Petya Dimitrova

The role of properdin in murine zymosan-induced arthritis.

Using properdin-deficient and wild-type mice, we have investigated the role of properdin in development and progression of zymosan-induced arthritis. At the initial phase of local, zymosan-induced inflammation, properdin-deficient and wild-type mice showed bone erosion, proteoglycan loss and cell infiltration. Compared to wild-type, properdin-deficient mice had reduced C5a and IL-6 but similar synovial TNF-alpha and sRANKL levels. Both groups showed a systemic immune response. Elevated IFN-gamma production and STAT1 signaling in splenocytes and a shift to Th1 response in popliteal lymph nodes were observed in properdin-deficient mice. Properdin-deficient mice had significantly less circulating zymosan-specific IgG antibodies than wild-type. In the chronic phase, the lack of properdin resulted in significant proteoglycan loss in the joints and lower cartilageneous STAT1 expression. The level of synovial C5a on day 30 was comparable in both groups, but C5aR staining was more apparent in the joints of properdin-deficient mice. Our data show that properdin is an important player in processes involved in inflammatory joint degradation.

Harpagoside: from Kalahari Desert to pharmacy shelf.

Harpagoside is an iridoid glycoside that was first isolated from Harpagophytum procumbens (devils claw, Pedaliaceae), a medicinal plant in which it is the major constituent of the iridoid pool. Both the pure compound and devils claw extracts have potent anti-rheumatic, anti-inflammatory and analgesic effects. According to the European Pharmacopoeia commercial devils claw products should contain at least 1.2% harpagoside. However, the compound has also been isolated from several other plant species and in vitro plant culture systems. Recent advances in knowledge of harpagoside distribution, biosynthesis/accumulation and pharmacology are summarized in this review. We also discuss the possible synergism and/or antagonism between major constituents in harpagoside-containing phytopharmaceutical products. Finally, future perspectives for its potential application are highlighted.

Inflammatory response in patients with active and inactive osteoarthritis

In the present study, we have investigated comparatively the inflammatory response of patients with active and inactive osteoarthritis. The sera from 31 healthy individuals, 37 patients with active OA, and 19 patients with inactive OA were assayed for TNF-α, IL-6, sRANKL, RANTES, and MRP8 using ELISA in order to evaluate their potential as markers of disease activity. Also, the spontaneous and LSP-induced release of TNF-α and IL-6 by peripheral blood neutrophils was determined. The activation of OA is associated with elevated TNF-α, IL-6, and RANTES serum levels while sRANKL and MRP8 appeared to be increased in both active and inactive OA. The neutrophil spontaneous and up-regulated by LPS cytokine release can contribute to the exacerbation of OA.

Abrogated RANKL expression in properdin-deficient mice is associated with better outcome from collagen-antibody-induced arthritis

IntroductionProperdin amplifies the alternative pathway of complement activation. In the present study, we evaluated its role in the development of collagen antibody-induced arthritis (CAIA).MethodsArthritis was induced by intraperitoneal injection of a collagen antibody cocktail into properdin-deficient (KO) and wild-type (WT) C57BL/6 mice. Symptoms of disease were evaluated daily. The degree of joint damage was assessed histologically and with immunostaining for bone-resorption markers. Phenotypes of cell populations, their receptor expression, and intracellular cytokine production were determined with flow cytometry. Osteoclast differentiation of bone marrow (BM) precursors was evaluated by staining for tartrate-resistant acid phosphatase (TRAP).ResultsProperdin-deficient mice developed less severe CAIA than did WT mice. They showed significantly improved clinical scores and downregulated expression of bone-resorption markers in the joints at day 10 of disease. The frequencies of Ly6G+CD11b+ cells were fewer in BM, blood, and synovial fluid (SF) of KO than of WT CAIA mice. The receptor activator of nuclear factor κB ligand (RANKL) was downregulated on arthritic KO neutrophils from BM and the periphery. Decreased C5a amounts in KO SF contributed to lower frequencies of CD5aR+-bearing neutrophils. In blood, surface C5aR was detected on KO Ly6G+ cells as a result of low receptor engagement. Circulating CD4+ T cells had an altered ability to produce interleukin (IL)-17 and interferon (IFN)-γ and to express RANKL. In KO CAIA mice, decreased frequencies of CD4+ T cells in the spleen were related to low CD86 expression on Ly6GhighCD11b+ cells. Arthritic KO T cells spontaneously secreted IFN-γ but not IL-17 and IL-6, and responded to restimulation with less-vigorous cytokine production in comparison to WT cells. Fewer TRAP-positive mature osteoclasts were found in KO BM cell cultures.ConclusionsOur data show that the active involvement of properdin in arthritis is related to an increased proinflammatory cytokine production and RANKL expression on immune cells and to a stimulation of the RANKL-dependent osteoclast differentiation.

Bone resorption and remodeling in murine collagenase-induced osteoarthritis after administration of glucosamine.

IntroductionGlucosamine is an amino-monosaccharide and precursor of glycosaminoglycans, major components of joint cartilage. Glucosamine has been clinically introduced for the treatment of osteoarthritis but the data about its protective role in disease are insufficient. The goal of this study was to investigate the effect of long term administration of glucosamine on bone resorption and remodeling.MethodsThe effect of glucosamine on bone resorption and remodeling was studied in a model of collagenase-induced osteoarthritis (CIOA). The levels of macrophage-inflammatory protein (MIP)-1α, protein regulated upon activation, normal T-cell expressed, and secreted (RANTES), soluble receptor activator of nuclear factor kappa-B ligand (RANKL), tumor necrosis factor (TNF)-α, and interleukin (IL)-6, 4 and 10 in synovial fluid were measured by enzyme-linked immunosorbent assay (ELISA). Cell populations in synovial extracts and the expression of RANKL, of receptors for TNF-α (TNF-αR) and interferon γ (IFN-γR) on clusters of differentiation (CD) three positive T cells were analyzed by flow cytometry. Transforming growth factor (TGF)-β3, bone morphogenetic protein (BMP)-2, phosphorylated protein mothers against decapentaplegic homolog 2 (pSMAD-2), RANKL and Dickkopf-1 protein (DKK-1) positive staining in CIOA joints were determined by immunohistochemistry.ResultsThe administration of glucosamine hydrochloride in CIOA mice inhibited loss of glycosaminoglycans (GAGs) and proteoglycans (PGs) in cartilage, bone erosion and osteophyte formation. It decreased the levels of soluble RANKL and IL-6 and induced IL-10 increase in the CIOA joint fluids. Glucosamine limited the number of CD11b positive Ly6G neutrophils and RANKL positive CD3 T cells in the joint extracts. It suppressed bone resorption via down-regulation of RANKL expression and affected bone remodeling in CIOA by decreasing BMP-2, TGF-β3 and pSMAD-2 expression and up-regulating DKK-1 joint levels.ConclusionsOur data suggest that glucosamine hydrochloride inhibits bone resorption through down-regulation of RANKL expression in the joints, via reduction of the number of RANKL positive CD3 T cells and the level of sRANKL in the joints extracts. These effects of glucosamine appear to be critical for the progression of CIOA and result in limited bone remodeling of the joints.

Properdin Deficiency in Murine Models of Nonseptic Shock

Hereditary properdin deficiency is linked to susceptibility to meningococcal disease (Neisseria meningitidis serotypes Y and W-135) with high mortality. Its relative contribution toward the outcome of nonseptic shock has not been investigated. Using properdin-deficient C57BL/6 mice and their littermates, this study examines their survival of zymosan-induced and LPS-induced shock. Properdin-deficient mice were more resistant to zymosan shock compared with wild-type mice, which showed greater impairment of end-organ function 24 h after zymosan injection, higher TNF-α production by alveolar and peritoneal macrophages, higher TNF-α, and, inversely, lower IL-10 levels in peritoneal lavage and circulation and higher plasma C5a levels. Properdin-deficient mice showed significantly higher mortality in LPS shock, elevated TNF-α, and, inversely, reduced IL-10 production by peritoneal macrophages as well as lower plasma C5a levels compared with wild-type littermates. NO production by peritoneal macrophages and plasma α1-antitrypsin levels at 24 h after the injection of LPS or zymosan were decreased in properdin-deficient mice in both models, and fewer histopathologic changes in liver were observed in properdin-deficient animals. This study provides evidence that properdin deficiency attenuates zymosan-induced shock and exacerbates LPS-induced shock.

Oxidative stress and chronic inflammation in osteoarthritis: can NRF2 counteract these partners in crime?

Osteoarthritis (OA) is an age‐related joint degenerative disease associated with pain, joint deformity, and disability. The disease starts with cartilage damage but then progressively involves subchondral bone, causing an imbalance between osteoclast‐driven bone resorption and osteoblast‐driven remodeling. Here, we summarize the data for the role of oxidative stress and inflammation in OA pathology and discuss how these two processes are integrated during OA progression, as well as their contribution to abnormalities in cartilage/bone metabolism and integrity. At the cellular level, oxidative stress and inflammation are counteracted by transcription factor nuclear factor erythroid p45–related factor 2 (NRF2), and we describe the regulation of NRF2, highlighting its role in OA pathology. We also discuss the beneficial effect of some phytonutrients, including the therapeutic potential of NRF2 activation, in OA.

TLR2 elicits IL-17-mediated RANKL expression, IL-17, and OPG production in neutrophils from arthritic mice.

We investigated the ability of neutrophils to express receptor activator of nuclear factor kappa-B ligand (RANKL), to secrete osteoprotegerin (OPG), and to produce IL-17. Arthritis was induced by intra-articular injection of zymosan, a ligand for Toll-like receptor 2 (TLR2). Frequencies of neutrophils in bone marrow (BM), blood and synovial fluid (SF), receptor expression, and cytokine production were evaluated by flow cytometry. 1A8 antibody (1A8 Ab) was used to deplete neutrophils in zymosan-injected SCID mice. IL-17, RANKL, and OPG amounts in SF, serum, or cell cultures were determined by ELISA. The development of arthritis was associated with increased secretion of IL-17, RANKL, and OPG in serum and SF, elevated frequencies of Ly6G+CD11b+ cells in BM, blood, and SF and upregulated RANKL expression. Both IL-17 and OPG were absent in serum and SF after neutrophil depletion; therefore we assume that they were released by neutrophils. In vitro blood Ly6G+CD11b+ cells from arthritic mice produced spontaneously IL-17, IFN-γ, and OPG and expressed RANKL. This phenotype was sustained by IL-17. TLR2 engagement increased IL-17 and IFN-γ production, potentiated IL-17-mediated RANKL expression, and inhibited OPG secretion. We conclude that TLR2 regulates the destructive potential of neutrophils and its targeting might limit joint alterations in arthritis.

Tyrphostin AG-490 inhibited the acute phase of zymosan-induced inflammation.

Tyrphostins, derivatives of benzylidene malononitrile are recognized as tyrosine kinase inhibitors that have been applied in some models of acute inflammatory conditions, like LPS and zymosan-induced shock. In the present study, we have investigated the effects of tyrphostin AG-490, on the development of multiple organ failure induced by i.p. injection of zymosan (1 mg/g body weight) in mice. Organ dysfunction and systemic inflammation was estimated 24 h after zymosan administration. Treatment of mice with AG-490 (dose, 5 mg/kg i.p. simultaneously with zymosan) decreased the number of cells and the level of NO in the peritoneal lavage. The substance attenuated the elevation of creatinine (indicator of renal failure), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and bilirubin (indicators for liver dysfunction) and prevented the accelerated coagulation time. The injection of zymosan resulted in a substantial increase in the serum level of TNF-alpha and IL-6, which was strongly inhibited by AG-490. Tyrphostin abolished the expression of iNOS and TNF-alphaR in the liver. Moreover, immunohistochemistry of liver showed decreased phosphorylation of Stat1 and Stat3. In conclusion, the administration of tyrphostin AG-490 in zymosan-induced nonseptic shock significantly improved the rate of survival and lead to less exerted signs of multiple organ failure.

Antiinflammatory properties of extracts and compounds isolated from Verbascum xanthophoeniceum Griseb.

Verbascum xanthophoeniceum Griseb. is an endemic plant of the Balkan region, a representative of the genus Verbascum used in traditional medicine for respiratory and inflammatory disorders. The objective of this study was to evaluate in vivo and in vitro the antiinflammatory action of crude extract, different fractions and pure compounds obtained from V. xanthophoeniceum Griseb. Bioactive metabolites were isolated by the use of low‐pressure chromatographic separation. Crude methanol extract (CME) was applied in a model of paw oedema and different fractions and substances were tested in vitro for their effect on NO and cytokine production by peritoneal macrophages, and on the COX‐1 and COX‐2 expression. The CME exerted inhibition on cobra venom factor (CVF)‐induced oedema in mice, in correlation with reduced alternative pathway (AP) complement activity. A highly suppressive effect was expressed by nigroside VI on IL‐6 and NO production and by forsythoside B on TNF‐α production. Leucosceptoside B lowered NO release and COX‐1 expression in macrophages. Verbascum xanthophoeniceum could serve as a promising source of active compounds with antiinflammatory action, particularly in complement‐mediated disorders. Copyright