A preliminary investigation of the contribution of different tenderness factors to beef loin, tri-tip and heel tenderness.

The objective of this study was to identify the relative contribution of tenderness factors for three beef muscles with similar tenderness ratings. Longissimus lumborum (LL), tensor fascia latae (TF) and gastrocnemius (GC) were collected from 10 USDA low Choice beef carcasses and assigned to a 5 or 21 days aging period (n = 60). Sarcomere length, troponin-T degradation, collagen content, mature collagen crosslink density, intramuscular lipid content and trained panel analysis were measured. Correlation and multivariate regression analysis indicated each muscle has a specific tenderness factor that contributed to the overall tenderness evaluated by trained panelists. The equations indicated LL tenderness was driven by lipid content (P < .05); TF tenderness was driven by collagen content (P < .05). GC tenderness was driven by proteolysis (P < .01), and only collagen content can be casually used as an overall tenderness predictor for all three cuts.

Correlation of serum protein biomarkers with disease activity in psoriatic arthritis.

Objective: To assess the correlation of serum protein biomarkers with disease activity across different domains of psoriatic arthritis (PsA).Material and methods: A cross-sectional cohort of 45 adult patients with PsA fulfilling the classification for psoriatic arthritis (CASPAR) criteria was recruited from University of California San Diego (UCSD) Arthritis Clinics. Clinical data and serum samples were collected and serum was analyzed for protein biomarkers hypothesized to be relevant to disease activity in PsA. Correlations were evaluated for clinical disease activity measures across disease domains.Results: Biomarkers with the highest correlation to the composite indices and disease domains were SAA, IL-6, YKL-40, and ICAM-1. In addition, several biomarkers were moderately correlated with individual composite indices and/or disease domains. Low or no correlation was observed with some biomarkers, e.g. MMP-3, MMP-1, EGF, VEGF, and IL-6R. In contrast, the correlation of all biomarkers with certain disease domains was low; specifically, pain, percent body surface area of psoriasis, and patient global assessment. The multi-biomarker disease activity score (MBDA) developed for rheumatoid arthritis (RA) showed high correlations with most composite indices and some disease domains in PsA.Conclusions: These data suggest biomarker analysis can reflect disease activity across disease domains in PsA. Certain domains would likely benefit from the evaluation of additional biomarkers.

Decreased photosynthetic rate under high temperature in wheat is due to lipid desaturation, oxidation, acylation, and damage of organelles.

BACKGROUND: High temperature is a major abiotic stress that limits wheat (Triticum aestivum L.) productivity. Variation in levels of a wide range of lipids, including stress-related molecular species, oxidative damage, cellular organization and ultrastructural changes were analyzed to provide an integrated view of the factors that underlie decreased photosynthetic rate under high temperature stress. Wheat plants of cultivar Chinese Spring were grown at optimum temperatures (25/15 °C, maximum/minimum) until the onset of the booting stage. Thereafter, plants were exposed to high temperature (35/25 °C) for 16 d. RESULTS: Compared with optimum temperature, a lower photosynthetic rate was observed at high temperature which is an interplay between thylakoid membrane damage, thylakoid membrane lipid composition, oxidative damage of cell organelle, and stomatal and non-stomatal limitations. Triacylglycerol levels were higher under high temperature stress. Polar lipid fatty acyl unsaturation was lower at high temperature, while triacylglycerol unsaturation was the same at high temperature and optimum temperature. The changes in lipid species indicates increases in activities of desaturating, oxidizing, glycosylating and acylating enzymes under high temperature stress. Cumulative effect of high temperature stress led to generation of reactive oxygen species, cell organelle and membrane damage, and reduced antioxidant enzyme activity, and imbalance between reactive oxygen species and antioxidant defense system. CONCLUSIONS: Taken together with recent findings demonstrating that reactive oxygen species are formed from and are removed by thylakoid lipids, the data suggest that reactive oxygen species production, reactive oxygen species removal, and changes in lipid metabolism contribute to decreased photosynthetic rate under high temperature stress.

The JAK inhibitor tofacitinib suppresses synovial JAK1-STAT signalling in rheumatoid arthritis.

OBJECTIVE: Tofacitinib is an oral Janus kinase (JAK) inhibitor for the treatment of rheumatoid arthritis (RA). The pathways affected by tofacitinib and the effects on gene expression in situ are unknown. Therefore, tofacitinib effects on synovial pathobiology were investigated. METHODS: A randomised, double-blind, phase II serial synovial biopsy study (A3921073; NCT00976599) in patients with RA with an inadequate methotrexate response. Patients on background methotrexate received tofacitinib 10 mg twice daily or placebo for 28 days. Synovial biopsies were performed on Days -7 and 28 and analysed by immunoassay or quantitative PCR. Clinical response was determined by disease activity score and European League Against Rheumatism (EULAR) response on Day 28 in A3921073, and at Month 3 in a long-term extension study (A3921024; NCT00413699). RESULTS: Tofacitinib exposure led to EULAR moderate to good responses (11/14 patients), while placebo was ineffective (1/14 patients) on Day 28. Tofacitinib treatment significantly reduced synovial mRNA expression of matrix metalloproteinase (MMP)-1 and MMP-3 (p<0.05) and chemokines CCL2, CXCL10 and CXCL13 (p<0.05). No overall changes were observed in synovial inflammation score or the presence of T cells, B cells or macrophages. Changes in synovial phosphorylation of signal transducer and activator of transcription 1 (STAT1) and STAT3 strongly correlated with 4-month clinical responses (p<0.002). Tofacitinib significantly decreased plasma CXCL10 (p<0.005) at Day 28 compared with placebo. CONCLUSIONS: Tofacitinib reduces metalloproteinase and interferon-regulated gene expression in rheumatoid synovium, and clinical improvement correlates with reductions in STAT1 and STAT3 phosphorylation. JAK1-mediated interferon and interleukin-6 signalling likely play a key role in the synovial response. TRIAL REGISTRATION NUMBER: NCT00976599.

Validated gene expression biomarker analysis for biopsy-based clinical trials in ulcerative colitis.

BACKGROUND: Accurate and reproducible measurement of expression of pro-inflammatory cytokines in colonic biopsies from patients with ulcerative colitis (UC) is essential for proof-of-concept and mechanism-of-action studies. Few studies have rigorously established the number of biopsies required for accurate and reproducible biomarker measurements. AIM: To validate methods for measuring changes in gene expression in colonic biopsy samples. METHODS: Twelve colonic biopsies were obtained from each of six healthy controls, six patients with inactive UC and seven patients with active UC. Mayo endoscopic scores were used as a clinical reference standard. Quantitative PCR was used to assess mRNA expression of eight known inflammatory genes. The power to detect a reduction in gene expression in active vs. inactive UC was calculated using a linear mixed effect model. RESULTS: mRNA analysis of colonic biopsies is a sensitive and feasible approach for measuring inflammatory gene expression in colonic biopsies. Inflammatory biomarkers correlate with Mayo endoscopic subscores for each colonic region. For most genes, three rectal biopsies from two to four patients are required to detect changes in gene expression corresponding to active vs. inactive UC to achieve a power of 80% with an alpha of 0.05. CONCLUSION: Our data suggest that systematic measurement of inflammatory biomarkers at the mRNA level can be a valuable tool for hypothesis testing, and assessment of clinical activity and response to therapy in ulcerative colitis.

Acid-sensing ion channel 3 expressed in type B synoviocytes and chondrocytes modulates hyaluronan expression and release.

BACKGROUND: Rheumatoid arthritis is an inflammatory disease marked by intra-articular decreases in pH, aberrant hyaluronan regulation and destruction of bone and cartilage. Acid-sensing ion channels (ASICs) are the primary acid sensors in the nervous system, particularly in sensory neurons and are important in nociception. ASIC3 was recently discovered in synoviocytes, non-neuronal joint cells critical to the inflammatory process. OBJECTIVES: To investigate the role of ASIC3 in joint tissue, specifically the relationship between ASIC3 and hyaluronan and the response to decreased pH. METHODS: Histochemical methods were used to compare morphology, hyaluronan expression and ASIC3 expression in ASIC3+/+ and ASIC3-/- mouse knee joints. Isolated fibroblast-like synoviocytes (FLS) were used to examine hyaluronan release and intracellular calcium in response to decreases in pH. RESULTS: In tissue sections from ASIC3+/+ mice, ASIC3 localised to articular cartilage, growth plate, meniscus and type B synoviocytes. In cultured FLS, ASIC3 mRNA and protein was also expressed. In FLS cultures, pH 5.5 increased hyaluronan release in ASIC3+/+ FLS, but not ASIC3-/- FLS. In FLS from ASIC3+/+ mice, approximately 50% of cells (25/53) increased intracellular calcium while only 24% (14/59) showed an increase in ASIC3-/- FLS. Of the cells that responded to pH 5.5, there was significantly less intracellular calcium increases in ASIC3-/- FLS compared to ASIC3+/+ FLS. CONCLUSION: ASIC3 may serve as a pH sensor in synoviocytes and be important for modulation of expression of hyaluronan within joint tissue.

Lipomatosis-associated inflammation and excess collagen may contribute to lower relative resting energy expenditure in women with adiposis dolorosa.

BACKGROUND: Adiposis dolorosa (AD) is a syndrome of obese and non-obese individuals whose hallmark is lipomatosis: unencapsulated painful fatty masses in subcutaneous fat. Lipomatosis may contain excess collagen and multi-nucleated giant (MNG) cells. Case reports suggest metabolic defects in AD. OBJECTIVES: (1) To determine whether women with AD have altered relative resting energy expenditure (REE per total body mass) compared with controls; and (2) to quantitate lipomatosis-associated collagen, MNGs and tissue and blood cytokines that may influence REE. METHODS: A total of 10 women with AD were compared with age, body mass index, fat and weight-matched control women. Adipose tissue was obtained from five women with AD and five controls and evaluated for collagen and macrophages/MNGs. Fat mass and fat-free mass were identified by dual X-ray absorptiometry. REE was by determined indirect calorimetry and related to mass. Adipokines and cytokines were evaluated in blood and tissue. RESULTS: Relative REE (REE per total body mass) was lower in women with AD compared with controls (P=0.007). Only lipomatosis (group) and total body mass were significant predictors of REE in forward stepwise regression (P<0.0001). Adipose interleukin (IL)-6 levels were elevated (P=0.03) and connective tissue was increased fourfold in lipomatosis compared with control tissue (P <0.0001). There was no difference in adipose tissue macrophages between groups; 30% of women with AD had MNG cells. Anti-inflammatory IL-13 levels were elevated (P=0.03), and cytokines important in the recruitment of monocytes, Fraktalkine (P=0.04) and macrophage inflammatory protein-1beta (P=0.009), were significantly lower in the blood of women with AD compared with controls. CONCLUSIONS: The lower relative REE in women with AD compared with controls was associated with increased connective (non-metabolic) tissue in the lipomatosis, and inflammation, although underlying metabolic defects may be important as well. Understanding the pathophysiology and metabolism of lipomatosis in AD may contribute to a better understanding of metabolism in non-lipomatosis obesity.

MKK3, an upstream activator of p38, contributes to formalin phase 2 and late allodynia in mice.

Spinal p38 mitogen activated (MAP) kinase plays a key role in chronic pain behavior. However, clinical development of p38 inhibitors has been hindered by significant toxicity. To evaluate alternative strategies of p38 regulation, we determined if known upstream activators of p38 (mitogen activated kinase kinase [MKK] 3 and MKK6), are involved in development and maintenance of pain and spinal p38 phosphorylation. Acute pain behaviors were not altered in MKK3 or MKK6 deficient mice. The phase 2 formalin response was delayed in MKK3-/- mice, but unchanged in magnitude, while the response remained normal in MKK6-/- mice. More striking, late formalin allodynia (3-18 days post-injection) was prominent in wild type and MKK6-/- mice, but was delayed for several days in MKK3-/- mice. In wild type, but not MKK3-/- mice, intraplantar formalin elicited increases in ipsilateral spinal MKK3/6 phosphorylation acutely and again at 9 days postinjection. Phosphorylation of MKK3/6 correlated with phase 2 formalin behavior. Wild type (WT) and MKK3-/- mice both expressed increases in spinal phosphorylated p38, however in WT mice this response began several days earlier, and was of higher magnitude and duration than in MKK3-/- mice. This phosphorylation correlated with the late allodynia. Phosphorylated MKK3/6 was detected only in astrocytes, given that phosphorylated p38 (P-p38) is usually not seen in astrocytes this argues for astrocytic release of soluble mediators that affect p38 phosphorylation in microglia. Taking these data together, MKK3, but not MKK6, is necessary for normal development of chronic pain behavior and phosphorylation of spinal p38.

Mode of action of abatacept in rheumatoid arthritis patients having failed tumour necrosis factor blockade: a histological, gene expression and dynamic magnetic resonance imaging pilot study.

OBJECTIVES: Abatacept is the only agent currently approved to treat rheumatoid arthritis (RA) that targets the co-stimulatory signal required for full T-cell activation. No studies have been conducted on its effect on the synovium, the primary site of pathology. The aim of this study was to determine the synovial effect of abatacept in patients with RA and an inadequate response to tumour necrosis factor alpha (TNFalpha) blocking therapy. METHODS: This first mechanistic study incorporated both dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI) and arthroscopy-acquired synovial biopsies before and 16 weeks after therapy, providing tissue for immunohistochemistry and quantitative real-time PCR analyses. RESULTS: Sixteen patients (13 women) were studied; all had previously failed TNFalpha-blocking therapy. Fifteen patients completed the study. Synovial biopsies showed a small reduction in cellular content, which was significant only for B cells. The quantitative PCR showed a reduction in expression for most inflammatory genes (Wald statistic of p<0.01 indicating a significant treatment effect), with particular reduction in IFNgamma of -52% (95% CI -73 to -15, p<0.05); this correlated well with MRI improvements. In addition, favourable changes in the osteoprotegerin and receptor activator of nuclear factor kappa B levels were noted. DCE-MRI showed a reduction of 15-40% in MRI parameters. CONCLUSION: These results indicate that abatacept reduces the inflammatory status of the synovium without disrupting cellular homeostasis. The reductions in gene expression influence bone positively and suggest a basis for the recently demonstrated radiological improvements that have been seen with abatacept treatment in patients with RA.

Synergistic benefit in inflammatory arthritis by targeting I kappaB kinase epsilon and interferon beta.

OBJECTIVES: The I kappaB kinase (IKK)-related kinase IKKepsilon regulates type I interferon expression and responses as well as proinflammatory mediator production. We examined the role of IKKepsilon in arthritis and its ability to enhance the therapeutic response to systemic interferon (IFN) beta therapy in passive murine K/BxN arthritis. METHODS: IKKepsilon(-/-), IFN alpha(approximately)beta R(-/-) and wild type mice were given K/BxN serum and treated with polyinosinic polycytidylic acid (poly(I:C)), IFN beta, or normal saline. Clinical response and histological scores were assessed. Gene expression in the paws was measured by quantitative PCR. Serum interleukin 1a receptor agonist (IL1Ra) and IL10 were measured by ELISA and multiplex bead array. RESULTS: Arthritis was almost completely blocked in wild type mice if arthritogenic K/BxN serum and the Toll-like receptor (TLR)3 ligand, poly(I:C), were coadministered at the onset of the model, but not in established disease. Mice deficient in IFN alpha(approximately)beta R had an accelerated course of arthritis, and did not respond to poly(I:C). IKKepsilon null mice had a modest decrease in clinical arthritis compared with heterozygous mice. Low doses of IFN beta that were ineffective in wild type mice significantly decreased clinical arthritis in IKKepsilon null mice. Articular chemokine gene expression was reduced in the IKKepsilon(-/-) mice with arthritis and secreted IL1Ra (sIL1Ra) mRNA was significantly increased. Serum levels of IL1Ra were increased in low dose IFN beta-treated IKKepsilon(-/-) mice. CONCLUSIONS: Subtherapeutic doses of IFN beta enhance the anti-inflammatory effects of IKKepsilon deficiency, possibly by increasing production of IL1Ra and unmasking the antichemokine effects. Combination therapy with low dose IFN beta and an IKKepsilon inhibitor might improve efficacy of either agent alone and offers a novel approach to RA.

Assessment of rituximab's immunomodulatory synovial effects (ARISE trial). 1: clinical and synovial biomarker results.

OBJECTIVE: Treatment with the anti-CD20 monoclonal antibody (mAb) rituximab is effective in rheumatoid arthritis (RA). Marked depletion of circulating B cells, seen in almost all patients, does not correlate with efficacy. The potential synovial immunomodulatory effects of rituximab have not been fully defined. METHODS: The ARISE trial is an open label, serial synovial biopsy (pre-treatment and 8 weeks) study of rituximab, given 1 g intravenously on days 0 and 14 without peri-infusional steroids, in active RA patients on concomitant methotrexate (MTX). Synovial tissue was analysed by immunohistochemistry with digital image analysis and gene expression by real-time PCR. RESULTS: The mean (SD) baseline DAS28 score was 6.5 (0.4), and mean MTX dose 17.3 mg/week. Of 13 patients, 11 had failed prior tumour necrosis factor (TNF) inhibitor therapy. With treatment, all patients experienced near complete depletion of circulating B cell numbers. During the 6 months after treatment, 7/13 patients achieved an American College of Rheumatology (ACR) 20% improvement (ACR20) response, 3/13 an ACR50 response and 2/13 an ACR70 response. There was a significant decrease in synovial B cells after treatment, but only a small trend towards greater reduction among clinical responders. Among the three patients with ACR50 responses there was a significant decrease in synovial immunoglobulin synthesis. CONCLUSIONS: These data suggest that unlike those in circulation, synovial B cells are decreased but are not eliminated by rituximab therapy. Patients with higher levels of response may have more consistent depletion of synovial B cells, and may also have an alteration in synovial B cell function, as indicated by decreases in synovial immunoglobulin synthesis. Thus, effects on synovial B cells may be necessary but not sufficient for inducing clinical efficacy. Other effects, such as on primary lymph organ B cell antigen presentation or cytokine production, may be operative.

Expression and regulation of cryopyrin and related proteins in rheumatoid arthritis synovium.

BACKGROUND: Rheumatoid arthritis (RA) synovium is characterised by enhanced NF-kappaB activity and proinflammatory cytokines. Cryopyrin (CIAS-1, NALP-3, PYPAF-1) has been shown to regulate NF-kappaB and caspase-1 activation. OBJECTIVE: To study the expression of cryopyrin, its effector molecule ASC, and its putative antagonist pyrin in RA and osteoarthritis (OA) synovium, and the main two cellular constituents of synovial lining, cultured fibroblast-like synoviocytes (FLS) and macrophages. METHODS: FLS and macrophages were cultured in the presence of inflammatory mediators. Real time polymerase chain reaction was used to quantify message levels in synovial biopsy specimens and cells. In situ hybridisation was employed to localise expression of cryopyrin mRNA. RESULTS: Cryopyrin mRNA was raised in RA synovium and detected in both lining and sublining regions. FLS from RA and OA tissue expressed low baseline levels of cryopyrin transcripts that were induced by tumour necrosis factor alpha (TNFalpha). In contrast, macrophages differentiated in vitro expressed relatively high cryopyrin levels, which were further induced by TNFalpha, but not by interleukin 1beta. ASC mRNA levels were comparable in RA and OA tissue, FLS, and macrophages, and were depressed by TNFalpha in macrophages. Pyrin expression was higher in RA synovium than in OA tissue, and virtually undetectable in FLS but high in macrophages where it was unchanged by TNFalpha treatment. CONCLUSION: These results suggest that enhanced cryopyrin levels in RA synovium are due to a greater numbers of tissue macrophages, and demonstrate transcriptional regulation of cryopyrin in a chronic inflammatory disease.

DNA mismatch repair enzyme expression in synovial tissue.

BACKGROUND: Oxidative stress in RA synovial tissue can cause DNA damage and suppress the DNA mismatch repair (MMR) system in cultured synoviocytes. This mechanism includes two enzyme complexes, hMutSalpha (hMSH2/hMSH6) and hMutSbeta (hMSH2/hMSH3). OBJECTIVE: To examine the expression and distribution of MMR enzymes in synovial tissues from patients with arthritis and from normal subjects. METHODS: Synovial tissues from patients with RA, osteoarthritis (OA), or normal subjects were analysed by immunohistochemistry using monoclonal antibodies to hMSH2, hMSH3, and hMSH6. MMR protein expression was evaluated by computer assisted digital image analysis. RESULTS: hMSH2, hMSH3, and hMSH6 were found in most synovial tissues evaluated, with greater levels in the intimal lining than sublining regions. In RA and OA, sublining perivascular staining for hMSH6 and hMSH3 was also prominent. Significantly higher sublining expression of hMSH2, hMSH3, and hMSH6 was seen in RA and OA than in normal synovium. Double label immunohistochemistry demonstrated that the main cells expressing MMR enzymes were CD68(+) and CD68(-) cells in the intimal lining. CONCLUSIONS: DNA MMR enzyme expression is greatest in the synovial intimal lining layer, where maximal oxidative stress in RA occurs. Although MMR enzyme expression is greater in RA than in normal tissue, this compensatory response cannot overcome the genotoxic environment, and DNA damage accumulates.

Glutathione peroxidase-1 deficiency leads to increased nuclear light scattering, membrane damage, and cataract formation in gene-knockout mice.

PURPOSE: Previous in vitro studies with transgenic and gene-knockout mice have shown that lenses with elevated levels of glutathione peroxidase (GPX)-1 activity are able to resist the cytotoxic effect of H(2)O(2), compared with normal lenses and lenses from GPX-1-deficient animals. The purpose of this study was to investigate the functional role of this enzyme in antioxidant mechanisms of lens in vivo by comparing lens changes of gene-knockout mice with age-matched control animals. METHODS: In vivo lens changes were monitored by slit lamp biomicroscopy, and enucleated lenses were examined under a stereomicroscope in gene-knockout animals and age-matched control animals ranging in age from 3 weeks to 18 months. Transmission (TEM) and confocal microscopy were performed on different regions of lenses after the mice were killed at various times. RESULTS: Slit lamp images showed an increase in nuclear light scattering (NLS) in gene-knockout mice compared with control animals. TEM revealed changes in the nucleus as early as 3 weeks of age by the appearance of waviness of fiber membranes. With increasing age, there was greater distortion of fiber membranes and distension of interfiber space at the apex of fiber cells compared with control mice. The changes in nuclear fiber membranes were even more dramatic, as observed by confocal microscopy, which was performed on thicker sections. In contrast to the changes in the lens nucleus, the morphology of the epithelium and superficial cortex remained unchanged in knockout animals during the same experimental period, consistent with slit lamp observations. Stereomicroscopy of ex vivo lenses demonstrated a significant increase in opacification in gene-knockout mice relative to control animals of the same age. This effect became evident in mice aged 5 to 9.9 months and persisted thereafter in older animals, resulting in mature cataracts after 15 months. CONCLUSIONS: The results demonstrate the critical role of GPX-1 in antioxidant defense mechanisms of the lens nucleus. The increased NLS appears to be associated with damage to fiber membranes in the nucleus, which is particularly susceptible to oxidative challenge because of the deficiency of GPX-1. It is suggested that the lens membrane changes in the knockout animals may be due to the formation of lipid peroxides, which serve as substrates for GPX-1. Cataract development in gene-knockout mice appeared to progress from focal opacities, apparent at an earlier age, to lamellar cataracts between 6 and 10 months, and finally to complete opacification in animals older than 15 months. This is the first reported phenotype in GPX-1-knockout mice.

Inhibitor of nuclear factor kappaB kinase beta is a key regulator of synovial inflammation.

OBJECTIVE: Inhibitor of nuclear factor kappaB kinase beta (IkappaB kinase beta, or IKKbeta) has emerged as a key regulator of the transcription factor nuclear factor kappaB (NF-kappaB). Since IKKbeta could have both pro- and antiinflammatory activity, we examined whether its constitutive activation was sufficient to cause a chronic inflammatory disease such as rheumatoid arthritis. METHODS: Normal Lewis rats were evaluated for paw swelling by plethysmometry and histologic assessment after intraarticular injection of an adenoviral construct encoding the IKKbeta wild-type gene (Ad.IKKbeta-wt); controls received an adenoviral construct encoding green fluorescent protein (Ad.GFP). The rats were killed after 7 days. Additionally, rats were killed 48 hours after intraarticular injection of Ad.IKKbeta-wt or Ad.GFP for studies of IKK activity and NF-kappaB binding. For studies of the effects of inhibition of IKKbeta activity, Lewis rats were immunized with Mycobacterium tuberculosis in mineral oil. The ankle joints were injected on day 12 with an adenoviral construct encoding IKKbeta K-->M (dominant negative, IKKbeta-dn) or Ad.GFP. We evaluated paw swelling and NF-kappaB expression on day 25. RESULTS: Intraarticular gene transfer of IKKbeta-wt into the joints of normal rats resulted in significant paw swelling and histologic evidence of synovial inflammation. Increased IKK activity was detectable in the IKKbeta-wt-injected ankle joints, coincident with enhanced NF-kappaB DNA binding activity. Intraarticular gene transfer of IKKbeta-dn significantly ameliorated the severity of adjuvant arthritis, accompanied by a significant decrease in NF-kappaB DNA expression in the joints of Ad.IKKbeta-dn-treated animals. CONCLUSION: IKKbeta plays a key role in rodent synovial inflammation. Intraarticular gene therapy to inhibit IKKbeta activity represents an attractive strategy for the treatment of chronic arthritis.

c-Jun N-terminal kinase is required for metalloproteinase expression and joint destruction in inflammatory arthritis.

Mitogen-activated protein kinase (MAPK) cascades are involved in inflammation and tissue destruction in rheumatoid arthritis (RA). In particular, c-Jun N-terminal kinase (JNK) is highly activated in RA fibroblast-like synoviocytes and synovium. However, defining the precise function of this kinase has been difficult because a selective JNK inhibitor has not been available. We now report the use of a novel selective JNK inhibitor and JNK knockout mice to determine the function of JNK in synoviocyte biology and inflammatory arthritis. The novel JNK inhibitor SP600125 (anthra[1,9-cd]pyrazol-6(2H)-one) completely blocked IL-1--induced accumulation of phospho-Jun and induction of c-Jun transcription in synoviocytes. Furthermore, AP-1 binding and collagenase mRNA accumulation were completely suppressed by SP600125. In contrast, complete inhibition of p38 had no effect, and ERK inhibition had only a modest effect. The essential role of JNK was confirmed in cultured synoviocytes from JNK1 knockout mice and JNK2 knockout mice, each of which had a partial defect in IL-1--induced AP-1 activation and collagenase-3 expression. Administration of SP600125 modestly decreased the rat paw swelling in rat adjuvant-induced arthritis. More striking was the near-complete inhibition of radiographic damage that was associated with decreased AP-1 activity and collagenase-3 gene expression. Therefore, JNK is a critical MAPK pathway for IL-1--induced collagenase gene expression in synoviocytes and in joint arthritis, indicating that JNK is an important therapeutic target for RA.

Physicochemical and microbiological properties of selected rice flour-based batters for fried chicken drumsticks.

Rice flour-based batter (RFBB) formulations for chicken drumstick coating were developed as an alternative for traditional wheat flour-based batter (WFBB). Physicochemical properties and storage stability of selected RFBB were evaluated and compared to WFBB. Batter pickup of RFBB formulated in combination with oxidized corn starch and methylcellulose (MC) was not significantly different from that of WFBB. In contrast, batters with only rice and corn flour (60:40% flour weight) exhibited significantly higher pickup. Rice flour batter with 15% oxidized corn starch had the lowest batter pickup. All RFBB exhibited (P < 0.05) lower oil absorption than WFBB. The TBA values of RFBB and WFBB increased (P < 0.05) with increased frozen storage time at -40 C for 90 d. The RFBB with MC exhibited the lowest TBA values, whereas WFBB had the highest values. Microstructural analysis revealed that freezing caused structural deterioration of all batters, but the RFBB with MC exhibited less freezing tolerance than other samples. The total plate counts of immediately fried or frozen fried chicken stored for 90 d were less than 1 log cfu/g sample. The RFBB with 5% oxidized corn starch and MC can replace WFBB on fried drumsticks. Additionally, RFBB results in a healthier product due to lower fat absorption.

Anti-inflammatory effects of ABT-702, a novel non-nucleoside adenosine kinase inhibitor, in rat adjuvant arthritis.

Adenosine (ADO) is a homeostatic inhibitory autocoid that is released at sites of inflammation and tissue injury, and exerts anti-inflammatory effects via multiple interactions at ADO receptor subtypes. Inhibition of ADO kinase (AK) increases extracellular ADO concentrations and AK inhibitors have demonstrated ADO-mediated anti-inflammatory effects in acute models of inflammation. To evaluate the potential utility of this approach in chronic inflammation, a novel, potent, and selective non-nucleoside AK inhibitor, ABT-702, was tested in the rat adjuvant arthritis model. Animals were immunized with complete Freund's adjuvant on day 0 and were treated with vehicle or ABT-702 (20 mg/kg/b.i.d. p.o.) beginning on day 8. ABT-702 significantly inhibited arthritis as determined by paw volume. In addition, histologic and radiographic evidence of bone and cartilage destruction was significantly decreased in the treated group. Coadministration of the ADO receptor antagonist theophylline attenuated the anti-inflammatory effects of ABT-702, suggesting that this action was mediated through endogenous ADO release. To evaluate the mechanism of chondroprotection, Northern blot and electrophoretic mobility shift assays were performed on joints samples. These studies demonstrated that ABT-702 suppressed collagenase and stromelysin gene expression in treated animals. In addition, the activator protein-1 and nuclear factor-kappaB binding activity was also decreased. Therefore, ABT-702 inhibited clinical, radiographic, and histologic evidence of chronic inflammatory arthritis. The mechanism of joint protection is likely related to suppressed transcription factor activation and matrix metalloproteinase gene expression.

Expression and regulation of inducible IkappaB kinase (IKK-i) in human fibroblast-like synoviocytes.

IkappaB kinase (IKK) plays a key role in the regulation of nuclear factor kappaB (NF-kappaB). We previously demonstrated the expression of two kinases, IKK1 and IKK2, in fibroblast-like synoviocytes (FLS) and determined their functional consequences for inflammatory gene expression in vitro and in vivo. Recently, a novel inducible IkappaB kinase has been described, namely, IKK-i or IKK-epsilon, which is functionally and structurally distinct from constitutively expressed IKK1 and IKK2. Therefore, we investigated the expression and regulation of this novel kinase in FLS from patients with rheumatoid arthritis and osteoarthritis. Interestingly, constitutive gene expression and protein expression were observed in all cell lines examined. TNFalpha stimulation for 24 h increased IKK-i expression 7.2 +/- 1.8-fold in FLS (P < 0.02). IL-1 also significantly increased IKK-i gene expression. Time course experiments demonstrated that IKK-i gene expression increased within 3 h of TNFalpha stimulation and persisted for at least 24 h. Dose-response studies showed that as little as 1 ng/ml of TNFalpha increased IKK-i gene expression. Constitutive IKK-1 gene expression was also noted in rheumatoid arthritis, osteoarthritis, and normal synovium. This is the first report demonstrating constitutive expression and cytokine regulation of this novel kinase in primary human synovial cells.

Adhesion of rice flour-based batter to chicken drumsticks evaluated by laser scanning confocal microscopy and texture analysis.

The convenience and appeal of battered or breaded products have resulted in a sales increase of 100% since 1980. Because of the rapid growth of the Asian-American population and increasing consumption of rice and rice products, rice flour is a logical alternative for wheat flour in traditional batter formulation. The effects of ingredients used in rice flour-based batters on adhesion characteristic for deep-fat fried chicken drumsticks were studied by laser scanning confocal microscopy (LSCM) and texture analysis. Raw chicken drumsticks were predusted with egg albumin powder before dipping into batters prepared from combinations of rice flour, yellow corn flour, oxidized cornstarch, methylcellulose, or xanthan gum. The drumsticks were fried at 175+/-5 C until the internal temperature reached at least 71 C. For LSCM, samples were fixed overnight and were sectioned by vibratome (200 microm) before viewing. Batter adhesion was determined using an attachment specifically designed for chicken drumsticks. Microstructural analysis showed that batter formulated with a 50:50 mixture of rice and corn flours adhered better to drumsticks than batter with other rice flour ratios. Xanthan gum (0.2%) or methylcellulose (0.3%) alone had poor adhesion to chicken skin. However, when combined with other ingredients, xanthan gum increased the amount of batter pick-up before frying by increasing viscosity. Egg albumin significantly facilitated batter adhesion. The results from texture analysis supported the microstructural studies. As rice flour ratio increased from 50 to 70%, the binding force decreased. Rice flour showed potential as an alternative to wheat flour for batter formulas when the appropriate levels of oxidized starch, xanthan gum, and methylcellulose were included in the formulation.