Multiplexed measurements of gene signatures in different analytes using the Nanostring nCounter™ Assay System

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Sputum RNA signature in allergic asthmatics following allergen bronchoprovocation test

Background: Inhaled allergen challenge is a validated disease model of allergic asthma offering useful pharmacodynamic assessment of pharmacotherapeutic effects in a limited number of subjects. Objectives: To evaluate whether an RNA signature can be identified from induced sputum following an inhaled allergen challenge, whether a RNA signature could be modulated by limited doses of inhaled fluticasone, and whether these gene expression profiles would correlate with the clinical endpoints measured in this study. Methods: Thirteen non-smoking, allergic subjects with mild-to-moderate asthma participated in a randomised, placebo-controlled, 2-period cross-over study following a single-blind placebo run-in period. Each period consisted of three consecutive days, separated by a wash-out period of at least 3 weeks. Subjects randomly received inhaled fluticasone ((FP) MDI; 500 mcg BID×5 doses in total) or placebo. On day 2, house dust mite extract was inhaled and airway response was measured by FEV1 at predefined time points until 7 h post-allergen. Sputum was induced by NaCl 4.5%, processed and analysed at 24 h pre-allergen and 7 and 24 h post-allergen. RNA was isolated from eligible sputum cell pellets (<80% squamous of 500 cells), amplified according to NuGEN technology, and profiled on Affymetrix arrays. Gene expression changes from baseline and fluticasone treatment effects were evaluated using a mixed effects ANCOVA model at 7 and at 24 h post-allergen challenge. Results: Inhaled allergen-induced statistically significant gene expression changes in sputum, which were effectively blunted by fluticasone (adjusted p<0.025). Forty-seven RNA signatures were selected from these responses for correlation analyses and further validation. This included Th2 mRNA levels for cytokines, chemokines, high-affinity IgE receptor FCER1A, histamine receptor HRH4, and enzymes and receptors in the arachidonic pathway. Individual messengers from the 47 RNA signatures correlated significantly with lung function and sputum eosinophil counts. Conclusion: Our RNA extraction and profiling protocols allowed reproducible assessments of inflammatory signatures in sputum including quantification of drug effects on this response in allergic asthmatics. This approach offers novel possibilities for the development of pharmacodynamic (PD) biomarkers in asthma

Properties of Calcium Phosphate/Hydrogel Bone Grafting Composite on the Model of Diaphyseal Rat Femur’s Defect: Experimental Study

Background. The problem of bone defects replacement is relevant nowadays, that is why many scientists create new synthetic bone substitutes, but the ideal material has not been found so far. The aims of the study: 1) to determine the suitability of the monocortical defect model in the rat femur diaphysis with additional prophylactic reinforcement with a bone plate for assessing the biological properties of implanted materials using the commercially available ChronOS material as an example; 2) to assess of the osteoconductive properties of composite materials based on poly(ethylene glycol)diacrylate and octacalcium phosphate with architecture Kelvin and gyroid types on the developed model. Methods. A prospective study, level of evidence II. A monocortical defect of the rat femoral diaphysis (length 7 mm) was produced under anaesthesia in aseptic conditions and fixed with a polyetheretherketone plate and six titanium screws. In the control group, the defect was left empty. In other groups, blocks of one of three materials were implanted сhronOS and composites of poly(ethylene glycol)diacrylate and octacalcium phosphate with 3D-printed Kelvin and gyroid architectures. After 3 and 6 weeks, the rats were sacrificed, and histological examination of the defect zone was performed. The amount of newly formed bone tissue was histometricly assessed, followed by statistical processing of the results. Results. All rats have reached the planned endpoint, and there were no infectious complications or loss of fixation. Histological examination of the defect zone revealed minimal bone growth in the Control group, rather slow bone formation in the Gyroid group, and statistically significantly more pronounced bone formation in the pores of the materials in the Kelvin and Chronos groups. Conclusions. Bone defect in this model was not spontaneously filled with bone tissue and allowed us to study the biological properties of bone substitutes (the ability to biodegrade and osteoconductive properties). The osteoconductive properties of a composite material based on poly(ethylene glycol)diacrylate and octacalcium phosphate with a Kelvin architecture are higher than with a gyroid architecture and are comparable to that of the сhronOS

Lung eQTLs to Help Reveal the Molecular Underpinnings of Asthma

Genome-wide association studies (GWAS) have identified loci reproducibly associated with pulmonary diseases; however, the molecular mechanism underlying these associations are largely unknown. The objectives of this study were to discover genetic variants affecting gene expression in human lung tissue, to refine susceptibility loci for asthma identified in GWAS studies, and to use the genetics of gene expression and network analyses to find key molecular drivers of asthma. We performed a genome-wide search for expression quantitative trait loci (eQTL) in 1,111 human lung samples. The lung eQTL dataset was then used to inform asthma genetic studies reported in the literature. The top ranked lung eQTLs were integrated with the GWAS on asthma reported by the GABRIEL consortium to generate a Bayesian gene expression network for discovery of novel molecular pathways underpinning asthma. We detected 17,178 cis- and 593 trans- lung eQTLs, which can be used to explore the functional consequences of loci associated with lung diseases and traits. Some strong eQTLs are also asthma susceptibility loci. For example, rs3859192 on chr17q21 is robustly associated with the mRNA levels of GSDMA (P = 3.55 × 10(-151)). The genetic-gene expression network identified the SOCS3 pathway as one of the key drivers of asthma. The eQTLs and gene networks identified in this study are powerful tools for elucidating the causal mechanisms underlying pulmonary disease. This data resource offers much-needed support to pinpoint the causal genes and characterize the molecular function of gene variants associated with lung diseases

Filgotinib modulates inflammation-associated peripheral blood protein biomarkers in adults with active rheumatoid arthritis and prior inadequate response to methotrexate

Introduction: Our aim was to evaluate protein biomarker changes related to the administration of filgotinib, a Janus kinase (JAK) 1 preferential inhibitor, in patients with moderately to severely active rheumatoid arthritis (RA) with inadequate response to methotrexate. Methods: Plasma and serum samples were collected from patients enrolled in FINCH 1 (NCT02889796), a Phase 3 trial. Patients with stable backgrounds of methotrexate were randomly assigned once-daily oral filgotinib 200 or 100 mg, subcutaneous adalimumab 40 mg every 2 weeks (W), or placebo. Up to 35 biomarkers were analyzed at baseline, W4, and W12 with enzyme-linked immunosorbent assays and chemiluminescence and electrochemiluminescence assays. Results: At baseline, 4 distinct biomarker clusters were identified. The strongest intragroup correlations were in bone-cartilage resorption/inflammation and JAK/signal transducer and activator of transcription (STAT) signaling activity. At baseline, significant positive correlations were identified for cytokines with patient-reported pain and with patient measures of fatigue. Filgotinib reduced levels of cytokines associated with inflammation and cell migration as early as W4 and through W12. Compared to adalimumab, filgotinib induced significant reductions in bone-related turnover biomarkers, N-telopeptide of type 1 collagen and C-telopeptide 1, as well as biomarkers associated with baseline disease activity. No baseline predictors of therapeutic response to filgotinib were identified. Conclusion: Filgotinib reduced peripheral protein biomarkers associated with JAK/STAT signaling, inflammatory signaling, immune cell migration, and bone resorption as soon as W4 in FINCH 1. Effects were dose dependent and consistent with the clinical efficacy of filgotinib observed in FINCH 1. The changes in peripheral biomarkers associated with filgotinib treatment in methotrexate-experienced patients are consistent with changes observed in both methotrexate-naïve and biologic disease-modifying antirheumatic drug–experienced RA populations. These data demonstrate dose-dependent effects of preferential JAK1 inhibition by filgotinib on peripheral blood protein biomarkers in methotrexate-experienced patients with RA

Six Genes Are Preferentially Transcribed by the Circulating and Sequestered Forms of Plasmodium falciparum Parasites That Infect Pregnant Women▿ †

In areas of stable malaria transmission, susceptibility to Plasmodium falciparum malaria increases during first pregnancy. Women become resistant to pregnancy malaria over successive pregnancies as they acquire antibodies against the parasite forms that sequester in the placenta, suggesting that a vaccine is feasible. Placental parasites are antigenically distinct and bind receptors, like chondroitin sulfate A (CSA), that are not commonly bound by other parasites. We used whole-genome-expression analysis to find transcripts that distinguish parasites of pregnant women from other parasites and employed a novel approach to define and adjust for cell cycle timing of parasites. Transcription of six genes was substantially higher in both placental parasites and peripheral parasites from pregnant women, and each gene encodes a protein with a putative export sequence and/or transmembrane domain. This cohort of genes includes var2csa, a member of the variant PfEMP1 gene family previously implicated in pregnancy malaria, as well as five conserved genes of unknown functions. Women in East Africa acquire antibodies over successive pregnancies against a protein encoded by one of these genes, PFD1140w, and this protein shows seroreactivity similar to that of VAR2CSA domains. These findings suggest that a suite of genes may be important for the genesis of the placental binding phenotype of P. falciparum and may provide novel targets for therapeutic intervention

Cilofexor in Patients with Compensated Cirrhosis Due to Primary Sclerosing Cholangitis: an Open-label Phase 1B Study

Objectives: This proof-of-concept, open-label phase 1b study evaluated the safety and efficacy of cilofexor, a potent selective farnesoid X receptor agonist, in patients with compensated cirrhosis due to primary sclerosing cholangitis (PSC). Methods: Escalating doses of cilofexor (30 mg [weeks 1–4], 60 mg [weeks 5–8], 100 mg [weeks 9–12]) were administered orally once daily over 12 weeks. The primary endpoint was safety. Exploratory measures included cholestasis and fibrosis markers, and pharmacodynamic biomarkers of bile acid homeostasis. Results: Eleven patients were enrolled (median age: 48 years; 55% men). The most common treatment-emergent adverse events (TEAEs) were pruritus (8/11 [72.7%]), fatigue, headache, nausea, and upper respiratory tract infection (2/11 [18.2%] each). Seven patients experienced a pruritus TEAE (one grade 3) considered drug related. One patient temporarily discontinued cilofexor owing to peripheral edema. There were no deaths, serious TEAEs, or TEAEs leading to permanent discontinuation. Median changes (interquartile ranges) from baseline to week 12 (predose, fasting) were −24.8% (−35.7, −7.4) for alanine transaminase, −13.0% (−21.9, −8.6) for alkaline phosphatase, −43.5% (−52.1, −30.8) for gamma-glutamyl transferase, −12.7% (−25.0, 0.0) for total bilirubin, and −21.2% (−40.0, 0.0) for direct bilirubin. Least-squares mean percentage change (95% confidence interval) from baseline to week 12 at trough was −55.3% (−70.8, −31.6) for C4 and −60.5% (−81.8, −14.2) for cholic acid. Fasting fibroblast growth factor 19 levels transiently increased after cilofexor administration. Conclusions: Escalating doses of cilofexor over 12 weeks were well tolerated and improved cholestasis markers in patients with compensated cirrhosis due to PSC (NCT04060147)