritant. We further examined cytokine mRNA expression levels by RNase protection assay, matrix metalloproteinase (MMP) activity by gelatin zymography, and epithelial cell apoptosis by active caspase3 immunohistochemistry in wild-type and Cd8-deficient mice exposed chronically to acrolein. RESULTS: These studies demonstrate that CD8(+) T cells are important mediators of macrophage accumulation in the lung and the progressive airspace enlargement in response to chronic acrolein exposures. The expression of several inflammatory cytokines (IP-10, IFN-gamma, IL-12, RANTES, and MCP-1), MMP2 and MMP9 gelatinase activity, and caspase3 immunoreactivity in pulmonary epithelial cells were attenuated in the Cd8-deficient mice compared to wild-type. CONCLUSIONS: These results indicate that CD8(+) T cells actively contribute to macrophage accumulation and the development of irritant-induced airspace enlargement.

The NKG2D-activating receptor mediates pulmonary clearance of Pseudomonas aeruginosa.

The NKG2D-activating receptor is expressed on cytotoxic lymphocytes and interacts with ligands expressed on the surface of cells stressed by pathogenic and nonpathogenic stimuli. In this study, we investigated the physiologic importance of NKG2D receptor-ligand interactions in response to acute pulmonary Pseudomonas aeruginosa infection. P. aeruginosa infection increased the expression of mouse NKG2D ligands (Rae1) in airway epithelial cells and alveolar macrophages in vivo and also increased the cell surface expression of human NKG2D ligands (ULBP2) on airway epithelial cells in vitro. NKG2D receptor blockade with a specific monoclonal antibody inhibited the pulmonary clearance of P. aeruginosa. NKG2D receptor blockade also resulted in decreased production of Th1 cytokines and nitric oxide in the lungs of P. aeruginosa-infected mice. Additionally, NKG2D receptor blockade reduced the epithelial cell sloughing that accompanies P. aeruginosa infection. Macrophage phagocytosis and bronchoalveolar lavage cellularity were not different in P. aeruginosa-infected mice with and without NKG2D receptor blockade. These results demonstrate the importance of NKG2D-mediated immune activation in the clearance of acute bacterial infection and suggest that epithelial cell-lymphocyte interactions mediate pulmonary cytokine production, epithelial cell integrity, and bacterial clearance.

NKG2D ligands are expressed on stressed human airway epithelial cells.

Immune surveillance of the airways is critical to maintain the integrity and health of the lung. We have identified a family of ligands expressed on the surface of stressed airway epithelial cells whose function is to bind the NKG2D-activating receptor found on several pulmonary lymphocytes, including natural killer cells, gammadelta(+) T cells, and CD8(+) T cells. We employed real-time PCR and flow cytometry in normal and transformed airway epithelial cell to demonstrate that major histocompatibility complex class I chain-related (MIC) B and the UL-16 binding protein (ULBP) ligands (ULBP1-4) are ubiquitously expressed at the mRNA level in all cell lines. MICA/B surface expression was present on 70% of transformed cell lines but was undetectable on primary cells. We demonstrate that MICA/B and ULBP 1, 2, 3, and 4 expression is rare or absent on the cell surface of unstimulated normal human bronchial epithelial cells although transcripts and intracellular proteins are present. Normal human bronchial epithelial cells exposed to 0.3 mM hydrogen peroxide exhibit an induction of all ligands examined on the cell surface. Surface expression is independent of changes in transcript level or total cellular protein and is mediated by the ERK family of mitogen-activated protein kinases. The induction of NKG2D ligands on stressed airway epithelial cells represents a potentially important mechanism of immune cell activation in regulation of pulmonary health and disease.

Nuclear factor-kappa B and apoptosis inducing factor activation by doxorubicin analog WP744 in SH-SY5Y neuroblastoma cells.

BACKGROUND: Neuroblastoma (NB) is one of the most common extracranial tumors in children. The chemotherapeutic doxorubicin (Dox) remains a mainstay for treatment. However, the emergence of drug resistance seriously limits treatment efficacy. Numerous Dox analogues have been designed to more potently kill drug naive as well as drug-resistant NB. We have shown that the Dox analogue WP744 has enhanced the killing activity of NB compared to Dox, but the mechanism(s) of action is unclear. MATERIALS AND METHODS: MTT assays were used to characterize the relative potencies of Dox and WP744 against SH-SY5Y NB cells. Western blotting was used to assess activation of caspases-3, -9, anti-poly (ADP-ribose) polymerase, p53, p21, AIF, IkappaBalpha, Bcl-2, Bcl-X(L), and cyclin D1. Nuclear factor (NF-kappaB) activation was assessed by electrophoretic mobility shift assay. RESULTS: After WP744 treatment, enhanced apoptosis and cell death were seen, associated with cleavage of caspases-3, -9, and anti-poly (ADP-ribose) polymerase, an increase in p53 protein levels, and the induction of p21. WP744 also induced translocation of apoptosis-inducing factor from mitochondria to nuclei. Most remarkably, WP744 was 50-fold more potent than Dox in activating NF-kappaB. WP744 treatment also resulted in the down-regulation of expression of downstream targets of NF-kappaB. CONCLUSIONS: WP744 is a novel Dox analogue that triggers apoptosis and cell killing by activation of proapoptotic mediators in NB cells. Enhanced cytotoxicity of this novel drug compared with Dox may be related to more effective activation of NF-kappaB and apoptosis-inducing factor in tumor cells.

WP744 is a novel anthracycline with enhanced activity against neuroblastoma.

BACKGROUND: Doxorubicin (Dox) is one of the most useful chemotherapeutic agents for patients with advanced neuroblastoma (NB). A series of Dox analogs with bulky substitutions at the C-4' at amino-sugar have been designed to impair interactions between the drug and P-glycoprotein (P-gp), a multidrug drug resistance (MDR) transporter. Two analogs, WP744 and WP769, were selected and their biological properties were compared with Dox and the daunorubicin-based bisintercalator WP631. These novel Dox analogs may have antitumor activity beyond MDR evasion. MATERIALS AND METHODS: MTT assays were used to determine the potency of three structurally altered Dox analogs against a panel of NB cell lines with and without amplification of the MYCN oncogene. Flow cytometry (FCM) was used to analyze apoptosis and cell death and phenotype cell lines for surface expression of the MDR protein P-gp. RESULTS: The 4'-O-benzylated Dox analogs WP744 and WP769 were 2 to 36 times more cytotoxic than Dox for the NB cell lines tested. The bis-intercalator WP631, despite its significantly greater affinity for DNA (>10,000-fold), was generally less potent against NB than Dox. In Tet21N cells, which conditionally express MYCN, greatly enhanced (nearly 6-fold) sensitivity to WP744 killing was seen when this oncogene was induced, while enhanced sensitivity to Dox was more modest (2-fold) under MYCN-induced conditions. Treatment with WP744 also resulted in enhanced apoptosis. Apoptosis, but not cell death, in response to either WP744 or Dox was inhibited by caspase inhibition, suggesting that cell death was not completely dependent upon apoptosis. P-gp expression was detectable on five NB cell lines. WP744 was more cytotoxic than Dox against both P-gp+ and P-gp- cells. CONCLUSIONS: These findings demonstrate that 4'-O-benzylation of the anthracycline molecule significantly enhances potency against NB independent of MYCN status, caspase activation, and MDR phenotype. However, WP744 demonstrated a unique synergy with MYCN for cell killing when this oncogene was specifically induced. WP744 may be more useful than conventional agents for the treatment of tumor clones that harbor defects in apoptotic pathways, in those with MYCN amplification, and in those with drug-resistant tumors.