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ID PMID Title PublicationDate abstract
18952102 Long-range enhancer differentially regulated by c-Jun and JunD controls peptidylarginine d 2008 Dec 31 Long-range cis elements are critical regulators of transcription, particularly for clustered paralogous genes. Such are the five PADI genes in 1p35-36 encoding peptidylarginine deiminases, which catalyze deimination, a Ca2+-dependent post-translational modification. Deimination has been implicated in the pathophysiology of severe human diseases such as multiple sclerosis and rheumatoid arthritis. The PADI genes present different expression patterns. PADI1-3 are expressed in the epidermis, with increased expression levels in the most differentiated keratinocytes. Previous studies on PADI proximal promoters failed to explain such specificity of expression. We identified a conserved intergenic sequence in the PADI locus (IG1), which may play a role in PADI transcriptional regulation. In this work, we identified two DNase I.hypersensitive sites located in IG1, PAD intergenic enhancer segment 1 (PIE-S1) and PIE-S2, which act in synergy as a bipartite enhancer of the PADI3 and probably PADI1 promoters in normal human epidermal keratinocytes differentiated by a high-calcium-containing medium (1.5 mM). PIE-S1 and PIE-S2 present all the hallmarks of transcriptional enhancers: orientation-independence, copy-number dependence and cell-type specificity. PIE-S1 and PIE-S2 comprise conserved putative binding sites for MIBP1/RFX1 and activator protein 1, respectively. Deletion mutant screening revealed that these sites are crucial for the enhancer activity. Furthermore, chromatin immunoprecipitation assays evidenced differential binding of JunD or c-Jun on the activator protein 1 site depending on the cell differentiation state. Our results reveal the molecular bases of the expression specificity of PADI1 and PADI3 during keratinocyte differentiation through a long-range enhancer and support a model of PADI gene regulation depending on c-Jun-JunD competition.
18778114 A PEGylated Fab' fragment against tumor necrosis factor for the treatment of Crohn disease 2008 Antibodies, having a high specificity for their particular target, are increasingly being used as therapeutic agents with functions including agonist, antagonist, and targeted drug delivery. The use of many biologic therapies, including antibody fragments, is generally limited by their rapid clearance from plasma. A commonly used approach to extend exposure to biologic therapies is the attachment of polyethylene glycol.Tumor necrosis factor (TNF)-alpha is a multifunctional cytokine involved in the regulation of immune responses. Elevated levels of TNFalpha are found in a wide range of diseases, including the chronic inflammatory conditions rheumatoid arthritis, psoriasis, and Crohn disease (CD). Anti-TNFalpha antibodies have proved highly efficacious in the treatment of these conditions. In addition, they have proved invaluable for investigating the role of TNFalpha in disease etiology. Based on evidence showing that neutralizing antibodies to TNFalpha were effective in animal models of CD, anti-TNFalpha antibody treatments were assessed in clinical trials. Interestingly, the anti-TNFalpha antibody etanercept proved ineffective at achieving remission in active CD despite potently neutralizing soluble TNFalpha. This indicated that an additional mode of action is also involved in the efficacy of the anti-TNFalpha agents adalimumab, certolizumab pegol, and infliximab in CD; one suggestion was apoptosis. However, etanercept, like adalimumab and infliximab, can induce apoptosis. Furthermore, certolizumab pegol (which has demonstrated efficacy in CD) does not cause complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, apoptosis, or necrosis of neutrophils, all measured in vitro. These functional differences observed with certolizumab pegol stem from its unique structure that does not include the crystallizable fragment (Fc) portion present in the other anti-TNFalpha agents, and the way in which it signals through membrane TNF. It is well established that bacteria are a major part of the inflammatory process in CD. The property identified that reflected the efficacies of the anti-TNFalpha agents etanercept, adalimumab, certolizumab pegol, and infliximab in CD was the ability to inhibit the cytokine production by monocytes that is induced by bacterial lipopolysaccharide. It may therefore be the case that this mode of action is important for efficacy in CD.
17579088 Apis mellifera venom and melittin block neither NF-kappa B-p50-DNA interactions nor the ac 2007 Jul 1 Many alternative treatment approaches, originating from Asia, are becoming increasingly popular in the Western hemisphere. Recently, an article published in a renowned journal reported that venom of apis mellifera (bee venom (BV)) and melittin mediate immune-modulating effects by blocking the activation of the transcription factor NF-kappaB. Such a modus operandi would corroborate the many claims of beneficial effects of BV treatment and give immediate credit to this form of therapy. Fibroblast-like synoviocytes from rheumatoid arthritis patients and dermal fibroblast cells and white blood cells from healthy volunteers were used to study the effects of BV and melittin on the activation of NF-kappaB and a series of genes that are markers of inflammation. EMSAs demonstrate that neither BV nor melittin blocked IL-1beta-induced NF-kappaB activation; neither did they affect phosphorylation or degradation of IkappaB. Contrary to published data, even high concentrations of BV and melittin were without any effect on NF-kappaB-p50-DNA interactions. More importantly, in fibroblast-like synoviocytes, but also in dermal fibroblasts as well as in mononuclear cells exposed to BV or melittin, mRNA levels of several proinflammatory genes are significantly increased, and Western blot data show elevated cyclooxygenase-2 protein levels. Furthermore, exposure to BV higher than 10 mug/ml resulted in disintegration of all cell types tested. In addition, large quantities of oxygen radicals are produced in a dose-dependent manner in leukocytes exposed to BV. Taken together, data presented in this work do not corroborate an earlier report regarding the effectiveness of BV as an inhibitor of the transcription factor NF-kappaB.
17538777 Effect of non-steroidal anti-inflammatory medications on the risk of amyotrophic lateral s 2007 Jun Inflammatory processes may be involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). We examined the association of non-steroidal anti-inflammatory drugs (NSAIDs) with the risk of ALS in case-control study of incident cases (n = 111) conducted within the Kaiser Permanente Medical Care Program of Northern California during the years 1996-2000. Controls (n = 258) randomly selected from the same population were frequency matched by age and gender to the ALS cases. Information regarding use of NSAIDs (non-aspirin and aspirin) and three classes of 'control' medications was collected by in-person structured interview. Subjects who used medication at least twice a week for at least a month were classified as 'ever users'. Multivariable logistic regression models were adjusted for age, gender, history of osteoarthritis/rheumatoid arthritis and pain, and other medication use. Overall, there was no association between NSAID use and ALS; however, some sex differences were noted for non-aspirin NSAID use. Among men, non-aspirin NSAID use was associated with a two-fold increased risk of ALS (adjusted odds ratio (OR) 2.0, 95% confidence interval (CI) 1.0-3.9), whereas among women, non-aspirin NSAID use was not associated with increased ALS risk (adjusted OR 0.5, 95% CI 0.2-1.2). ALS risk was not associated with aspirin use or with 'control' medications. This study did not find any evidence to suggest that NSAID use reduces the risk of ALS. The observed sex differences with non-aspirin NSAID use could be due to chance or an unmeasured confounder.
18172821 An efficient and economic high-throughput cell screening model targeting the glucocorticoi 2008 Jan AIM: To discover compounds or proteins that can efficiently bind the glucocorticoid receptor (GR) and trigger the transcription of target genes, resulting in clinical improvement of diseases such as rheumatoid arthritis, asthma, inflammatory bowel disease, a high-throughput drug screening cell model using green fluorescent protein 4 (GFP4) as a marker expressed in response to GR activation has been established and evaluated. METHODS: Eight repeats of the glucocorticoid response element (GRE) were cloned into the Peak12SxSynGFP4 vector, and the resulting recombinant plasmid Peak12GRE8 x SxSynGFP4 was stably transfected into the 293E cells. The stable and sensitive cell line 293E/GRE8 x /GFP4 was selected by dexamethasone (DEX) using fluorescent microscopy and fluorescence-activated cell sorting. DEX induction and phorbol myristate acetate (PMA) inhibition of the green fluorescence intensity of the cell line were tested. RESULTS: The expression of GFP4 in the cell line was under the control of GRE, up-regulated by DEX treatment and down-regulated by phorbol myristate acetate (PMA). The up-regulation of the GFP4 expression was DEX concentration-dependent, with an EC(50) at approximately 5 x 10(- 8) M. The down-regulation of the GFP4 expression was phorbol myristate acetate (PMA) concentration-dependent, with an IC(50) at approximately 3 x 10(- 6) gl - 1. The expression of GFP4 was effectively activated when cells were treated with triamcinolone acetonide. CONCLUSION: This drug screening cell line can be used for GR-targeted high-throughput drug screening for the treatment of inflammatory diseases.
17920527 Inhibitory effects of Stewartia koreana on osteoclast differentiation and bone resorption. 2007 Dec 5 Osteoclasts are responsible for bone lysis in several bone diseases such as osteoporosis and rheumatoid arthritis. Natural products from plants have been invaluable source in discovery of compounds for new therapies. In this study, we screened plant products for potential application to therapy for bone loss using a primary osteoclastogenesis culture system and found that extract of Stewartia koreana (SKE) had a strong inhibitory effect on osteoclast formation. To gain molecular insights, we examined the effect of SKE on signaling pathways and transcription factors stimulated by the osteoclast differentiation factor RANKL. SKE suppressed the induction of c-Fos and NFATc1 by RANKL. However, SKE did not inhibit NF-kappaB activation by RANKL. Among the MAPKs stimulated by RANKL, SKE significantly reduced the activation of ERK and p38. Therefore, the anti-osteoclastogenic effect of SKE is likely to be elicited by interference with RANKL signaling to ERK and p38, which mediate the induction of c-Fos and subsequently that of NFATc1. Consistent with the in vitro effect on osteoclast differentiation, SKE showed a great inhibitory effect on in vivo bone loss in LPS-challenged mice. Taken together, we demonstrated that SKE has inhibitory effects on osteoclast differentiation in vitro and confirmed its in vivo efficacy in prevention of inflammatory bone loss.
17912515 Are radiographs needed when MR imaging is performed for non-acute knee symptoms in patient 2007 Dec OBJECTIVE: The objective was to determine the value of radiographs in young adults with non-acute knee symptoms who are scheduled for magnetic resonance imaging (MRI). MATERIALS AND METHODS: Nine hundred and sixty-one consecutive patients aged between 16 and 45 years with knee symptoms of at least 4 weeks' duration were prospectively included in three participating hospitals. After applying exclusion criteria, 798 patients remained. Exclusion criteria were previous knee surgery (including arthroscopy) or MRI, history of rheumatoid arthritis, clinical diagnosis of retropatellar chondromalacia, contra-indication for MRI and recent trauma. We identified two groups: group A with no history of trauma (n = 332), and group B with an old (>4 weeks) history of trauma (n = 466). Patients had a standardized history taken, and underwent a physical exam, antero-posterior (AP) and lateral radiographs and MRI. We evaluated the radiographs and MRI for osseous lesions, articular surface lesions, fractures, osteoarthritis, loose bodies, bone marrow edema and incidental findings. Subsequently, patients with osseous abnormalities (Kellgren grade 1 and 2 excluded) on radiographs and a matched control group was evaluated again using MRI without radiographs. RESULTS: Median duration of symptoms was 20 weeks. In group A, radiographs showed 36 osseous abnormalities in 332 patients (10.8%). Only 13 of these, all Kellgren grade 1 osteoarthritis, were not confirmed on MRI. MRI showed 72 (21.7%) additional abnormalities not confirmed on radiographs. In group B, radiographs showed 40 osseous abnormalities (8.6%) in 466 patients. Only 15 of these, all Kellgren grade 1 osteoarthritis, were not confirmed on MRI. MRI showed 194 (41.6%) additional abnormalities not confirmed on radiographs. The second evaluation of MRI without radiographs in 34 patients was identical to the first MRI evaluation. Common lesions were significantly more often diagnosed with MRI than with radiographs. CONCLUSION: Radiographs should not be obtained routinely when MRI is being performed in young adults with non-acute knee complaints because the yield and added value to MRI are low.
17353164 Effects of tumor necrosis factor antagonist treatment on hepatitis C-related immunological 2006 Dec BACKGROUND: Chronic hepatitis C infection is frequently associated with a mixed cryoglobulinaemia and circulating auto-antibodies, especially anti-smooth muscle cells (SMA) and anti-liver/kidney/microsome type 1 (LKM-1) anti-tissue antibodies. Treatments with TNF antagonists favour the emergence of auto-antibodies, and particularly anti-dsDNA antibodies. OBJECTIVE: To determine the impact of TNF antagonists on hepatitis C-related immune abnormalities. METHODS: We prospectively monitored for 14 weeks, six patients with actively replicating chronic hepatitis C, initiating an anti-TNF treatment for an associated rheumatoid arthritis. RESULTS: Anti-nuclear and anti-dsDNA antibodies were induced in two and three patients, respectively. Treatment had no impact on the production of antibodies against extractable nuclear antigens, and it did not induce anti-tissues antibodies in any patient. Cryoglobulinaemia appeared in 2/6 patients, and it persisted in 2 others. No patient developed any news signs of autoimmunity. HCV viraemia remained unchanged. CONCLUSIONS: Induction of auto-antibodies by TNF antagonist treatments does not involve anti-tissues antibodies, even in patients with actively replicating chronic hepatitis C prone to produce anti-SMA and anti-LKM-1 antibodies. In contrast, TNF antagonists may favour emergence of cryoglobulinaemia in such patients.
17330802 Interleukin-18 induces apoptosis in human articular chondrocytes. 2007 May Elevated levels of the pro-inflammatory cytokine, interleukin-18 (IL-18) have recently been demonstrated in osteoarthritic cartilage. However, the effects of IL-18 on chondrocyte signalling and matrix biosynthesis are poorly understood. Therefore, the present study was undertaken to further characterize the impact of IL-18 on human articular chondrocyte in vitro. Human articular chondrocytes were stimulated with various concentrations of recombinant human IL-18 (1, 10, 100 ng/ml) for 0, 4, 8, 12, 24, 48, 72 h in vitro. The effects of IL-18 on the cartilage-specific matrix protein collagen type II, the cytoskeletal protein vinculin, the cell matrix signal transduction receptor beta-integrin, key signalling proteins of the MAPKinase pathway (such as SHC (Sarc Homology Collagen) and activated MAPKinase [ERK-1/-2]), the pro-inflammatory enzyme cyclo-oxygenase-2 (COX-2) and the apoptosis marker activated caspase-3 were evaluated by Western blot analysis and immunofluorescence labelling. Morphological features of IL-18 stimulated chondrocytes were estimated by transmission electron microscopy. IL-18 lead to inhibition of collagen type II-deposition, decreased beta-integrin receptor and vinculin synthesis, SHC and MAPKinase activation, increased COX-2 synthesis and activation of caspase-3 in chondrocytes in a time- and dose-dependent manner. Furthermore, chondrocytes treated with IL-18 exhibited typical morphological features of apoptosis as revealed by transmission electron microscopy. Taken together, the results of the present study underline key catabolic events mediated by IL-18 signalling in chondrocytes such as loss of cartilage-specific matrix and apoptosis. Inhibition of MAPKinase signalling is hypothesized to contribute to these features. Future therapeutics targeting IL-18 signalling pathways may be beneficial in rheumatoid arthritis and osteoarthritis therapy.
17051397 Atlantoaxial fixation using the polyaxial screw-rod system. 2007 Apr The aim of this study is to evaluate the first results of the atlantoaxial fixation using polyaxial screw-rod system. Twenty-eight patients followed-up 12-29 months (average 17.1 months) were included in this study. The average age was 59.5 years (range 23-89 years). The atlantoaxial fusion was employed in 20 patients for an acute injury to the upper cervical spine, in 1 patient with rheumatoid arthritis for atlantoaxial vertical instability, in 1 patient for C1-C2 osteoarthritis, in 2 patients for malunion of the fractured dens. Temporary fixation was applied in two patients for type III displaced fractures of the dens and in two patients for the atlantoaxial rotatory dislocation. Retrospectively, we evaluated operative time, intraoperative bleeding and the interval of X-ray exposure. The resulting condition was subjectively evaluated by patients. We evaluated also the placement, direction and length of the screws. Fusion or stability in the temporary fixation was evaluated on radiographs taken at 3, 6, 12 weeks and 6 and 12 months after the surgery. As concerns complications, intraoperatively we monitored injury of the nerve structures and the vertebral artery. Monitoring of postoperative complications was focused on delayed healing of the wound, breaking or loosening of screws and development of malunion. Operative time ranged from 35 to 155 min, (average 83 min). Intraoperative blood loss ranged from 50 to 1,500 ml (average 540 ml). The image intensifier was used for a period of 24 s to 2 min 36 s (average 1 min 6 s). Within the postoperative evaluation, four patients complained of paresthesia in the region innervated by the greater occipital nerve. A total of 56 screws were inserted into C1, their length ranged from 26 to 34 mm (average, 30.8 mm). All screws were positioned correctly in the C1 lateral mass. Another 56 screws were inserted into C2. Their length ranged from 28 to 36 mm (average 31.4 mm). Three screws were malpositioned: one screw perforated the spinal canal and two screws protruded into the vertebral artery canal. C1-C2 stability was achieved in all patients 12 weeks after the surgery. No clinically manifested injury of the vertebral artery or nerve structures was observed in any of these cases. As for postoperative complications, we recorded wound dehiscence in one patient. The Harms C1-C2 fixation is a very effective method of stabilizing the atlantoaxial complex. The possibility of a temporary fixation without damage to the atlantoaxial joints and of reduction after the screws and rods had been inserted is quite unique.
16855176 Impact of thiopurine methyltransferase activity and 6-thioguanine nucleotide concentration 2006 Jun As azathioprine is one of the standard immunosuppressive drugs used for treatment of patients with different chronic inflammatory diseases, the effect of the azathioprine metabolizing enzyme thiopurine methyltransferase (TPMT) activity on incidence of adverse events (AE) was examined. In addition, potential correlations between the concentration of the azathioprine metabolite 6-thioguanine nucleotide (6-TGN) in erythrocytes (RBC) and inflammatory disease activity as well as hematological AE were investigated. TPMT activities were investigated prospectively in 139 patients (35 male, 104 female) with chronic inflammatory diseases [systemic lupus erythematosus (SLE, 38), progressive systemic sclerosis (PSS, 13), Wegener's granulomatosis (4), rheumatoid arthritis (RA, 5), and other chronic inflammatory diseases (79)]. In addition, 6-TGN concentrations were investigated in a second cohort of 58 patients (17 patients with SLE, 5 with PSS, 5 with vasculitides, 4 with undifferentiated connective tissue diseases, 1 with dermatomyositis, 1 with Sjögren's syndrome, 1 with RA, 20 with Crohn's disease, and 4 with ulcerative colitis) prior to and during therapy with azathioprine. The distribution of activities of TPMT in 139 patients showed a normal Gaussian distribution in the Caucasian population. Within the group of 96 patients taking azathioprine, known azathioprine-related AE could be observed: minor AE (sickness, rash, and increase in cholestasis parameters) in 11 patients (11.4%), and severe AE (bone marrow toxicity) in 7 patients (7.3%). Below a "cutoff" value of 11.9 nmol/mL RBC x h of TPMT activity, AE were significantly more frequent. In the second cohort of patients, no significant correlations could be observed between 6-TGN concentrations and parameters of disease activity. Reduced activity of TPMT in patients with chronic inflammatory diseases requiring immunosuppressive therapy with azathioprine, especially below a distinct cutoff, appears to inherit a substantial risk for development of AE.
16398566 Role of Toll-like receptors in infection and immunity: clinical implications. 2006 The remarkable discovery of the Toll-like receptors (TLRs) over the past 5 years has opened up an entirely new era in the understanding of the molecular events that initiate the inflammatory response. These type 1 transmembrane receptors are expressed on a large number of immune cells as well as epithelial cells and play an essential role in the activation of the innate immune response to microbial pathogens. They impact on adaptive immune reactions and contribute to the initiation and maintenance of the inflammatory response to a multitude of potential microbial pathogens through recognition of pathogen-associated molecular patterns. TLRs also interact with a variety of endogenous human ligands and influence the activity of a wide range of tissues and cell processes. Among the common and important processes in which TLRs play a role are asthma, acute respiratory distress syndrome, cardiac ischaemia, coronary artery disease, ventricular remodelling, vascular collapse, inflammatory bowel disease, acute tubular necrosis, psoriasis, rheumatoid arthritis, pre-term birth, fertility, cancer angiogenesis and transplant rejection. From this strikingly diverse list, many important opportunities for disease modification through TLR manipulation can be imagined. Their role as potential targets for therapeutic intervention is just beginning to be appreciated, and the current status of these treatment strategies is reviewed in this article.
18831740 Obesity and osteoarthritis in knee, hip and/or hand: an epidemiological study in the gener 2008 Oct 2 BACKGROUND: Obesity is one of the most important risk factors for osteoarthritis (OA) in knee(s). However, the relationship between obesity and OA in hand(s) and hip(s) remains controversial and needs further investigation. The purpose of this study was to investigate the impact of obesity on incident osteoarthritis (OA) in hip, knee, and hand in a general population followed in 10 years. METHODS: A total of 1854 people aged 24-76 years in 1994 participated in a Norwegian study on musculoskeletal pain in both 1994 and 2004. Participants with OA or rheumatoid arthritis in 1994 and those above 74 years in 1994 were excluded, leaving n = 1675 for the analyses. The main outcome measure was OA diagnosis at follow-up based on self-report. Obesity was defined by a body mass index (BMI) of 30 and above. RESULTS: At 10-years follow-up the incidence rates were 5.8% (CI 4.3-7.3) for hip OA, 7.3% (CI 5.7-9.0) for knee OA, and 5.6% (CI 4.2-7.1) for hand OA. When adjusting for age, gender, work status and leisure time activities, a high BMI (> 30) was significantly associated with knee OA (OR 2.81; 95%CI 1.32-5.96), and a dose-response relationship was found for this association. Obesity was also significantly associated with hand OA (OR 2.59; 1.08-6.19), but not with hip OA (OR 1.11; 0.41-2.97). There was no statistically significant interaction effect between BMI and gender, age or any of the other confounding variables. CONCLUSION: A high BMI was significantly associated with knee OA and hand OA, but not with hip OA.
20641803 (99m)Tc-Ethylenediaminediacetic acid/hydrazinonicotinic acid-cyclo(Arg-Gly-Asp-D-Try-Lys). 2004 Integrins are a family of heterodimeric glycoproteins on cell surfaces that mediate diverse biological events involving cell–cell and cell–matrix interactions (1). Integrins consist of an α and a β subunit and are important for cell adhesion and signal transduction. The α(v)β(3) integrin is the most prominent receptor affecting tumor growth, tumor invasiveness, metastasis, tumor-induced angiogenesis, inflammation, osteoporosis, and rheumatoid arthritis (2-7). Expression of the α(v)β(3) integrin is strong on tumor cells and activated endothelial cells, whereas expression is weak on resting endothelial cells and most normal tissues. Antagonists of α(v)β(3) are being studied as antitumor and antiangiogenic agents, and the agonists of α(v)β(3) are being studied as angiogenic agents for coronary angiogenesis (6, 8, 9). A tripeptide sequence consisting of Arg-Gly-Asp (RGD) has been identified as a recognition motif used by extracellular matrix proteins (vitronectin, fibrinogen, laminin, and collagen) to bind to a variety of integrins, including α(v)β(3). Various radiolabeled antagonists have been introduced for imaging of tumors and tumor angiogenesis (10). Most cyclic RGD peptides are composed of five amino acids. Haubner et al. (11) reported that various cyclic RGD peptides exhibit selective inhibition of binding to α(v)β(3) (inhibition concentration (IC(50)), 7–40 nM) but not to integrins α(v)β(5) (IC(50), 600–4,000 nM) or α(IIb)β(3) (IC(50), 700–5,000 nM). Various radiolabeled cyclic RGD peptides have been found to have high accumulation in tumors in nude mice (12). Hydrazinonicotinic acid (HYNIC) is a coupling agent for (99m)Tc labeling of peptides that can achieve high specific activities without affecting the receptor-binding ability of the amino acid sequence. Decristoforo et al. (13) reported the success of radiolabeling cyclo(Arg-Gly-Asp-D-Tyr-Lys) (c(RGDyK)) by conjugation of HYNIC to the ε amino group of Lys with ethylenediaminediacetic acid (EDDA) and tris(hydroxymethyl)-methylglycine (tricine) as coligand. The (99m)Tc-EDDA/HYNIC-c(RGDyK) peptide showed high tumor accumulation in nude mice bearing M21 human melanoma tumors and A549 human lung carcinoma tumors.
18583119 Novel leads from Heliotropium ovalifolium, 4,7,8-trimethoxy-naphthalene-2-carboxylic acid 2008 Dec From our screening program, we identified the anti-inflammatory effects of the extracts of Heliotropium ovalifolium in its ability to inhibit specific cytokines. The H. ovalifolium extract was found to be moderately active with an IC(50) equaling 10 microg/ml for inhibition of interleukin-6 (IL-6) in a human monocytic cell line. Interleukin-6 is a pleiotropic cytokine with implications in the regulation of the immune response, inflammation and hematopoiesis. This prompted us to examine and identify the active molecules that are responsible for the bioactivity in THP-1 cells. Bioassay guided fractionation identified two compounds 4,7,8-trimethoxy-naphthalene-2-carboxylic acid and 6-hydroxy-5,7-dimethoxy-naphthalene-2-carbaldehyde with an IC(50) of 2.4 and 2.0 microM for IL-6 inhibition and an IC(50) of 15.6 and 7.0 microM for tumor necrosis factor-alpha (TNF-alpha) inhibition in THP-1 cells. The protein expression data were supported by the inhibitory effect on mRNA gene expression. The compounds isolated from H. ovalifolium were also non-toxic in human peripheral blood monocytes from normal donors and the activity profile was similar to that obtained on THP-1 cells. Thus, we believe that these scaffolds may be of interest to develop leads for treating rheumatoid arthritis, psoriasis, ulcerative colitis, Crohn's disease and other inflammatory disorders. However, more detailed investigations need to be carried out to explain the efficacy of these compounds as drugs.
18573345 Non-invasive, ultra-sensitive, high-throughput assays to quantify rare biomarkers in the b 2008 Sep Many diseases are easier to treat and control when detected at an early stage of disease progression. Often, disease-related antigens or biomarkers are shed from the primary site and present in the blood. Unfortunately, there are very few tests capable of detecting these rare biomarkers in the blood. A blood test would be very useful to diagnose the disease earlier, monitor effectiveness of treatments, predict recurrence, and monitor recurrence. There is certainly a need to develop assays that are ultra-sensitive, non-invasive, and high-throughput. Here we describe several highly sensitive immunological assays we have developed to detect rare serum antigens. Initially we created an assay named immuno-detection amplified by T7 RNA polymerase (IDAT). To enhance the effectiveness and streamline the procedure, this assay was amended to the facile amplification system termed fluorescent amplification catalyzed by T7 polymerase technique (FACTT). These assays have been used to analyze the tumor antigen HER2 and the prion protein PrPSc. They can also be applied to other tumor markers or antigens from a variety of diseases such as cardiovascular disease, rheumatoid arthritis, Alzheimer's disease, Parkinson's disease, and hepatitis. These tests are not limited to testing only serum, but may also be applicable to detecting biomarkers in tissue, saliva, urine, cerebrospinal fluid, etc. Clearly, the FACTT-based technology represents an important step in the detection of rare molecules in fluids or tissues for a variety of diseases.
17602748 Reverse signaling initiated from GITRL induces NF-kappaB activation through ERK in the inf 2008 Jan Glucocorticoid-induced TNF receptor family related protein ligand (GITRL) is known to interact with its cognate receptor GITR. In order to investigate the potential role of GITRL in the pro-inflammatory activation of macrophages and the signaling pathway induced by GITRL, we stimulated the macrophage cell line, THP-1, and primary macrophages with an anti-GITRL monoclonal antibody or a GITR:Fc fusion protein and analyzed the cellular responses. The stimulation of GITRL induced the expression of pro-inflammatory cytokines and matrix metalloproteinase (MMP)-9 and up-regulated ICAM-1 expression levels, which was responsible for enhanced cellular aggregation and adhesion to extracellular matrix proteins. The activation of these pro-inflammatory mediators required the activation of ERK1/2 mitogen-activated protein kinase (MAPK) and negatively regulated by p38 MAPK and JNK. Immunofluorescence analysis detected nuclear translocation of the NF-kappaB p50 subunit and this was blocked by ERK inhibitor, indicating that GITRL stimulation induced ERK1/2 phosphorylation and subsequent activation of NF-kappaB. Furthermore, the expression of GITRL and GITR was detected in macrophages in inflammatory disease specimens such as atherosclerotic plaques and synovial tissues of rheumatoid arthritis. These observations raise the possibility that the GITRL-mediated inflammatory activation of macrophages is involved in the pathogenesis of inflammatory diseases.
17599281 Allose gallates suppress expression of pro-inflammatory cytokines through attenuation of N 2007 Jul Gallotannins are plant-derived, water-soluble polyphenols with wide-ranging biological activities. Mast cell-mediated allergic inflammation is known to cause many diseases such as asthma, sinusitis, and rheumatoid arthritis. Mast cells induce synthesis and production of pro-inflammatory cytokines including tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6 with immune regulatory properties. Expression of inflammatory cytokines is mainly regulated by a transcription factor, nuclear factor (NF)-kappaB. In the present study, the effect of eight gallotannins on the level of pro-inflammatory cytokines and NF-kappaB activation was investigated in human mast cell line (HMC-1). HMC-1 cells were sensitized by phorbol 12-myristate 13-acetate (PMA) and calcium ionophore (A23187). Among the eight gallotannins from EUPHORBIA species, three gallotannins such as 1,2,3,4,6-penta- O-galloyl-beta-D-glucose, 1,2,6-tri-O-galloyl-beta-D-allopyanose, and 1,2,3,6-tetra-O-galloyl-beta-D-allopyranose suppressed the gene expression and secretion of pro-inflammatory cytokines in a dose-dependent manner. In addition, these three gallotannins blocked the activation of NF-kappaB as indicated by an NF-kappaB-dependent gene reporter assay. We conclude that these gallotannins may have potential for the treatment of inflammatory diseases through the down-regulation of NF-kappaB-mediated activation of mast cells.
17557415 A genomewide single-nucleotide-polymorphism panel for Mexican American admixture mapping. 2007 Jun For admixture mapping studies in Mexican Americans (MAM), we define a genomewide single-nucleotide-polymorphism (SNP) panel that can distinguish between chromosomal segments of Amerindian (AMI) or European (EUR) ancestry. These studies used genotypes for >400,000 SNPs, defined in EUR and both Pima and Mayan AMI, to define a set of ancestry-informative markers (AIMs). The use of two AMI populations was necessary to remove a subset of SNPs that distinguished genotypes of only one AMI subgroup from EUR genotypes. The AIMs set contained 8,144 SNPs separated by a minimum of 50 kb with only three intermarker intervals >1 Mb and had EUR/AMI FST values >0.30 (mean FST = 0.48) and Mayan/Pima FST values <0.05 (mean FST < 0.01). Analysis of a subset of these SNP AIMs suggested that this panel may also distinguish ancestry between EUR and other disparate AMI groups, including Quechuan from South America. We show, using realistic simulation parameters that are based on our analyses of MAM genotyping results, that this panel of SNP AIMs provides good power for detecting disease-associated chromosomal segments for genes with modest ethnicity risk ratios. A reduced set of 5,287 SNP AIMs captured almost the same admixture mapping information, but smaller SNP sets showed substantial drop-off in admixture mapping information and power. The results will enable studies of type 2 diabetes, rheumatoid arthritis, and other diseases among which epidemiological studies suggest differences in the distribution of ancestry-associated susceptibility.
17533220 Mascot file parsing and quantification (MFPaQ), a new software to parse, validate, and qua 2007 Sep Proteomics strategies based on nanoflow (nano-) LC-MS/MS allow the identification of hundreds to thousands of proteins in complex mixtures. When combined with protein isotopic labeling, quantitative comparison of the proteome from different samples can be achieved using these approaches. However, bioinformatics analysis of the data remains a bottleneck in large scale quantitative proteomics studies. Here we present a new software named Mascot File Parsing and Quantification (MFPaQ) that easily processes the results of the Mascot search engine and performs protein quantification in the case of isotopic labeling experiments using either the ICAT or SILAC (stable isotope labeling with amino acids in cell culture) method. This new tool provides a convenient interface to retrieve Mascot protein lists; sort them according to Mascot scoring or to user-defined criteria based on the number, the score, and the rank of identified peptides; and to validate the results. Moreover the software extracts quantitative data from raw files obtained by nano-LC-MS/MS, calculates peptide ratios, and generates a non-redundant list of proteins identified in a multisearch experiment with their calculated averaged and normalized ratio. Here we apply this software to the proteomics analysis of membrane proteins from primary human endothelial cells (ECs), a cell type involved in many physiological and pathological processes including chronic inflammatory diseases such as rheumatoid arthritis. We analyzed the EC membrane proteome and set up methods for quantitative analysis of this proteome by ICAT labeling. EC microsomal proteins were fractionated and analyzed by nano-LC-MS/MS, and database searches were performed with Mascot. Data validation and clustering of proteins were performed with MFPaQ, which allowed identification of more than 600 unique proteins. The software was also successfully used in a quantitative differential proteomics analysis of the EC membrane proteome after stimulation with a combination of proinflammatory mediators (tumor necrosis factor-alpha, interferon-gamma, and lymphotoxin alpha/beta) that resulted in the identification of a full spectrum of EC membrane proteins regulated by inflammation.