In Vitro Inhibition of Xanthine Oxidase Purified from Arthritis Serum Patients by Nanocurcumin and Artemisinin Active Compounds.

Curcumin and artemisinin are commonly used in traditional East Asian medicine. In this study, we investigated the inhibitory effects of these active compounds on xanthine oxidase (XO) using allopurinol as a control. XO was purified from the serum of arthritis patients through ammonium sulfate precipitation (65%) and ion exchange chromatography on diethylaminoethyl (DEAE)-cellulose. The specific activity of the purified enzyme was 32.5 U/mg protein, resulting in a 7-fold purification with a yield of 66.8%. Molecular docking analysis revealed that curcumin had the strongest interaction energy with XO, with a binding energy of -9.28 kcal/mol. The amino acid residues Thr1077, Gln762, Phe914, Ala1078, Val1011, Glu1194, and Ala1079 were located closer to the binding site of curcumin than artemisinin, which had a binding energy of -7.2 kcal/mol. In vitro inhibition assays were performed using nanocurcumin and artemisinin at concentrations of 5, 10, 15, 20, and 25 µg/mL. Curcumin inhibited enzyme activity by 67-91%, while artemisinin had a lower inhibition ratio, which ranged from 40-70% compared to allopurinol as a control.

Local steroid activation is a critical mediator of the anti-inflammatory actions of therapeutic glucocorticoids.

OBJECTIVES: The enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) plays a well-characterised role in the metabolism and activation of endogenous glucocorticoids (GCs). However, despite its potent upregulation at sites of inflammation, its role in peripheral metabolism and action of therapeutic GCs remains poorly understood. We investigated the contribution of 11ß-HSD1 to the anti-inflammatory properties of the active GC corticosterone, administered at therapeutic doses in murine models of polyarthritis. METHODS: Using the tumour necrosis factor-tg and K/BxN serum-induced models of polyarthritis, we examined the anti-inflammatory properties of oral administration of corticosterone in animals with global, myeloid and mesenchymal targeted transgenic deletion of 11ß-HSD1. Disease activity and joint inflammation were scored daily. Joint destruction and measures of local and systemic inflammation were determined by histology, micro-CT, quantitative RT-PCR, fluorescence activated cell sorting and ELISA. RESULTS: Global deletion of 11ß-HSD1 resulted in a profound GC resistance in animals receiving corticosterone, characterised by persistent synovitis, joint destruction and inflammatory leucocyte infiltration. This was partially reproduced with myeloid, but not mesenchymal 11ß-HSD1 deletion, where paracrine GC signalling between cell populations was shown to overcome targeted deletion of 11ß-HSD1. CONCLUSIONS: We identify an entirely novel component of therapeutic GC action, whereby following their systemic metabolism, they require peripheral reactivation and amplification by 11ß-HSD1 at sites of inflammation to deliver their anti-inflammatory therapeutic effects. This study provides a novel mechanistic understanding of the anti-inflammatory properties of therapeutic GCs and their targeting to sites of inflammation in polyarthritis.

Matrix-degrading protease ADAMTS-5 cleaves inter-α-inhibitor and releases active heavy chain 2 in synovial fluids from arthritic patients.

Destruction of the cartilage matrix in joints is an important feature of arthritis. Proteolytic degradation of cartilage glycoproteins can contribute to the loss of matrix integrity. Human inter-α-inhibitor (IαI), which stabilizes the extracellular matrix, is composed of the light-chain serine proteinase inhibitor bikunin and two homologous heavy chains (HC1 and HC2) covalently linked through chondroitin 4-sulfate. Inflammation promotes the transfer of HCs from chondroitin 4-sulfate to hyaluronan by tumor necrosis factor-stimulated gene-6 protein (TSG-6). This reaction generates a covalent complex between the heavy chains and hyaluronan that can promote leukocyte invasion. This study demonstrates that both IαI and the HC-hyaluronan complex are substrates for the extracellular matrix proteases ADAMTS-5 and matrix metalloprotease (MMP) -3, -7, and -13. The major cleavage sites for all four proteases are found in the C terminus of HC2. ADAMTS-5 and MMP-7 displayed the highest activity toward HC2. ADAMTS-5 degradation products were identified in mass spectrometric analysis of 29 of 33 arthropathic patients, indicating that ADAMTS-5 cleavage occurs in synovial fluid in arthritis. After cleavage, free HC2, together with TSG-6, is able to catalyze the transfer of heavy chains to hyaluronan. The release of extracellular matrix bound HC2 is likely to increase the mobility of the HC2/TSG-6 catalytic unit and consequently increase the rate of the HC transfer reaction. Ultimately, ADAMTS-5 cleavage of HC2 could alter the physiological and mechanical properties of the extracellular matrix and contribute to the progression of arthritis.

Detecting Basal Myeloperoxidase Activity in Living Systems with a Near-Infrared Emissive "Turn-On" Probe.

Detecting myeloperoxidase (MPO) activity in living organisms is important because MPO contributes to the pathogenesis of many diseases such as rheumatoid arthritis and other inflammatory diseases, artherosclerosis, neurodegenerative disease, and some cancers. However, rapid and effective methods for the detection of basal MPO activity in living systems have not yet been reported. Herein, we report a near-infrared (NIR) emissive "turn-on" probe FD-301 that can specifically bind to MPO and accurately measure MPO activity in living cells and in vivo via a rapid response to initial hypochlorous acid (HOCl), produced by MPO. Notably, FD-301 could detect the basal level of MPO activity in human promyelocytic leukemia cells (HL-60) and could discriminate between MPO high-expression and low-expression cells. Furthermore, FD-301 was successfully applied to in vivo imaging of MPO in MPO-dependent diseases, such as arthritis and inflammatory bowel disease.

Respective contribution of cytosolic phospholipase A2α and secreted phospholipase A2 IIA to inflammation and eicosanoid production in arthritis.

Phospholipase A2s (PLA2) play a key role in generation of eicosanoids. Cytosolic PLA2α (cPLA2α) is constitutively expressed in most cells, whereas IIA secreted PLA2 (sPLA2-IIA) is induced during inflammation and is present at high levels in the synovial fluid of rheumatoid arthritis patients. In mice, both cPLA2α and sPLA2-IIA have been implicated in autoimmune arthritis; however, the respective contribution of these two enzymes to the pathogenesis and production of eicosanoids is unknown. We evaluated the respective role of cPLA2α and sPLA2-IIA with regard to arthritis and eicosanoid profile in an in vivo model of arthritis. While arthritis was most severe in mice expressing both enzymes, it was abolished when both cPLA2α and sPLA2-IIA were lacking. cPLA2α played a dominant role in the severity of arthritis, although sPLA2-IIA sufficed to significantly contribute to the disease. Several eicosanoids were modulated during the course of arthritis and numerous species involved sPLA2-IIA expression. This study confirms the critical role of PLA2s in arthritis and unveils the distinct contribution of cPLA2α and sPLA2-IIA to the eicosanoid profile in arthritis.

LOXL3 Function Beyond Amino Oxidase and Role in Pathologies, Including Cancer.

Lysyl oxidase like 3 (LOXL3) is a copper-dependent amine oxidase responsible for the crosslinking of collagen and elastin in the extracellular matrix. LOXL3 belongs to a family including other members: LOX, LOXL1, LOXL2, and LOXL4. Autosomal recessive mutations are rare and described in patients with Stickler syndrome, early-onset myopia and non-syndromic cleft palate. Along with an essential function in embryonic development, multiple biological functions have been attributed to LOXL3 in various pathologies related to amino oxidase activity. Additionally, various novel roles have been described for LOXL3, such as the oxidation of fibronectin in myotendinous junction formation, and of deacetylation and deacetylimination activities of STAT3 to control of inflammatory response. In tumors, three distinct roles were described: (1) LOXL3 interacts with SNAIL and contributes to proliferation and metastasis by inducing epithelial-mesenchymal transition in pancreatic ductal adenocarcinoma cells; (2) LOXL3 is localized predominantly in the nucleus associated with invasion and poor gastric cancer prognosis; (3) LOXL3 interacts with proteins involved in DNA stability and mitosis completion, contributing to melanoma progression and sustained proliferation. Here we review the structure, function and activity of LOXL3 in normal and pathological conditions and discuss the potential of LOXL3 as a therapeutic target in various diseases.

Matrix metalloproteinase collagenolysis in health and disease.

The proteolytic processing of collagen (collagenolysis) is critical in development and homeostasis, but also contributes to numerous pathologies. Mammalian interstitial collagenolytic enzymes include members of the matrix metalloproteinase (MMP) family and cathepsin K. While MMPs have long been recognized for their ability to catalyze the hydrolysis of collagen, the roles of individual MMPs in physiological and pathological collagenolysis are less defined. The use of knockout and mutant animal models, which reflect human diseases, has revealed distinct collagenolytic roles for MT1-MMP and MMP-13. A better understanding of temporal and spatial collagen processing, along with the knowledge of the specific MMP involved, will ultimately lead to more effective treatments for cancer, arthritis, cardiovascular conditions, and infectious diseases. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman.

[Age-related traits of cytogenetic abnormalities in synovial cells of knee-joint in residents of the North of Siberia with arthritis of different etiology depending on the GSTM1 gene polymorphism of glutathion-S-transferase.]

It was studied the frequency of cells with cytogenetic abnormalities in the synovial fluid cells of the knee joint in patients of different age groups suffering from chronic arthritis associated with Lyme borreliosis (CAALB) or post-traumatic arthritis (PTA), depending on the polymorphism of the GSTM1 gene of glutathione-S-transferase. The study included 135 residents of the north of the Tomsk and Tyumen regions, 68 of whom suffered from CAALB, and the rest of the 67 patients who made up the control group were diagnosed with PTA. The results of this study have demonstrated that there are significant age-related differences in the frequency of cytogenetic abnormalities of the synovial fluid cells of the knee joint between young and elderly patients of СAALB. The integrative assessment of clinical and cytogenetic parameters in the group of elderly СAALB patients with mutant GSTM1 (0/0) allele, as compared with the other groups, enable to conclude that there are significant positive correlations between the indices of the severity disruption of articular locomotor function and the frequency of synovial fluid cells with trisomy of chromosome 7.

ADAMTS-12: a multifaced metalloproteinase in arthritis and inflammation.

ADAMTS-12 is a member of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family of proteases, which were known to play important roles in various biological and pathological processes, such as development, angiogenesis, inflammation, cancer, arthritis, and atherosclerosis. In this review, we briefly summarize the structural organization of ADAMTS-12; concentrate on the emerging role of ADAMTS-12 in several pathophysiological conditions, including intervertebral disc degeneration, tumorigenesis and angioinhibitory effects, pediatric stroke, gonad differentiation, trophoblast invasion, and genetic linkage to schizophrenia and asthma, with special focus on its role in arthritis and inflammation; and end with the perspective research of ADAMTS-12 and its potential as a promising diagnostic and therapeutic target in various kinds of diseases and conditions.

Positional identification of Ncf1 as a gene that regulates arthritis severity in rats.

The identification of genes underlying quantitative-trait loci (QTL) for complex diseases, such as rheumatoid arthritis, is a challenging and difficult task for the human genome project. Through positional cloning of the Pia4 QTL in rats, we found that a naturally occurring polymorphism of Ncf1 (encoding neutrophil cytosolic factor 1, a component of the NADPH oxidase complex) regulates arthritis severity. The disease-related allele of Ncf1 has reduced oxidative burst response and promotes activation of arthritogenic T cells. Pharmacological treatment with substances that activate the NADPH oxidase complex is shown to ameliorate arthritis. Hence, Ncf1 is associated with a new autoimmune mechanism leading to severe destructive arthritis, notably similar to rheumatoid arthritis in humans.

PI3K p110delta regulates T-cell cytokine production during primary and secondary immune responses in mice and humans.

We have previously described critical and nonredundant roles for the phosphoinositide 3-kinase p110delta during the activation and differentiation of naive T cells, and p110delta inhibitors are currently being developed for clinical use. However, to effectively treat established inflammatory or autoimmune diseases, it is important to be able to inhibit previously activated or memory T cells. In this study, using the isoform-selective inhibitor IC87114, we show that sustained p110delta activity is required for interferon-gamma production. Moreover, acute inhibition of p110delta inhibits cytokine production and reduces hypersensitivity responses in mice. Whether p110delta played a similar role in human T cells was unknown. Here we show that IC87114 potently blocked T-cell receptor-induced phosphoinositide 3-kinase signaling by both naive and effector/memory human T cells. Importantly, IC87114 reduced cytokine production by memory T cells from healthy and allergic donors and from inflammatory arthritis patients. These studies establish that previously activated memory T cells are at least as sensitive to p110delta inhibition as naive T cells and show that mouse models accurately predict p110delta function in human T cells. There is therefore a strong rationale for p110delta inhibitors to be considered for therapeutic use in T-cell-mediated autoimmune and inflammatory diseases.

Genetic sphingosine kinase 1 deficiency significantly decreases synovial inflammation and joint erosions in murine TNF-alpha-induced arthritis.

Sphingosine kinase 1 (SphK1) is an enzyme that converts sphingosine to bioactive sphingosine-1-phosphate. Recent in vitro data suggest a potential role of SphK1 in TNF-alpha-mediated inflammation. Our aims in this study were to determine the in vivo significance of SphK1 in TNF-alpha-mediated chronic inflammation and to define which pathogenic mechanisms induced by TNF-alpha are SphK1 dependent. To pursue these aims, we studied the effect of SphK1 deficiency in an in vivo model of TNF-alpha-induced chronic inflammatory arthritis. Transgenic hTNF-alpha mice, which develop spontaneous inflammatory erosive arthritis beginning at 14-16 wk, were crossed with SphK1 null mice (SphK1(-/-)), on the C57BL6 genetic background. Beginning at 4 mo of age, hTNF/SphK1(-/-) mice had significantly less severe clinically evident paw swelling and deformity, less synovial and periarticular inflammation, and markedly decreased bone erosions as measured quantitatively through micro-CT images. Mechanistically, the mice lacking SphK1 had less articular cyclooxygenase 2 protein and fewer synovial Th17 cells than did hTNF/SphK1(+/+) littermates. Microarray analysis and real-time RT-PCR of the ankle synovial tissue demonstrated that hTNF/SphK1(-/-) mice had increased transcript levels of suppressor of cytokine signaling 3 compared with hTNF/SphK1(+/+) mice, likely also contributing to the decreased inflammation in the SphK1-deficient mice. Finally, significantly fewer mature osteoclasts were detected in the ankle joints of hTNF/SphK1(-/-) mice compared with hTNF/SphK1(+/+) mice. These data indicate that SphK1 plays a key role in hTNF-alpha-induced inflammatory arthritis via impacting synovial inflammation and osteoclast number.

The role of human xanthine oxidoreductase (HXOR), anti-HXOR antibodies, and microorganisms in synovial fluid of patients with joint inflammation.

This work is to investigate the levels of human xanthine oxidoreductase (HXOR), its antibodies, and microorganisms in synovial fluid of patients with untreated rheumatoid joint diseases. Synovial fluids were collected from sixty-four patients with rheumatoid joint diseases. Sixty-four age-matched individuals were included as control. Xanthine oxidoreductase (XOR) proteins level and anti-XOR antibodies were determined in the blood and synovial fluid, using human XOR as antigen, by enzyme-linked immunosorbent (ELISA) assay. Synovial fluids were cultured for bacteria and fungi. The titers of XOR protein in the synovial fluid of patients with rheumatoid arthritis were 90.43 ± 23.37 µg/ml (mean ± SD, n = 29) and up to 62.42 ± 8.74 µg/ml (mean ± SD, n = 35) in other joint inflammation. Anti-HXOR antibodies titers in patients were 167.72 ± 23.64 µg/ml, n = 64, which was significantly higher in rheumatoid arthritis patients. The results indicated that anti-HXOR antibodies in synovial fluids have a protective role as high concentrations against XOR were detected in inflammatory arthritis. These antibodies play a role in eliminating XOR from synovial fluids. However, immune complex formation could activate complement and participate in propagating the inflammatory cycle. Synovial aspirate ordinary microbial cultures were negative for any bacteria or fungi, but that does not exclude organisms of special culture requirements.

Natural history of alkaptonuria revisited: analyses based on scoring systems.

Increased circulating homogentisic acid in body fluids occurs in alkaptonuria (AKU) due to lack of enzyme homogentisate dioxygenase leading in turn to conversion of HGA to a pigmented melanin-like polymer, known as ochronosis. The tissue damage in AKU is due to ochronosis. A potential treatment, a drug called nitisinone, to decrease formation of HGA is available. However, deploying nitisinone effectively requires its administration at the most optimal time in the natural history. AKU has a long apparent latent period before overt ochronosis develops. The rate of change of ochronosis and its consequences over time following its recognition has not been fully described in any quantitative manner. Two potential tools are described that were used to quantitate disease burden in AKU. One tool describes scoring the clinical features that includes clinical assessments, investigations and questionnaires in 15 patients with AKU. The second tool describes a scoring system that only includes items obtained from questionnaires in 44 people with AKU. Analysis of the data reveals distinct phases of the disease, a pre-ochronotic phase and an ochronotic phase. The ochronotic phase appears to demonstrate an earlier slower progression followed by a rapidly progressive phase. The rate of change of the disease will have implications for monitoring the course of the disease as well as decide on the most appropriate time that treatment should be started for it to be effective either in prevention or arrest of the disease.

Isolation and characterization of human monoclonal antibodies specific to MMP-1A, MMP-2 and MMP-3.

Matrix metalloproteinases (MMPs), a group of more than 20 zinc-containing endopeptidases, are up-regulated in many diseases, but the use of MMP inhibitors for therapeutic purposes has often been disappointing, possibly for the limited specificity of the drugs used in clinical trials. In principle, individual MMPs could be specifically drugged by monoclonal antibodies, either by inhibition of their catalytic activity or by antibody-based pharmacodelivery strategies. In this article we describe the isolation and affinity maturation of recombinant antibodies (SP1, SP2, SP3) specific to the murine catalytic domains of MMP-1A, MMP-2 and MMP-3. These novel reagents allowed a systematic comparative immunofluorescence analysis of the expression patterns of their cognate antigens in a variety of healthy, cancerous and arthritic murine tissues. While all three MMPs were strongly expressed in tumor and arthritis specimens, MMP-1A was completely undetectable in the normal tissues tested, while MMP-2 and MMP-3 exhibited a weak expression in certain normal tissues (e.g., liver). The new antibodies may serve as building blocks for the development of antibody-based therapy strategies in mouse models of pathology.

ADAMTS5 is the major aggrecanase in mouse cartilage in vivo and in vitro.

Aggrecan is the major proteoglycan in cartilage, endowing this tissue with the unique capacity to bear load and resist compression. In arthritic cartilage, aggrecan is degraded by one or more 'aggrecanases' from the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of proteinases. ADAMTS1, 8 and 9 have weak aggrecan-degrading activity. However, they are not thought to be the primary aggrecanases because ADAMTS1 null mice are not protected from experimental arthritis, and cleavage by ADAMTS8 and 9 is highly inefficient. Although ADAMTS4 and 5 are expressed in joint tissues, and are known to be efficient aggrecanases in vitro, the exact contribution of these two enzymes to cartilage pathology is unknown. Here we show that ADAMTS5 is the major aggrecanase in mouse cartilage, both in vitro and in a mouse model of inflammatory arthritis. Our data suggest that ADAMTS5 may be a suitable target for the development of new drugs designed to inhibit cartilage destruction in arthritis, although further work will be required to determine whether ADAMTS5 is also the major aggrecanase in human arthritis.

The nuclear membrane organization of leukotriene synthesis.

Leukotrienes (LTs) are signaling molecules derived from arachidonic acid that initiate and amplify innate and adaptive immunity. In turn, how their synthesis is organized on the nuclear envelope of myeloid cells in response to extracellular signals is not understood. We define the supramolecular architecture of LT synthesis by identifying the activation-dependent assembly of novel multiprotein complexes on the outer and inner nuclear membranes of mast cells. These complexes are centered on the integral membrane protein 5-Lipoxygenase-Activating Protein, which we identify as a scaffold protein for 5-Lipoxygenase, the initial enzyme of LT synthesis. We also identify these complexes in mouse neutrophils isolated from inflamed joints. Our studies reveal the macromolecular organization of LT synthesis.

Cartilage matrix depletion by rheumatoid synovial cells in tissue culture.

Articular cartilage fragments were added to monolayer cultures of synovial membrane cells. After 3 wk of incubation, the cartilage fragments were examined histologically for metachromasia and basophilia, and for fluorescent staining using a rabbit antiserum to cartilage protein-polysaccharide. Cartilage incubated with cells derived from rheumatoid synovial membranes showed striking loss of metachromasia and basophilia as well as diminished to absent fluorescent staining. Cartilage fragments incubated with cells from normal synovia, or with cells from the synovial membrane of a patient with Reiter's syndrome, did not show these changes and resembled control cartilage incubated in tissue culture medium alone. It appears, therefore, that rheumatoid synovial cells in tissue culture are able to deplete the matrix of articular cartilage.

Increased plasma myeloperoxidase levels in systemic lupus erythematosus.

The objective of the study was to quantify plasma myeloperoxidase (MPO) levels in systemic lupus erythematosus (SLE) patients and to evaluate a correlation between MPO levels and disease activity. 71 female SLE patients and 70 controls were studied. Patients were divided into two groups: Group I (n = 48) with SLEDAI-2K score 0-5 and Group II (n = 23) with SLEDAI-2K score > or = 6. Mann-Whitney test and Spearman rank correlation were used. Two-sided P values < 0.05 were considered significant and P values > or = 0.05 and < 0.08 were considered as a tendency. The median age of patients and controls were comparable and the mean disease duration was 99.2 +/- 61.7 months. MPO levels were higher in patients than controls [5.99 (4.38-8.64) vs. 5.00 (3.33-7.08) ng/ml, P = 0.02]. We did not find correlation between MPO levels and SLEDAI-2k (r = 0.07, P = 0.58). MPO levels were not affected by treatment with prednisone, cyclophosphamide or azathioprine, however, a tendency of lower levels was observed among patients under antimalarial drugs. There was no significant difference in MPO plasma levels between Group I and Group II (5.83 vs. 6.02 ng/ml, P = 0.99). MPO levels were higher in patients with arthritis than in those without arthritis (8.15 vs. 5.56 ng/ml, P = 0.010). No difference was observed among patients with and without other organs/systems involvement. SLE patients presented increased MPO plasma levels than healthy controls. Despite the lack of correlation between MPO plasma levels and disease activity, the higher MPO levels in patients with articular involvement suggests MPO may play a different role in the inflammatory process of some SLE manifestations.