External quality assurance program for diagnostic complement laboratories: evaluation of the results of the past seven years.

Introduction: The complement external quality assurance (EQA) program was first organized in 2010 by a group of researchers working in diagnostic complement laboratories. Starting in 2016, INSTAND e.V., a German, non-profit interdisciplinary scientific medical society dedicated to providing expert EQA programs for medical laboratories, started organizing the EQAs for complement diagnostic laboratories together with the same group of experienced scientists and doctors who also work as EQA experts. The aim of the current work is to provide descriptive analysis of the past seven years' complement EQA results and evaluate timeline changes in proficiency testing. Methods: Each year, in March and October, blinded samples (normal, pathological) were sent to the participating diagnostic laboratories, where complement parameters were evaluated exactly as in daily routine samples. Since no reference method/target values exist for these parameters, and participants used different units for measurement, the reported results were compared to the stable mean (Algorithm A) of the participants using the same method/measurement units. A reported result was qualified as "passed" if it fell into the 30-50% evaluation/target range around the mean of reported results (depending on the given parameter). Results: While the number of participating laboratories has increased in the past years (from around 120 to 347), the number of complement laboratories providing multiple determinations remained mostly unchanged (around 30 worldwide). C3, C4, C1-inhibitor antigen and activity determinations provided the best proficiency results, with >90% passing quotas in the past years, independent of the applied method. Determination of the functional activity of the three activation pathways was good in general, but results showed large variance, especially with the pathological samples. Complement factor C1q and regulators FH and FI are determined by only a few laboratories, with variable outcomes (in general in the 85-90% pass range). Activation products sC5b-9 and Bb were determined in 30 and 10 laboratories, respectively, with typical passing quotas in the 70-90% range, without a clear tendency over the past years. Conclusion: With these accumulated data from the past seven years, it is now possible to assess sample-, method-, and evaluation related aspects to further improve proficiency testing and protocolize diagnostic complement determinations.

The Effect of the Particle Size Reduction on the Biorelevant Solubility and Dissolution of Poorly Soluble Drugs with Different Acid-Base Character.

Particle size reduction is a commonly used process to improve the solubility and the dissolution of drug formulations. The solubility of a drug in the gastrointestinal tract is a crucial parameter, because it can greatly influence the bioavailability. This work provides a comprehensive investigation of the effect of the particle size, pH, biorelevant media and polymers (PVA and PVPK-25) on the solubility and dissolution of drug formulations using three model compounds with different acid-base characteristics (papaverine hydrochloride, furosemide and niflumic acid). It was demonstrated that micronization does not change the equilibrium solubility of a drug, but it results in a faster dissolution. In contrast, nanonization can improve the equilibrium solubility of a drug, but the selection of the appropriate excipient used for nanonization is essential, because out of the two used polymers, only the PVPK-25 had an increasing effect on the solubility. This phenomenon can be explained by the molecular structure of the excipients. Based on laser diffraction measurements, PVPK-25 could also inhibit the aggregation of the particles more effectively than PVA, but none of the polymers could hold the nanonized samples in the submicron range until the end of the measurements.

Drug-survival profiling of second-line biologic therapy in rheumatoid arthritis: Choice of another tumour necrosis factor inhibitor or a biologic of different mode of action?

OBJECTIVES: To assess the best choice of second-line therapy between tumour necrosis factor-inhibitor (TNFi) and biologics of different-mode-of-action (BDMA-rituximab/tocilizumab/abatacept) in rheumatoid arthritis (RA) by evaluating drug-survival following discontinuation of the first-line TNFi. METHODS: This retrospective drug-survival study was performed across two different hospitals by conventional-statistics and machine-learning approach. RESULTS: From a total of 435 patients, 213 (48.9%; TNFi = 122, BDMA = 91) discontinued their second-line biologic {median drug-survival: TNFi, 27 months [95% confidence interval (95%CI) 22-32] vs BDMA, 37 months (95%CI 32-52)}. As a second-line biologic, BDMA was likely to reduce the risk of treatment-discontinuation [hazard-ratio (HR) 0.63, 95%CI 0.48-0.83] compared to TNFi, but only in seropositive-patients (HR 0.52, 95%CI 0.38-0.73), not in seronegative-RA. Drug-survival benefit of BDMA over TNFi was not observed if the seropositive-patients were previously exposed to monoclonal-TNFi (HR 0.77, 95%CI 0.49-1.22) versus soluble-TNFi (etanercept/biosimilars) or if the first-line TNFi was terminated within 23.9 months of initiation (HR 0.97, 95%CI 0.56-1.68). CONCLUSIONS: BDMA, as a second-line biologic, is more likely to be sustained in seropositive-patients, particularly without prior exposure to monoclonal-TNFi. The drug-survival benefit of BDMA was not observed in seronegative-patients or if the first-line TNFi was stopped within 2 years.

Novel genetic sex markers reveal unexpected lack of, and similar susceptibility to, sex reversal in free-living common toads in both natural and anthropogenic habitats.

Anthropogenic environmental changes are affecting biodiversity and microevolution worldwide. Ectothermic vertebrates are especially vulnerable because environmental changes can disrupt their sexual development and cause sex reversal, a mismatch between genetic and phenotypic sex. This can potentially lead to sex-ratio distortion and population decline. Despite these implications, there is scarce empirical knowledge on the incidence of sex reversal in nature. Populations in anthropogenic environments may be exposed to sex-reversing stimuli more frequently, which may lead to higher sex-reversal rate or, alternatively, these populations may adapt to resist sex reversal. We developed PCR-based genetic sex markers for the common toad (Bufo bufo) to assess the prevalence of sex reversal in wild populations living in natural, agricultural and urban habitats, and the susceptibility of the same populations to two ubiquitous oestrogenic pollutants in a common garden experiment. We found negligible sex-reversal frequency in free-living adults despite the presence of various endocrine-disrupting pollutants in their breeding ponds. Individuals from different habitat types showed similar susceptibility to sex reversal in the laboratory: all genetic males developed female phenotype when exposed to 1 µg L-1 17α-ethinylestradiol (EE2) during larval development, whereas no sex reversal occurred in response to 1 ng L-1 EE2 and a glyphosate-based herbicide with 3 µg L-1 or 3 mg L-1  glyphosate. The latter results do not support that populations in anthropogenic habitats would have either increased propensity for or higher tolerance to chemically induced sex reversal. Thus, the extremely low sex-reversal frequency in wild toads compared to other ectothermic vertebrates studied before might indicate idiosyncratic, potentially species-specific resistance to sex reversal.

Associations of vascular and bone status in arthritis patients.

Cardiovascular (CV) disease and osteoporosis (OP) have been associated with rheumatoid arthritis (RA) and ankylosing spondylitis (AS). Bone and vascular biomarkers and parameters along with the effect of 1-year anti-TNF therapy on these markers were assessed in order to determine correlations between vascular pathophysiology and bone metabolism in RA and AS. Thirty-six patients treated with etanercept or certolizumab pegol and 17 AS patients treated with ETN were included in a 12-month follow-up study. Bone and vascular markers were previously assessed by ELISA. Bone density was measured by DXA and quantitative CT (QCT). Flow-mediated vasodilation (FMD), common carotid intima-media thickness (IMT) and pulse-wave velocity (PWV) were assessed by ultrasound. Multiple correlation analyses indicated associations between bone and vascular markers. Osteoprotegerin, sclerostin and cathepsin K were significantly associated with FMD, IMT and PWV, respectively (p < 0.05). Moreover, total and trabecular BMD determined by QCT inversely correlated with IMT (p < 0.05). On the other hand, among vascular parameters, platelet-derived growth factor BB and IMT correlated with DXA femoral and QCT total BMD, respectively (p < 0.05). In the RM-ANOVA analysis, anti-TNF treatment together with baseline osteocalcin, procollagen 1 N-terminal propeptide (P1NP) or vitamin D3 levels determined one-year changes in IMT (p < 0.05). In the MANOVA analysis, baseline disease activity indices (DAS28, BASDAI), the one-year changes in these indices, as well as CRP exerted effects on multiple correlations between bone and vascular markers (p < 0.05). As the pattern of interactions between bone and vascular biomarkers differed between baseline and after 12 months, anti-TNF therapy influenced these associations. We found a great number of correlations in our RA and AS patients undergoing anti-TNF therapy. Some of the bone markers have been associated with vascular pathophysiology, while some vascular markers correlated with bone status. In arthritis, systemic inflammation and disease activity may drive both vascular and bone disease.

Evaluation of Biodegradable PVA-Based 3D Printed Carriers during Dissolution.

The presence of additive manufacturing, especially 3D printing, has the potential to revolutionize pharmaceutical manufacturing owing to the distinctive capabilities of personalized pharmaceutical manufacturing. This study's aim was to examine the behavior of commonly used polyvinyl alcohol (PVA) under in vitro dissolution conditions. Polylactic acid (PLA) was also used as a comparator. The carriers were designed and fabricated using computer-aided design (CAD). After printing the containers, the behavior of PVA under in vitro simulated biorelevant conditions was monitored by gravimetry and dynamic light scattering (DLS) methods. The results show that in all the dissolution media PVA carriers were dissolved; the particle size was under 300 nm. However, the dissolution rate was different in various dissolution media. In addition to studying the PVA, as drug delivery carriers, the kinetics of drug release were investigated. These dissolution test results accompanied with UV spectrophotometry tracking indirectly determine the possibilities for modifying the output of quality by computer design.

Comparative Evaluation of Pellet Cushioning Agents by Various Imaging Techniques and Dissolution Studies.

Most of the commercially available pharmaceutical products for oral administration route are marketed in the tablet dosage forms. However, compression of multiparticulate systems is a challenge for the pharmaceutical research and industry, especially if the individual unit is a coated particle, as the release of the active ingredient depends on the integrity of the coating. In the present study, polymer-coated pellets tableted with different types of excipients (powder, granules, pellets) then were investigated by various tablet-destructive (microscopic) and tablet non-destructive (microfocus X-ray; microCT) imaging methods. The information obtained from the independent evaluation of the in vitro drug release profiles model is confirmed by the results obtained by image analysis, regardless of whether X-ray or stereomicroscopic images of the coated, tableted pellets were used for image analysis. The results of this study show that the novel easy-to-use, fast, and non-destructive MFX method is a good alternative to the already used microscopic image analysis methods regarding the characterization of particulates, compressed into tablets.

Angiogenesis in Inflammatory Arthritis.

BACKGROUND: Angiogenesis is the outgrowth of new blood vessels from existing ones and is an early occurrence in inflamed joint tissue. It is governed by a tightly controlled balance of pro- and anti-angiogenic stimuli, which promote or inhibit generation and proliferation of new endothelial cells, vascular morphogenesis, and vessel remodeling. At the beginning, capillary formation is crucial in maintaining the supply of various nutrients as well as oxygen to the inflamed tissue. Local and systemic expression of angiogenic factors may indicate a constant remodeling of synovial vasculature. Redox signaling is closely related to angiogenesis and can alter angiogenic responses of synovial cells. In this review we discuss key issues about the endothelial pathology in inflammatory arthritis followed by a review of angiogenic processes and main angiogenic mediators. We discuss the hypoxia-vascular endothelial growth factor (VEGF)-Ang/Tie2 system and its related therapeutic implications in detail with further review of various mediator protein targets and intracellular regulatory pathway targets with their current and potential future role in preclinical or clinical setting whilst ameliorating inflammation.

Autoimmune and angiogenic biomarkers in autoimmune atherosclerosis.

Several inflammatory, proteolytic, angiogenic and bone-associated factors play a role in the development of autoimmune, accelerated atherosclerosis in rheumatic diseases. Some of these may serve as biomarkers of vascular pathology and may be useful in the follow-up of vascular damage and outcome. Multi-biomarker profiles rather than a single markers would likely be optimal in this respect.

Optimization of Quality Attributes and Atomic Force Microscopy Imaging of Reconstituted Nanodroplets in Baicalin Loaded Self-Nanoemulsifying Formulations.

The objective of the study was to develop baicalin loaded liquid self-nanoemulsifying drug delivery systems (BSNEDDS) and to characterize them by physicochemical methods in order to optimize the composition and quality attributes. Atomic force microscopy (AFM) was utilized to evaluate the morphological characteristics and size distribution of reconstituted nanoemulsion droplets with a new sample preparation method for the elucidation of individual nanodroplets without any signs of coalescence. Response surface methodology and desirability approach was used to select the optimized composition related to droplet size, zeta-potential, polydispersity index (PDI), and turbidity characteristics. Droplet size distribution measured by dynamic light scattering method was highly desirable with 52.87 ± 0.5322 nm, which was confirmed by AFM imaging. The optimized formula contains Peceol® (14.29%, w/w), Kolliphor® EL (57.14%, w/w), and Transcutol® P (28.57%, w/w). Long-term stability analysis did not show any significant change in droplet size or PDI over the investigated period. More than 40.5-times solubility improvement was achieved with the optimized BSNEDDS correlated to solubility of baicalin in distilled water. In vitro dissolution studies at pH 1.2 and pH 6.8 were performed and revealed that the optimized BSNEDDS formula showed pH independent drug dissolution, and 100% of incorporated baicalin dissolved within five minutes in rapidly dispersing nanodroplets.

Study on the dissolution improvement of albendazole using reconstitutable dry nanosuspension formulation.

The aim of the study was to improve the solubility and dissolution rate of the poorly water soluble drug albendazole via surfactant assisted media milling process. Preparation of a nanosuspension and then post-processing with a solidification technique applied to improve the applicability of nanosuspension in a solid dosage forms carrier. The dry nanosuspension was obtained using microcrystalline cellulose as solid carrier after tray drying at 40 °C. Both reconstitution from the solid carrier and dissolution profile studies were investigated in biorelevant Artificial Rumen Fluid (ARF) at pH = 6.50 and dissolution media at pH = 1.20 and pH = 6.80. Reconstitution studies have demonstrated that the mean hydrodynamic diameter values of albendazole crystals released from the dry suspension were nanosized (intensity weighted hydrodynamic diameter values: 200.40 ±â€¯2.318 nm in ARF at pH = 6.50, 197.17 ±â€¯0.208 nm in dissolution medium at pH = 6.80). Thermodynamic solubility studies have indicated a 2.98 times increase in water solubility (144.41 ±â€¯0.09 µg/ml milled, 48.38 ±â€¯0.01 µg/ml unmilled, 8.21 ±â€¯0.02 µg/ml albendazole powder) in ARF at pH = 6.50, and 2.33 times in dissolution medium at pH = 6.8: (146.27 ±â€¯0.28 µg/ml milled, 62.71 ±â€¯0.04 µg/ml unmilled, 9.00 ±â€¯0.01 µg/ml albendazole powder), and 13.65% increase at pH = 1.20 (1728.31 ±â€¯3.31 µg/ml milled, 1559.41 ±â€¯0.40 µg/ml unmilled, 1520.70 ±â€¯1.39 µg/ml albendazole powder), dissolution rates have also increased. Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM) imaging investigations detected no albendazole nanocrystals on the surface of the carrier, which demonstrated the incorporation of albendazole into the microcrystalline cellulose solid carrier structure.

Oxidative stress impairs energy metabolism in primary cells and synovial tissue of patients with rheumatoid arthritis.

BACKGROUND: In this study, we examined the effect of oxidative stress on cellular energy metabolism and pro-angiogenic/pro-inflammatory mechanisms of primary rheumatoid arthritis synovial fibroblast cells (RASFC) and human umbilical vein endothelial cells (HUVEC). METHODS: Primary RASFC and HUVEC were cultured with the oxidative stress inducer 4-hydroxy-2-nonenal (4-HNE), and extracellular acidification rate, oxygen consumption rate, mitochondrial function and pro-angiogenic/pro-inflammatory mechanisms were assessed using the Seahorse analyser, complex I-V activity assays, random mutation mitochondrial capture assays, enzyme-linked immunosorbent assays and functional assays, including angiogenic tube formation, migration and invasion. Expression of angiogenic growth factors in synovial tissue (ST) was assessed by IHC in patients with rheumatoid arthritis (RA) undergoing arthroscopy before and after administration of tumour necrosis factor inhibitors (TNFi). RESULTS: In RASFC and HUVEC, 4-HNE-induced oxidative stress reprogrammed energy metabolism by inhibiting mitochondrial basal, maximal and adenosine triphosphate-linked respiration and reserve capacity, coupled with the reduced enzymatic activity of oxidative phosphorylation complexes III and IV. In contrast, 4-HNE elevated basal glycolysis, glycolytic capacity and glycolytic reserve, paralleled by an increase in mitochondrial DNA mutations and reactive oxygen species. 4-HNE activated pro-angiogenic responses of RASFC, which subsequently altered HUVEC invasion and migration, angiogenic tube formation and the release of pro-angiogenic mediators. In vivo markers of angiogenesis (vascular endothelial growth factor, angiopoietin 2 [Ang2], tyrosine kinase receptor [Tie2]) were significantly associated with oxidative damage and oxygen metabolism in the inflamed synovium. Significant reduction in ST vascularity and Ang2/Tie2 expression was demonstrated in patients with RA before and after administration of TNFi. CONCLUSIONS: Oxidative stress promotes metabolism in favour of glycolysis, an effect that may contribute to acceleration of inflammatory mechanisms and subsequent dysfunctional angiogenesis in RA.

Controlled Release Oral Delivery of Apigenin Containing Pellets with Antioxidant Activity.

BACKGROUND: Drug delivery of phytochemicals has gained interest recently due to their remarkable health effects. Apigenin, a plant flavonoid, has antioxidant, anti-inflammatory and anticancer activities but its delivery is challenging. It could be absorbed through the whole intestine, however, it has poor bioavailability due to its low aqueous solubility. In Europe, the daily intake was estimated to be as low as 3 ± 1 mg. Pellets offer several advantages such as improved bioavailability and various resultant drug release profiles can be obtained by simply mixing pellets with different coatings. OBJECTIVE: The objective of our study was to develop a carrier system containing 20 mg apigenin thus enhancing intake and to offer reduction of oxidative stress which can cause inflammation in the intestine. METHOD: The apigenin powder was dispersed in aqueous solution of binding material and layered onto the inert cores in a fluidized bed apparatus. The layered cores were further coated with enteric polymers and the process parameters were optimized. RESULTS: The prepared pellets met with the requirements and have good physical characteristic. 10% (w/w) Eudragit® L was suitable for enteric coating with a complete release at pH 6.8 within 1 hour. 15% (w/w) Eudragit® FS coating ensured acid resistance ability and colonic delivery. The therapeutic efficiency was confirmed with antioxidant activity measurement by using DPPH* assay. CONCLUSION: Enteric coated spheres allow targeted delivery into the intestine and colon thus reaching the main absorption site. Pellets were proved to be an optimal delivery system for apigenin thus providing enhanced apigenin intake.

Targeting of proangiogenic signalling pathways in chronic inflammation.

Angiogenesis is de novo capillary outgrowth from pre-existing blood vessels. This process not only is crucial for normal development, but also has an important role in supplying oxygen and nutrients to inflamed tissues, as well as in facilitating the migration of inflammatory cells to the synovium in rheumatoid arthritis, spondyloarthritis and other systemic autoimmune diseases. Neovascularization is dependent on the balance of proangiogenic and antiangiogenic mediators, including growth factors, cytokines, chemokines, cell adhesion molecules and matrix metalloproteinases. This Review describes the various intracellular signalling pathways that govern these angiogenic processes and discusses potential approaches to interfere with pathological angiogenesis, and thereby ameliorate inflammatory disease, by targeting these pathways.

Comparative evaluation of the effect of cyclodextrins and pH on aqueous solubility of apigenin.

The aqueous solubility of a flavonoid, apigenin, was studied in the presence of first generation cyclodextrins (α-CyD, ß-CyD, γ-CyD), ionic and nonionic synthetic derivatives of ß-CyD, namely SBE-ß-CyD, HP-ß-CyD and RM-ß-CyD at various physiological pH. The order of solubility enhancement was as follows: RM-ß-CyD>SBE-ß-CyD>γ-CyD>HP-ß-CyD>ß-CyD>α-CyD. The phase solubility diagrams of HP-ß-CyD and SBE-ß-CyD indicated Higuchi AL subtype behavior, suggesting 1:1 stoichiometry of the complex. In contrast, AP subtype, so higher order complex formation can be assumed in the case of RM-ß-CyD and γ-CyD. The formation of inclusion complexes has been confirmed by absorption and fluorescence spectroscopic measurements. Increased antioxidant activity was observed due to the inclusion complexes. These results prove that synthetic derivatives of ß-CyD will be potentially useful excipients in the development of drug delivery systems for healthcare products containing flavonoids.

Hypoxia and STAT3 signalling interactions regulate pro-inflammatory pathways in rheumatoid arthritis.

OBJECTIVE: To examine the effect of hypoxia on Signal Transducer and Activator of Transcription 3 (STAT3)-induced pro-inflammatory pathways in rheumatoid arthritis (RA). METHODS: Detection of phospho-STAT3 was assessed in RA synovial tissue and fibroblasts (RASFC) by immunohistology/immunofluorescence. Primary RASFCs and a normal synoviocyte cell line (K4IM) were cultured under hypoxic and normoxic conditions±Stat3-siRNA, HIF-siRNA or WP1066 (JAK2-inhibitor). HIF1α, p-STAT3, p-STAT1 and Notch-1IC protein expression were analysed by western blot. Functional mechanisms were quantified by invasion chamber, matrigel and migration assays. IL-6, IL-8, IL-10 and matrixmetalloproteinases (MMP)-3 were quantified by ELISA. Notch-1 receptor, its DLL-4 ligand and downstream target genes (hrt-1, hrt-2) were quantified by real-time PCR. The effect of WP1066 on spontaneous secretion of pro/anti-inflammatory cytokines and Notch signalling was examined in RA synovial explants ex vivo. RESULTS: p-STAT3 was increased in RA synovium compared with control (p<0.05). Hypoxia induced p-STAT3, p-STAT1 and HIF1α expression, an effect blocked by Stat3-siRNA and WP1066. Hypoxia-induced cell invasion, migration and cytokine production were inhibited by Stat3-siRNA (p<0.05) and WP1066 (p<0.05). While HIF1α siRNA inhibited hypoxia-induced p-STAT3 detection, Stat3-siRNA also inhibited hypoxia-induced HIF1α. Furthermore, hypoxia-induced Notch-1IC, DLL4, hrt-1 and -2 expression were significantly inhibited by WP1066 (p<0.05). Finally, in RA synovial explant cultures ex vivo, WP1066 decreased spontaneous secretion of IL-6, IL-8 and MMP3 (p<0.05), Notch-1 mRNA (p<0.05) and induced IL-10 (p<0.05). CONCLUSIONS: This is the first study to provide evidence of a functional link between HIF1α, STAT3 and Notch-1 signalling in the regulation of pro-inflammatory mechanisms in RA, and further supports a role for STAT blockade in the treatment of RA.

Microstructural analysis of the fast gelling freeze-dried sodium hyaluronate.

Although sodium hyaluronate is a very unstable and heat sensitive molecule, it can remain relatively stable during the freeze-drying process. Aqueous sodium hyaluronate (NaHA) gels were prepared and the obtained samples were freeze-dried. The freeze-dried NaHA samples showed fast gelling ability meanwhile preserved their initial viscoelasticity even after reconstitution. The microstructure of gels obtained from raw substance and freeze-dried NaHA samples was characterized with positron annihilation lifetime spectroscopy and X-ray diffraction patterns while their functionality-related macrostructural properties were tested based on their rheological behavior. The presence of phosphate salts improved the formation of ordered supramolecular structure retaining water in the free volume holes of the polymer chains characterized with decreased ortho-positronium lifetime values. This property may be advantageous in the development of a freeze-dried NaHA injection dosage form.

Protection promoted by pGP3 or pGP4 against Chlamydia muridarum is mediated by CD4(+) cells in C57BL/6N mice.

Urogenital tract infection with Chlamydia trachomatis is a leading cause of sexually transmitted infections. There is currently no commercially available vaccine against C. trachomatis. The highly conserved plasmid of chlamydiae has been considered to be a virulence factor and the plasmid proteins have important roles in the Chlamydia-specific immune response. This study was designed to evaluate the efficacy of vaccination with plasmid proteins in the prevention of C. muridarum lung infection in a mouse model. C57BL/6N mice were immunised 3 times subcutaneously with recombinant pGP3 or pGP4 and infected with C. muridarum. Immunisation of the mice with recombinant pGP3 or pGP4 protein caused a significantly lower chlamydial burden in the lungs of the infected mice; the lower IFN-γ level indicated a reduced extent of inflammation. In vitro or in vivo neutralisation of C. muridarum with sera obtained from immunised mice did not reduce the number of viable C. muridarum in the lungs of mice. However, adoptive transfer of the CD4(+) spleen cells isolated from the immunised mice resulted in a significantly reduced bacterial burden. Our results indicate that it is not the pGP3- and pGP4-specific antibodies, but the CD4(+) cells that are responsible for the protective effect of the immune response to plasmid proteins.

Redox-mediated angiogenesis in the hypoxic joint of inflammatory arthritis.

OBJECTIVE: Inflammatory arthritis is associated with joint inflammation, synovial tissue proliferation, and degradation of articular cartilage and bone. Angiogenesis is an early and fundamental component of synovial inflammation. Oxygen metabolism is recognized as an important mediator of joint vascular remodeling. The aim of this study was to determine whether in vivo synovial hypoxia (tissue PO2 [tPO2 ]) and tumor necrosis factor (TNF) blocking therapy alter synovial vascular expression of NADPH oxidase (NOX) and how this action regulates angiogenic mechanisms. METHODS: NOX-2 protein and messenger RNA expression was examined in patients with inflammatory arthritis before and after receiving TNF inhibitor (TNFi) therapy and in mice with collagen-induced arthritis (CIA). Proangiogenic processes were assessed in human microvascular endothelial cells (HMVECs) following culture with NOX-2 activators (TNFα and 4-hydroxynonenal), small interfering RNA (siRNA) for NOX, and the inhibitor diphenyleneiodonium (DPI) under conditions of normoxia or 3% hypoxia. RESULTS: We demonstrated significantly increased NOX-2 expression in the joints of patients with inflammatory arthritis and the joints of mice with CIA as compared to controls. NOX-2 expression was higher in patients with synovial tPO2 levels <3% than in those with tPO2 levels >3% (P < 0.05), and correlated with in vivo macroscopic/microscopic measures of angiogenesis, such as vascularity and levels of vascular endothelial growth factor, angiopoietin 2, factor VIII, neural cell adhesion molecule, and α-smooth muscle actin (P < 0.05 for all). A decrease in NOX-2 expression was paralleled by an increase in in vivo tPO2 levels only in those patients who were defined as TNFi responders. In vitro NOX-2 activators and 3% hypoxia significantly promoted HMVEC migration, angiogenic tube formation, and secretion of proangiogenic mediators, effects that were blocked by siRNA for NOX-2 or the NOX-2 inhibitor DPI. CONCLUSION: We demonstrated that hypoxia activates NOX-2 protein expression, and NOX-2-induced oxidative stress may be an initiating factor in driving angiogenesis.