Measurement of Neutrophil Extracellular Traps as a Biomarker for the Differential Diagnosis Between Antineutrophil Cytoplasmic Antibody-Positive Individuals With Antineutrophil Cytoplasmic Antibody-Associated Vasculitis and Nonautoimmune Diseases.

BACKGROUND/OBJECTIVE: Neutrophil extracellular traps (NETs) have a correlation with disease activity in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). However, it is not known whether there is an association between NETs and the presence of ANCA in other diseases. This study aimed to assess the occurrence of NETs in individuals with ANCA and whether serum NET quantitation is capable of distinguishing them with regard to the diagnosis. METHODS: This was a cross-sectional, observational study. From the positive ANCA by indirect immunofluorescence, 94 individuals were divided into groups: AAV, infectious diseases, and neoplastic diseases. Healthy controls served for comparisons. Neutrophil extracellular traps were evaluated through the investigation of NET remnants, by detecting cell-free DNA bound to proteins such as histone, myeloperoxidase, and neutrophil elastase (NE). RESULTS: In patients with perinuclear ANCA (p-ANCA) the detection of NETs by NE was able to distinguish AAV from infection/neoplasia and healthy controls. Receiver operating characteristic curves for serum NETs by NE in patients with p-ANCA were drawn in 2 situations: AAV versus infection/neoplasia, showing a sensitivity of 0.65 and specificity of 0.84, with an area under the curve of 65%; and AAV versus controls, showing a sensitivity of 0.84 and a specificity of 0.88, with an area under the curve of 96%. CONCLUSIONS: For p-ANCA-positive individuals, we found higher serum NETs detected by NE-DNA in those with chronic infectious and neoplastic diseases than in AAV individuals and healthy controls. This allows us to infer that the evaluation of serum NETs may be of value as a biomarker for differential diagnosis.

Neutrophil extracellular traps mediate bone erosion in rheumatoid arthritis by enhancing RANKL-induced osteoclastogenesis.

BACKGROUND AND PURPOSE: Rheumatoid arthritis (RA) is a chronic autoimmune disease that can cause bone erosion due to increased osteoclastogenesis. Neutrophils involvement in osteoclastogenesis remains uncertain. Given that neutrophil extracellular traps (NETs) can act as inflammatory mediators in rheumatoid arthritis, we investigated the role of NETs in stimulating bone loss by potentiating osteoclastogenesis during arthritis. EXPERIMENTAL APPROACH: The level of NETs in synovial fluid from arthritis patients was assessed. Bone loss was evaluated by histology and micro-CT in antigen-induced arthritis (AIA)-induced WT mice treated with DNase or in Padi4-deficient mice (Padi4flox/flox LysMCRE ). The size and function of osteoclasts and the levels of RANKL and osteoprotegerin (OPG) released by osteoblasts that were incubated with NETs were measured. The expression of osteoclastogenic marker genes and protein levels were evaluated by qPCR and western blotting. To assess the participation of TLR4 and TLR9 in osteoclastogenesis, cells from Tlr4-/- and Tlr9-/- mice were cultured with NETs. KEY RESULTS: Rheumatoid arthritis patients had higher levels of NETs in synovial fluid than osteoarthritis patients, which correlated with increased levels of RANKL/OPG. Moreover, patients with bone erosion had higher levels of NETs. Inhibiting NETs with DNase or Padi4 deletion alleviated bone loss in arthritic mice. Consistently, NETs enhanced RANKL-induced osteoclastogenesis that was dependent on TLR4 and TLR9 and increased osteoclast resorptive functions in vitro. In addition, NETs stimulated the release of RANKL and inhibited osteoprotegerin in osteoblasts, favouring osteoclastogenesis. CONCLUSIONS AND IMPLICATIONS: Inhibiting NETs could be an alternative strategy to reduce bone erosion in arthritis patients.

Neutrophil extracellular traps in rheumatoid arthritis and periodontitis: Contribution of PADI4 gene polymorphisms.

AIM: We sought to investigate the release of neutrophil extracellular traps (NETs) in neutrophils from individuals with rheumatoid arthritis (RA) and controls and compare the presence of NETs in gingival tissues according to periodontal status. Also, the association between single nucleotide polymorphisms (SNPs) of the peptidyl arginine deaminase type 4 (PADI4) gene and the GTG haplotype with RA, periodontitis and NETs was evaluated in vitro. MATERIALS AND METHODS: Peripheral neutrophils were isolated by density gradient, and NET concentration was determined by the PicoGreen method. Immunofluorescence was studied to identify NETs by co-localization of myeloperoxidase (MPO)-citrullinated histone H3 (H3Cit). Genotyping for SNPs (PADI4_89; PADI4_90; PADI4_92; and PADI4_104) was performed in 87 individuals with RA and 111 controls. RESULTS: The release of NETs in vitro was significantly higher in individuals with RA and periodontitis and when stimulated with Porphyromonas gingivalis. Gingival tissues from subjects with RA and periodontitis revealed increased numbers of MPO-H3Cit-positive cells. Individuals with the GTG haplotype showed a higher release of NETs in vitro and worse periodontal parameters. CONCLUSIONS: The release of NETs by circulating neutrophils is associated with RA and periodontitis and is influenced by the presence of the GTG haplotype.

Neutrophil virucidal activity against SARS-CoV-2 is mediated by NETs.

BACKGROUND: The inflammation in the lungs and other vital organs in COVID-19 are characterized by the presence of neutrophils and high concentration of neutrophil extracellular traps (NETs), which also seems to mediate host tissue damage. However, it is not known whether NETs could have virucidal activity against SARS-CoV-2. METHODS: We investigated whether NETs could prevent SARS-CoV-2 replication in neutrophils and epithelial cells, and what the consequence of NETs degradation in K18-humanized ACE2 transgenic mice infected with SARS-CoV-2. RESULTS: Here, by immunofluorescence microscopy we observed that viral particles co-localize with NETs in neutrophils isolated from COVID-19 patients or from healthy individuals and infected in vitro. The inhibition of NETs production increased virus replication in neutrophils. In parallel, we observed that NETs inhibited virus abilities to infect and replicate in epithelial cells after 24 h of infection. Degradation of NETs with DNase I prevented their virucidal effect in vitro. Using K18-humanized ACE2 transgenic mice we observed a higher viral load in animals treated with DNase I. On the other hand, the virucidal effect of NETs was not dependent on neutrophil elastase or myeloperoxidase activity. CONCLUSION: Our results provide evidence of the role of NETosis as a mechanism of SARS-CoV-2 viral capture and inhibition.

Comparative study of the synovial levels of RANKL and OPG in rheumatoid arthritis, spondyloarthritis and osteoarthritis.

INTRODUCTION: In chronic arthropathies, there are several mechanisms of joint destruction. In recent years, studies have reported the implication of receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG) in the process of activation and differentiation of osteoclasts, a key cell in the development of bone erosion. The RANKL/OPG ratio is increased in the serum of patients with malignant diseases and lytic bone disease, as well as rheumatoid arthritis (RA). The objective of this study was to measure and compare the concentrations of OPG and RANKL in the synovial fluid (SF) of patients with rheumatoid arthritis, spondyloarthritis (SpA) and osteoarthritis (OA). METHODS: This was an observational and cross-sectional study with 83 patients, 33 with RA, 32 with SpA and 18 with OA, followed up regularly in the outpatient clinics of the Rheumatology Department of the Clinics Hospital of the Ribeirão Preto Medical School-USP. All patients were assessed for indications for arthrocentesis by the attending physicians at the time of SF collection and were evaluated for demographic variables and medication use. Disease activity was assessed in individuals with RA and SpA. The quantification of SF OPG and RANKL levels was performed by ELISA, and the correlations of the results with clinical, laboratory and radiological parameters were assessed. RESULTS: We found no statistically significant difference in the RANKL and OPG levels among the groups. Patients with RA showed a positive correlation between the SF cell count and RANKL level (r = 0.59; p < 0.05) and the RANKL/OPG ratio (r = 0.55; p < 0.05). Patients with OA showed a strong correlation between C-reactive protein (CRP) and the RANKL/OPG ratio (r = 0.82; p < 0.05). There was no correlation between the OPG and RANKL levels and markers of inflammatory activity or the disease activity index in patients with RA or SpA. CONCLUSION: Within this patient cohort, the RANKL/OPG ratio was correlated with the SF cell count in patients with RA and with serum CRP in patients with OA, which may suggest a relationship with active inflammation and more destructive joint disease.

Methotrexate and Non-Surgical Periodontal Treatment Change the Oral-Gut Microbiota in Rheumatoid Arthritis: A Prospective Cohort Study.

This study evaluated the changes in the composition of oral-gut microbiota in patients with rheumatoid arthritis (RA) caused by methotrexate (MTX) and non-surgical periodontal treatment (NSPT). Assessments were performed at baseline (T0), 6 months after MTX treatment (T1), and 45 days after NSPT (T2). The composition of the oral and gut microbiota was assessed by amplifying the V4 region of the 16S gene from subgingival plaques and stools. The results of the analysis of continuous variables were presented descriptively and non-parametric tests and Spearman's correlation were adopted. A total of 37 patients (27 with periodontitis) were evaluated at T0; 32 patients (24 with periodontitis) at T1; and 28 patients (17 with periodontitis) at T2. MTX tended to reduce the alpha diversity of the oral-gut microbiota, while NSPT appeared to increase the number of different species of oral microbiota. MTX and NSPT influenced beta diversity in the oral microbiota. The relative abundance of oral microbiota was directly influenced by periodontal status. MTX did not affect the periodontal condition but modified the correlations that varied from weak to moderate (p < 0.05) between clinical parameters and the microbiota. MTX and NSPT directly affected the composition and richness of the oral-gut microbiota. However, MTX did not influence periodontal parameters.

Comparative study of the synovial levels of RANKL and OPG in rheumatoid arthritis, spondyloarthritis and osteoarthritis

Abstract Introduction In chronic arthropathies, there are several mechanisms of joint destruction. In recent years, studies have reported the implication of receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG) in the process of activation and differentiation of osteoclasts, a key cell in the development of bone erosion. The RANKL/OPG ratio is increased in the serum of patients with malignant diseases and lytic bone disease, as well as rheumatoid arthritis (RA). The objective of this study was to measure and compare the concentrations of OPG and RANKL in the synovial fluid (SF) of patients with rheumatoid arthritis, spondyloarthritis (SpA) and osteoarthritis (OA). Methods This was an observational and cross-sectional study with 83 patients, 33 with RA, 32 with SpA and 18 with OA, followed up regularly in the outpatient clinics of the Rheumatology Department of the Clinics Hospital of the Ribeirão Preto Medical School-USP. All patients were assessed for indications for arthrocentesis by the attending physicians at the time of SF collection and were evaluated for demographic variables and medication use. Disease activity was assessed in individuals with RA and SpA. The quantification of SF OPG and RANKL levels was performed by ELISA, and the correlations of the results with clinical, laboratory and radiological parameters were assessed. Results We found no statistically significant difference in the RANKL and OPG levels among the groups. Patients with RA showed a positive correlation between the SF cell count and RANKL level (r = 0.59; p < 0.05) and the RANKL/ OPG ratio (r = 0.55; p < 0.05). Patients with OA showed a strong correlation between C-reactive protein (CRP) and the RANKL/OPG ratio (r = 0.82; p < 0.05). There was no correlation between the OPG and RANKL levels and markers of inflammatory activity or the disease activity index in patients with RA or SpA. Conclusion Within this patient cohort, the RANKL/OPG ratio was correlated with the SF cell count in patients with RA and with serum CRP in patients with OA, which may suggest a relationship with active inflammation and more destructive joint disease.

Enhancing untargeted metabolomics using metadata-based source annotation.

Human untargeted metabolomics studies annotate only ~10% of molecular features. We introduce reference-data-driven analysis to match metabolomics tandem mass spectrometry (MS/MS) data against metadata-annotated source data as a pseudo-MS/MS reference library. Applying this approach to food source data, we show that it increases MS/MS spectral usage 5.1-fold over conventional structural MS/MS library matches and allows empirical assessment of dietary patterns from untargeted data.

Diagnostic Agreement Among General Practitioners, Residents, and Senior Rheumatologists for Rheumatic Diseases.

OBJECTIVE: The aim of this study was to evaluate the concordance of the diagnoses made by senior rheumatologists and those made by residents in rheumatology and by general practitioners (GPs). METHODS: In this cohort, 497 patients referred by GPs from August 1, 2018 to December 16, 2019 were evaluated first by a second-year resident in rheumatology. After clinical rounds, the diagnoses by senior rheumatologists were assumed as the criterion standard and defined the prevalence of the rheumatic diseases, divided into 5 groups: rheumatoid arthritis, spondyloarthritis, other connective tissue diseases and vasculitis, nonautoimmune rheumatic diseases, and nonrheumatic diseases. The follow-up ended on November 30, 2020. We calculated sensibility, specificity, positive predictive value, negative predictive value, and κ coefficient of the diagnosis by GPs and residents. RESULTS: The diagnoses were changed for 58% of the referral letters. Diseases of low complexity, such as fibromyalgia and osteoarthritis, accounted for 50% of the diagnoses. Compared with senior rheumatologists, residents in rheumatology had κ > 0.6 for all the groups, whereas GPs had κ < 0.5, with the worst performance for nonautoimmune rheumatic disease (κ = -0.18) and nonrheumatic disease (κ = 0.15). In terms of level of complexity, 46% of the letters were inappropriate. CONCLUSIONS: We found a poor level of diagnostic agreement between GPs and the rheumatology team. General practitioners had difficulties diagnosing and treating rheumatic diseases, referring patients that should be treated in the primary level of health care. One year of training in rheumatology made residents' skills comparable to those of senior rheumatologists.

Are neutrophil extracellular traps the link for the cross-talk between periodontitis and rheumatoid arthritis physiopathology?

OBJECTIVES: Neutrophil extracellular traps (NETs) play a role in the pathogenesis of periodontitis and rheumatoid arthritis (RA). However, it remains poorly understood whether NETs participate in the cross-talk between periodontitis and RA. Herein, we investigated the production of NETs in individuals with periodontitis and RA and its association with clinical parameters. The impact of periodontal therapy on RA and NET release was also assessed. METHODS: The concentration of NETs and cytokines was determined in the saliva and plasma of individuals with early RA (n = 24), established RA (n = 64) and individuals without RA (n = 76). The influence of periodontitis on the production of NETs and cytokines was also evaluated. RESULTS: Individuals with early RA had a higher concentration of NETs in saliva and plasma than individuals with established RA or without RA. Periodontitis resulted in an increase in the concentration of NETs of groups of individuals without RA and with early RA. The proportion of individuals with high concentrations of IL-6, IL-10 and GM-CSF was higher among individuals with periodontitis than among individuals without periodontitis. The concentrations of TNF-α, IL-6, IL-17/IL-25 and IL-28A were particularly high in individuals with early RA. Worse periodontal clinical parameters, RA onset and RA activity were significantly associated with circulating NETs. Periodontal therapy was associated with a reduction in the concentration of NETs and inflammatory cytokines and amelioration in periodontitis and RA. CONCLUSION: This study reveals that NETs are a possible link between periodontitis and RA, with periodontal therapy resulting in a dramatic switch in circulating NET levels.

Is there an Inflammation Role for MYD88 in Rheumatoid Arthritis?

Rheumatoid arthritis (RA) is an autoimmune and inflammatory disease with strong genetic influence, especially upon immune response components. Several cytokines from the toll-like receptors activation pathway display recognized role for RA establishment. However, few studies have verified the role of key mediators such as MYD88 gene and its genetic variants. In the present study, we aim to evaluate the rs6853 functional single-nucleotide variation (SNV) role in RA etiopathogenesis, clinical severity status, and its impact in MYD88 mRNA levels and IL-lß protein levels. For the association study, a total of 423 RA patients and 346 health individuals, enrolled as control, from Northeast and Southeast Brazil were genotyped using specific Taqman probe. For the gene expression assays, we performed a MYD88 rs6853 genotype-guided monocyte cell culture divided into non-stimulated and lypopolysaccharides (LPS)-stimulated cells from healthy individuals. MYD88 gene expression was measured using primer specifics while IL-1ß levels were evaluated by ELISA. We observed that A allele and AA genotype were associated to an increased risk to RA development (OR = 1.60; 95% CI 1.24-2.08; p = 0.0004/OR = 2.83; 95% CI 1.25-6.41; p = 0.0152). The AA genotype exhibited lower MYD88 mRNA levels than GG genotype in non-stimulated monocyte cell culture (FC - 3.83; p = 0.003). Additionally, we verified an increase of IL-1ß levels when AA genotype non-stimulated monocytes were compared to AA genotype LPS-stimulates (p = 0.021). In summary, MYD88 rs6853 polymorphism associated to RA development in our Brazilian cohort and showed influence upon MYD88 mRNA levels' expression and IL-lß production.

Neutrophil extracellular traps mediate joint hyperalgesia induced by immune inflammation.

OBJECTIVE: To evaluate the role of neutrophil extracellular traps (NETs) in the genesis of joint hyperalgesia using an experimental model of arthritis and transpose the findings to clinical investigation. METHODS: C57BL/6 mice were subjected to antigen-induced arthritis (AIA) and treated with Pulmozyme (PLZ) to degrade NETs or Cl-amidine to inhibit NET production. Oedema formation, the histopathological score and mechanical hyperalgesia were evaluated. NETs were injected intra-articularly in wild type (WT), Tlr4-/-, Tlr9-/-, Tnfr1-/- and Il1r-/- mice, and the levels of cytokines and Cox2 expression were quantified. NETs were also quantified from human neutrophils isolated from RA patients and individual controls. RESULTS: AIA mice had increased NET concentration in joints, accompanied by increased Padi4 gene expression in the joint cells. Treatment of AIA mice with a peptidyl arginine deiminase 4 inhibitor or with PLZ inhibited the joint hyperalgesia. Moreover, the injection of NETs into joints of naïve animals generated a dose-dependent reduction of mechanical threshold, an increase of articular oedema, inflammatory cytokine production and cyclooxygenase-2 expression. In mice deficient for Tnfr1, Il1r, Tlr4 and Tlr9, joint hyperalgesia induced by NETs was prevented. Last, we found that neutrophils from RA patients were more likely to release NETs, and the increase in synovial fluid NET concentration correlated with an increase in joint pain. CONCLUSION: The findings indicate that NETs cause hyperalgesia possibly through Toll-like receptor (TLR)-4 and TLR-9. These data support the idea that NETs contribute to articular pain, and this pathway can be an alternative target for the treatment of pain in RA.

Systemic Lupus Erythematosus Activity Affects the Sinusoidal Uptake Transporter OATP1B1 Evaluated by the Pharmacokinetics of Atorvastatin.

The present study assessed the effect of systemic lupus erythematosus (SLE) activity, a chronic and inflammatory autoimmune disease, on the sinusoidal uptake transporter OATP1B1 using atorvastatin (ATV) as a probe drug. Fifteen healthy subjects, 13 patients with controlled SLE (SLEDAI 0-4), and 12 patients with uncontrolled SLE (SLEDAI from 6 to 15), all women, were investigated. Apparent total clearance of midazolam (MDZ), a marker of CYP3A4 activity, did not vary among the three investigated groups. The controlled and uncontrolled SLE groups showed higher plasma concentrations of MCP-1 and TNF-α, while the uncontrolled SLE group also showed higher plasma concentrations of IL-10. The uncontrolled SLE group showed higher area under the curve (AUC) for ATV (60.47 (43.76-83.56) vs. 30.56 (22.69-41.15) ng⋅hour/mL) and its inactive metabolite ATV-lactone (98.74 (74.31-131.20) vs. 49.21 (34.89-69.42) ng⋅hour/mL), and lower apparent total clearance (330.7 (239.30-457.00) vs. 654.5 (486.00-881.4) L/hour) and apparent volume of distribution (2,609 (1,607-4,234) vs. 7,159 (4,904-10,450) L), when compared to the healthy subjects group (geometric mean and 95% confidence interval). The pharmacokinetics of ATV and its metabolites did not differ between the healthy subject group and the patients with controlled SLE group. In conclusion, uncontrolled SLE increased the systemic exposure to both ATV and ATV-lactone, inferring inhibition of OATP1B1 activity, once in vivo CYP3A4 activity assessed by oral clearance of MDZ was unaltered. The inflammatory state, not the disease itself, was responsible for the changes described in the uncontrolled SLE group as a consequence of inhibition of OATP1B1, because systemic exposure to ATV and its metabolites were not altered in patients with controlled SLE.

Plasma mevalonic acid exposure as a pharmacodynamic biomarker of fluvastatin/atorvastatin in healthy volunteers.

Fluvastatin and atorvastatin are inhibitors of hydroxy-methylglutaryl-CoA (HMG-CoA) reductase, the enzyme that converts HMG-CoA to mevalonic acid (MVA). The present study reports for the first time the analysis of mevalonolactone (MVL) in plasma samples by UPLC-MS/MS as well as the use of MVA, analyzed as MVL, as a pharmacodynamics parameter of fluvastatin in multiple oral doses (20, 40 or 80 mg/day for 7 days) and atorvastatin in a single oral dose (20, 40 or 80 mg) in healthy female volunteers. this study presents the use of MVL exposure as a pharmacodynamics biomarker of fluvastatin in multiple oral doses (20, 40 or 80 mg/day for 7 days) or atorvastatin in a single oral dose (20, 40 or 80 mg) in healthy volunteers (n = 30). The administration of multiple doses of fluvastatin (n = 15) does not alter the values (geometric mean and 95 % CI) of AUC0-24 h of MVL [72.00 (57.49-90.18) vs 65.57 (51.73-83.12) ng∙h/mL], but reduces AUC0-6 h [15.33 (11.85-19.83) vs 8.15 (6.18-10.75) ng∙h/mL] by approximately 47 %, whereas single oral dose administration of atorvastatin (n = 15) reduces both AUC0-24 h [75.79 (65.10-88.24) vs 32.88 (27.05-39.96) ng∙h/mL] and AUC0-6 h [17.07 (13.87-21.01) vs 7.01 (5.99-8.22) ng∙h/mL] values by approximately 57 % and 59 %, respectively. In conclusion, the data show that the plasma exposure of MVL represents a reliable pharmacodynamic parameter for PK-PD (pharmacokinetic-pharmacodynamic) studies of fluvastatin in multiple doses and atorvastatin in a single dose.

Rheumatoid arthritis downregulates the drug transporter OATP1B1: Fluvastatin as a probe.

AIMS: Rheumatoid arthritis (RA) is a long term autoimmune inflammatory disease characterized by high autoantibody production and cytokine release, especially IL-6 and TNF-α. Some clinical studies have shown the effect of RA on CYP metabolism. However, the effect of RA on the drug transporter OATP1B1 remains a gap. METHODS: Patients with RA under pharmacological treatment (n = 10) and healthy volunteers (n = 15) treated for seven consecutive days with racemic fluvastatin (20, 40, or 80 mg/24 h) were investigated. Serial blood samples were collected during the last dose interval. All participants were assessed for cytokine profile and CYP2C9 genotype. RESULTS: Patients with RA showed increased plasma concentrations of IFN-γ and TNF-α up to two and four times, respectively, when compared to healthy volunteers, whereas CYP2C9 activity based on genotype was considered normal or slightly reduced for both investigated groups. When compared to healthy volunteers, patients with RA presented higher values (median and 25th-75th percentiles) of normalized AUC for 20 mg dose (250 [114-405] vs. 96.7 [78.1-131] ng h mL-1 for (-)-3S,5R-fluvastatin and 163 [96.9-325] vs. 83.1 [61.7-107] ng⋅h⋅mL-1 for (+)-3R,5S-fluvastatin) and lower values of CL/F (40.9 [24.5-89.1] vs. 103 [75.9-128] L⋅h-1 for (-)-3S,5R-fluvastatin and 61.4 [30.6-103] vs. 120 [93.0-162] L⋅h-1 for (+)-3R,5S-fluvastatin) and V/F (73.0 [28.5-117] vs. 143 [108-221] L for (-)-3S,5R-fluvastatin and 93.9 [32.7-116] vs. 153 [122-234] L for (+)-3R,5S-fluvastatin) for both enantiomers. CONCLUSION: The lower values of CL/F and V/F for both fluvastatin enantiomers in RA patients suggest that the inflammatory disease downregulates the sinusoidal drug transporter OATP1B1, the rate-determining step in the hepatic clearance of fluvastatin.

Immunomodulating action of the 3-phenylcoumarin derivative 6,7-dihydroxy-3-[3',4'-methylenedioxyphenyl]-coumarin in neutrophils from patients with rheumatoid arthritis and in rats with acute joint inflammation.

OBJECTIVE: To examine whether free (3-PD-5free) and/or liposomal (3-PD-5lipo) 6,7-dihydroxy-3-[3',4'-methylenedioxyphenyl]-coumarin (3-PD-5) (1) modulate the effector functions of neutrophils from patients with rheumatoid arthritis under remission (i-RA) and with active disease (a-RA), in vitro; and (2) exert anti-inflammatory effect in a rat model of zymosan-induced acute joint inflammation. METHODS AND RESULTS: Incorporation of 3-PD-5 into unilamellar liposomes of soya phosphatidylcholine and cholesterol was efficient (57.5 ± 7.9%) and yielded vesicles with low diameter (133.7 ± 18.4 nm), polydispersity index (0.39 ± 0.06), and zeta potential (- 1.22 ± 0.34 mV). 3-PD-5free (1 µM) and 3-PD-5lipo (3 µM) equally suppressed elastase release and reactive oxygen species generation in neutrophils from healthy subjects and i-RA and a-RA patients, stimulated with immune complexes. 3-PD-5free (20 µM) suppressed the release of neutrophil extracellular traps and chemotaxis in vitro, without clear signs of cytotoxicity. 3-PD-5lipo (1.5 mg/kg, i.p.) diminished joint edema and synovial infiltration of total leukocytes and neutrophils, without changing the synovial levels of TNF-α, IL-1ß, and IL-6. CONCLUSION: Altogether, the results reported herein indicate that 3-PD-5 is a promising modulator of the early stages of acute joint inflammation that can help to diminish not only excessive neutrophil infiltration in the synovia but also neutrophil activation and its outcomes in RA patients.

T-cell specific upregulation of Sema4A as risk factor for autoimmunity in systemic lupus erythematosus and rheumatoid arthritis.

The aim of the present study was to evaluate the impact of SEMA4A genetic variants on expression of sema4A protein and its relation to autoimmunity development in Systemic Lupus Erythematosus and Rheumatoid Arthritis patients. A total of 541 SLE patients, 390 RA patients and 607 healthy individuals were genotyped. We also assessed SEMA4A mRNA expression from whole blood cells and the in vitro protein production from resting and activated T lymphocytes as well as mature dendritic cells from healthy individuals stratified according to their genotypes for SLE/RA associated SEMA4A variants. Our results showed that T/T genotype for rs3738581 SNP is associated with both RA and SLE development (p = .000053, OR = 2.35; p = .0019, OR = 2.07, respectively; statistical power = 100%) and also to an increased in vitro sema4A production in active T lymphocytes. Our findings are indicative of a T cell-specific upregulation of sema4A in the presence of T/T genotype, being a risk factor for SLE and RA.

Simultaneous analysis of the total plasma concentration of atorvastatin and its five metabolites and the unbound plasma concentration of atorvastatin: Application in a clinical pharmacokinetic study of single oral dose.

Atorvastatin (ATV) and its two active metabolites, o-hydroxy atorvastatin acid (o-OH-ATV) and p-hydroxy atorvastatin acid (p-OH-ATV) are responsible for its HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme-A) reductase inhibitory activity, while its corresponding inactive lactone forms (LAC) are related to the manifestation of myopathy. The present study reports the development and validation of a method for the simultaneous analysis of ATV and its five metabolites (o-OH-ATV, p-OH-ATV, ATV-LAC, o-OH-ATV-LAC, p-OH-ATV-LAC) as total plasma concentration and ATV as unbound plasma concentration using UPLC-MS/MS. The method was applied in a pharmacokinetic study following administration of a single oral 20, 40 or 80 mg ATV dose in healthy volunteers (n = 15). ATV and its five metabolites were separated on a C18 column using as mobile phase a mixture of 0.2% formic acid and acetonitrile (55:45, v/v) at a flow of 0.4 mL/min. The method showed linearity from 25 pg/mL to 200 ng/mL plasma as total concentration and from 6.25 pg to 25 ng/mL plasma ultrafiltrate as ATV unbound concentration. The coefficients of variation and the relative standard errors of the accuracy and precision analyses were <15%. The method allowed quantification of plasma concentrations of ATV and its five metabolites up to 36 h after 20, 40 or 80 mg ATV administration. The pharmacokinetic parameters dose normalized to 20 mg are presented as follow (n = 15, mean): unbound fraction 9.38%, maximum plasma concentration 9.52 ng/mL, time to reach maximum plasma concentration 0.98 h, apparent total clearance 742.90 L/h, apparent distribution volume 9005 L, and AUC metabolite/ATV ratios 0.06 for p-OH-ATV, 0.94 for o-OH-ATV, 1.43 for ATV-LAC, 0.25 for p-OH-ATV-LAC and 1.75 for o-OH-ATV-LAC. In conclusion, the methods for simultaneous analysis of ATV and its five metabolites as total plasma concentration and ATV as the unbound plasma concentration showed sensitivity, linearity, precision and accuracy compatible with application in pharmacokinetic studies of single oral dose of 20, 40 or 80 mg ATV.

UPLC-MS/MS method for gemfibrozil determination in plasma with application to a pharmacokinetic study in healthy Brazilian volunteers.

Gemfibrozil (GFZ) is a derivative of fibric acid and is used in the treatment of dyslipidemia. GFZ may affect the metabolism of various drugs, including statins, by inhibiting the sinusoidal influx transporter OATP1B1 and also CYP2C9 and CYP2C8 enzymes. This study presents the development and validation of a rapid, simple, sensitive and reproducible method of GFZ analysis in human plasma using UPLC-MS/MS. The method was applied in a pharmacokinetic study following administration of multiple doses of 600 mg GFZ every 12 h in healthy volunteers (n = 15). GFZ was separated on a C18 column using a mixture of 0.01% formic acid and acetonitrile (40:60, v/v) as the mobile phase at a flow rate of 0.4 mL/min. The method showed linearity in the range from 0.01 µg/mL to 100 µg/mL plasma. The coefficients of variation and the relative standard errors of the accuracy and precision analyses were <15%. The method allowed quantification of plasma concentrations of GFZ in the dose interval of the sixth day of administration of multiple oral doses of GFZ every 12 h. The pharmacokinetic parameters are presented as mean (95% CI): area under the plasma concentration versus time curve 88.84 (72.72-104.96) µg·h/mL, steady state mean plasma concentration 7.40 (6.06-8.75) µg/mL, minimum plasma concentration 1.24 (0.87-1.61) µg/mL, maximum plasma concentration 26.73 (21.31-32.15) µg/mL, time to reach maximum plasma concentration 2.28 (1.42-3.13) h, elimination half-life 2.81 (2.22-3.40) h, apparent total clearance 7.72 (5.85-9.58) L/h, apparent distribution volume 33.97 (18.41-49.53) L. In conclusion, the method for analysis of GFZ in human plasma showed sensitivity, linearity, precision and accuracy compatible with application in pharmacokinetic studies of multiple oral dose of 600 mg GFZ every 12 h.