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To further validate a serum proteomics panel for predicting radiographic (structural) knee OA progression.

In the past three decades, extraordinary advances have been made in the understanding of the pathogenesis of, and treatment options for, inflammatory arthritides, including rheumatoid arthritis and spondyloarthritis. The use of methotrexate and subsequently biologic therapies (such as TNF inhibitors, among others) and oral small molecules have substantially improved clinical outcomes for many patients with inflammatory arthritis; for others, however, these agents do not substantially improve their symptoms. The emerging field of pharmacomicrobiomics, which investigates the effect of variations within the human gut microbiome on drugs, has already provided important insights into these therapeutics. Pharmacomicrobiomic studies have demonstrated that human gut microorganisms and their enzymatic products can affect the bioavailability, clinical efficacy and toxicity of a wide array of drugs through direct and indirect mechanisms. This discipline promises to facilitate the advent of microbiome-based precision medicine approaches in inflammatory arthritis, including strategies for predicting response to treatment and for modulating the microbiome to improve response to therapy or reduce drug toxicity.

The lack of disease modifying drugs in Osteoarthritis (OA) may be attributed to the difficulty in robust response based on patient-reported outcomes (PROs) linked to drug mechanism of action. Joint tissue turnover biomarkers are associated with disease progression. A subset of patients has elevated serum levels of CRP metabolite (CRPM). This explorative study investigates the associations between PROs and joint tissue turnover markers in patients with high or low CRPM.

In the last decade, there has been renewed interest in three-year MD pathway programs. In 2015, with support from the Josiah Macy Jr., Foundation, eight North American medical schools with three-year accelerated medical pathway programs formed the Consortium of Accelerated Medical Pathway Programs (CAMPP). The schools are two campuses of the Medical College of Wisconsin; McMaster University Michael G. DeGroote School of Medicine; Mercer University School of Medicine; New York University School of Medicine; Penn State College of Medicine; Texas Tech University Health Sciences Center School of Medicine; University of California, Davis School of Medicine; and University of Louisville School of Medicine. These programs vary in size and medical specialty focus but all include the reduction of student debt from savings in tuition costs. Each school’s mission to create a three-year pathway program differs; common themes include the ability to train physicians to practice in underserved areas or to allow students for whom the choice of specialty is known to progress more quickly. Compared with McMaster, these programs are small, but most capitalize on training and assessing competency across the undergraduate medical education–graduate medical education continuum and include conditional acceptance into an affiliated residency program. This article includes an overview of each CAMPP school with attention to admissions, curriculum, financial support, and regulatory challenges associated with the design of an accelerated pathway program. These programs are relatively new, with a small number of graduates; this article outlines opportunities and challenges for schools considering the development of accelerated programs.

The role of the gut microbiome in animal models of inflammatory and autoimmune disease is now well established. The human gut microbiome is currently being studied as a potential modulator of the immune response in rheumatic disorders. However, the vastness and complexity of this host–microorganism interaction is likely to go well beyond taxonomic, correlative observations. In fact, most advances in the field relate to the functional and metabolic capabilities of these microorganisms and their influence on mucosal immunity and systemic inflammation. An intricate relationship between the microbiome and the diet of the host is now fully recognized, with the microbiota having an important role in the degradation of polysaccharides into active metabolites. This Review summarizes the current knowledge on the metabolic role of the microbiota in health and rheumatic disease, including the advances in pharmacomicrobiomics and its potential use in diagnostics, therapeutics and personalized medicine.

Since the early 1900s, the role of periodontal disease in the pathogenesis of rheumatoid arthritis has been a matter of intense research. The last decade has witnessed many advances supporting a link between periodontitis, the presence of specific bacterial species (i.e. Porphyromonas gingivalis) and their effects in immune response. This review will examine available evidence on the individuals.

Despite the progress toward understanding the molecular pathogenesis of rheumatoid arthritis (RA), its cause remains elusive. Genes are important but rather insufficient to explain the majority of RA cases. This review describes the novel data supporting the microbiome and its interactions with the human host as potential en(‘in’)vironmental factors in RA pathogenesis.

Identification of patients at risk for incident disease or disease progression in osteoarthritis remains challenging, as radiography is an insensitive reflection of molecular changes that presage cartilage and bone abnormalities. Thus there is a widely appreciated need for biochemical and imaging biomarkers. We describe recent developments with such biomarkers to identify osteoarthritis patients who are at risk for disease progression.

Humans are not (and have never been) alone. From the moment we are born, millions of micro-organisms populate our bodies and coexist with us rather peacefully for the rest of our lives. This microbiome represents the totality of micro-organisms (and their genomes) that we necessarily acquire from the environment. Micro-organisms living in or on us have evolved to extract the energy they require to survive, and in exchange they support the physiological, metabolic and immune capacities that have contributed to our evolutionary success. Although currently categorized as an autoimmune disorder and regarded as a complex genetic disease, the ultimate cause of rheumatoid arthritis (RA) remains elusive. It seems that interplay between predisposing genetic factors and environmental triggers is required for disease manifestation. New insights from DNA sequence-based analyses of gut microbial communities and a renewed interest in mucosal immunology suggest that the microbiome represents an important environmental factor that can influence autoimmune disease manifestation. This Review summarizes the historical clues that suggest a possible role for the microbiota in the pathogenesis of RA, and will focus on new technologies that might provide scientific evidence to support this hypothesis.

Osteoarthritis (OA) is often a progressive and disabling disease, which occurs in the setting of a variety of risk factors – such as advancing age, obesity and trauma – that collude to incite a cascade of pathophysiological events within joint tissues. An important emerging theme in OA is a broadening of focus from a disease of cartilage to one of the ‘whole joint.’ The synovium, bone and cartilage are each involved in pathological processes that lead to progressive joint degeneration. Additional themes that have emerged over the past decade are novel mechanisms of cartilage degradation and repair, the relationship between biomechanics and biochemical pathways, the importance of inflammation and the role of genetics. In this article, we review the molecular, clinical and imaging evidence that synovitis is not an ‘incidental finding of OA’, but plays a significant role in disease pathogenesis, and could therefore represent a target for future treatments.

Background: Elevated levels of periostin (Postn) in the cartilage and bone are associated with osteoarthritis (OA). However, it remains unknown whether Postn loss-of-function can delay or prevent the development of OA. In this study, we sought to better understand the role of Postn in OA development and assessed the functional impact of Postndeficiency on post-traumatic and age-related OA in mice. Methods: The effects of Postndeficiency were studied in two murine experimental OA models using Postn−/−(n= 32) and littermate wild-type (wt) mice (n= 36). Post-traumatic OA was induced by destabilization of the medial meniscus (DMM) in 10-week-old mice (n= 20); age-related OA was analyzed in 24-month-old mice (n= 13). Cartilage degeneration was assessed histologically using the OARSI scoring system, and synovitis was evaluated by measuring the synovial lining cell layer and the cells density in the synovial stroma. Bone changes were measured by μCT analysis. Serum levels of Postn were determined by ELISA. Expression of Postn and collagenase-3 (MMP-13) was measured by immunostaining. RNA-seq was performed on chondrocytes isolated from 21-day old Postn−/−(n= 3) and wtmice (n= 3) to discover genes and pathways altered by Postnknockout. Results: Postn −/−mice exhibited significantly reduced cartilage degeneration and OARSI score relative to wtmice in post-traumatic OA after 8 weeks (maximum: 2.37 ± 0.74 vs. 4.00 ± 1.20, P= 0.011; summed: 9.31 ± 2.52 vs. 21.44 ± 6.01, P= 0.0002) and spontaneous OA (maximum: 1.93 ± 0.45 vs. 3.58 ± 1.16, P= 0.014; summed: 6.14 ± 1.57 vs. 11.50 ± 3.02, P= 0.003). Synovitis was significantly lower in Postn−/−mice than wtonly in the DMM model (1.88 ± 1.01 vs. 3.17 ± 0.63; P= 0.039). Postn−/−mice also showed lower trabecular bone parameters such as BV/TV, vBMD, Tb.Th, and Tb.N and high Tb. Sp in both models. Postn−/−mice had negligible levels of serum Postn compared with wt. Immunofluorescent studies of cartilage indicated that Postn−/−mice expressed lower MMP-13 levels than wt mice. RNA-seq revealed that cell-cell-adhesion and cell-differentiation processes were enriched in Postn−/−mice, while those related to cell-cycle and DNA-repair were enriched in wtmice. Conclusions: Postndeficiency protects against DMM-induced post-traumatic and age-related spontaneous OA. RNA-seq findings warrant further investigations to better understand the mechanistic role of Postn and its potential as a therapeutic target in OA.

To examine cyclooxygenase 2 inhibitor (coxib) utilization by rheumatologists for patients receiving nonsteroidal antiinflammatory drugs (NSAIDs) prior to the coxib market withdrawals.A prospective study of patients with rheumatoid arthritis enrolled in the Consortium of Rheumatology Researchers of North America registry was performed.Of 1,833 patients receiving prescription NSAIDs, 1,380 (75.3%) received gastroprotection, defined as either coxib monotherapy and/or gastroprotective agent (GPA) cotherapy, and 1,207 (65.8%) received coxibs. The distribution of gastroprotective strategies included 860 (46.9%) patients who were prescribed coxib monotherapy, 347 (18.9%) prescribed dual coxib plus GPA cotherapy, 173 (9.4%) prescribed a nonselective NSAID (NS‐NSAID) plus GPA cotherapy, and 453 (24.7%) prescribed an NS‐NSAID without GPA cotherapy. For patients with 0, 1, and ≥2 identifiable gastrointestinal (GI) risk factors, coxib prescribing rates as a proportion of NSAID agents were 64.1%, 66.4%, and 68.6%, respectively; among dual aspirin/NSAID users, coxib prescribing rates were 66.2%, 78.3%, and 68.5% of NSAID prescriptions, respectively.The majority of NSAID users were prescribed a gastroprotective strategy, primarily attributable to coxib utilization. Coxib utilization rates were consistently high across all levels of GI risk, including patients without identifiable risk factors. These data indicate that rheumatologists broadly adopted the coxib class of NSAIDs in a nonselective manner with respect to underlying GI risk and concomitant aspirin use. As novel therapeutic classes are introduced, early evaluation of prescribing patterns using arthritis registries can determine the appropriateness of prescribing patterns and may improve patient outcomes.

Osteoarthritis (OA) can be a progressive, disabling disease, leading to diminished quality of life, and, for over 500,000 individuals annually in the US, total joint replacement. The etiology of OA will vary among individuals, with potential roles for systemic factors (such as genetics and obesity) as well as for local biomechanical factors (such as muscle weakness, joint laxity and traumatic injury). Joint deterioration occurs over extended periods of time, and the diverse molecular mechanisms that mediate pathogenic events of early, mid and late disease are not yet fully understood. The success of biologic therapies in the treatment of rheumatoid arthritis has demonstrated that the blockade of a single dominant cytokine or regulatory molecule can prevent cartilage destruction in a complex disease, and has raised expectations that mechanism-based treatments could also be developed for patients with OA. In this review, we will address the biological mechanisms that mediate structural damage in OA and examine current targets that are candidates for disease modification. The challenges to drug development and the obstacles to disease modification strategies will also be addressed.

To evaluate the effects of cardiovascular comorbidities and aspirin coprescription on cyclooxygenase (COX)‐2 inhibitor (coxib) prescribing patterns among rheumatologists.A prospective cohort study was carried out with rheumatoid arthritis and osteoarthritis patients in the Consortium of Rheumatology Researchers of North America registry. Medication and comorbidity data were obtained prospectively from physician and patient questionnaires between March 2002 and September 2003. Multivariate adjusted associations between coxib use and specific cardiovascular variables, including aspirin use, were examined.A total of 3,522 arthritis patients were included. COX inhibitors, including coxibs, nonselective nonsteroidal antiinflammatory drugs (NSAIDs), and meloxicam, were prescribed to a larger proportion of osteoarthritis patients (68.4%) than rheumatoid arthritis patients (47.1%) in our study (P < 0.001). COX inhibitors were prescribed to the majority of aspirin users (51.5%) and a similar proportion of nonusers (49.8%). In multivariate analyses, independent predictors of coxib use versus nonselective NSAID use included diagnoses of osteoarthritis (odds ratio [OR] 2.52, 95% confidence interval [95% CI] 1.81–3.52) and diabetes (OR 1.63, 95% CI 1.06–2.51). Conversely, aspirin use independently predicted selection of a nonselective NSAID rather than a coxib (OR 0.73, 95% CI 0.55–0.98). Neither a history of myocardial infarction nor stroke predicted utilization of a coxib. Similarly, cardiovascular variables did not predict the use of rofecoxib versus celecoxib.Our data indicate that COX inhibitor coprescription among aspirin users is frequent. Despite cardiovascular concerns regarding the coxibs, our data suggest that aspirin use, but not cardiovascular comorbidities, predicted the selection of nonselective NSAIDs over coxibs.

The introduction of tumor necrosis factor-α antagonists in 1998 has had a significant impact on the treatment of rheumatoid arthritis. However, as use of these agents has increased worldwide, infrequent adverse events that were not apparent in pivotal controlled clinical trials required for registration have emerged.

No abstract.

Nitric oxide (NO) is synthesized via the oxidation of arginine by a family of nitric oxide synthases (NOS), which are either constitutive (ie. endothelial (ec)NOS and neuronal (nc)NOS) or inducible (iNOS). The production of nitric oxide plays a vital role in the regulation of physiological processes, host defence, inflammation and immunity. Pro-inflammatory effects include vasodilation, oedema, cytotoxicity and the mediation of cytokine-dependent processes that can lead to tissue destruction. Nitric oxide-dependent tissue injury has been implicated in a variety of rheumatic diseases, including systemic lupus erythematosus (SLE), rheumatoid arthritis and osteoarthritis. Conversely, the production of NO by endothelial cell NOS may serve a protective, or anti-inflammatory, function by preventing the adhesion and release of oxidants by activated neutrophils in the microvasculature. In this chapter we describe the multifaceted role of nitric oxide in inflammation and address the potential therapeutic implications of NOS inhibition.

No abstract.

To identify extracellular and intraarticular matrix components that are differentially expressed in normal and osteoarthritis (OA)–affected cartilage and to investigate their functions with respect to regulation of mediators of inflammation.Differential‐display reverse transcriptase–polymerase chain reaction (RT‐PCR) analysis of a pool of messenger RNA (mRNA) from 10 human OA cartilage samples and 5 normal cartilage samples was performed using arbitrary primers. Confirmatory analysis of the up‐regulated transcripts of fibronectin (FN) and osteopontin (OPN) was performed by RT‐PCR of individual RNA samples from a separate set of donors. The effect of recombinant OPN (or anti‐OPN antiserum) on chondrocyte function was examined by analyzing the spontaneous or interleukin‐1 (IL‐1)–induced release of nitric oxide (NO) and prostaglandin E2 (PGE2) from human OA‐affected cartilage under ex vivo conditions.Up‐regulation (300–700%) of FN and OPN mRNA was observed in human OA‐affected cartilage as compared with normal cartilage. Functional analysis of the role of OPN in OA cartilage showed that 1) Addition of 1 μg/ml (20 nM) of recombinant OPN to human OA‐affected cartilage under ex vivo conditions inhibited spontaneous and IL‐1β–induced NO and PGE2 production, and 2) neutralization of intraarticular OPN with anti‐OPN antiserum augmented NO production.The data indicate that one of the functions of intraarticular OPN, which is overexpressed in OA cartilage, is to act as an innate inhibitor of IL‐1, NO, and PGE2 production. These findings suggest that the production of pleiotropic mediators of inflammation that influence cartilage homeostasis, such as NO and PGE2, is regulated by the interaction of chondrocytes with differentially expressed proteins within the extracellular matrix.

Interleukin 1 (IL-1), produced by both synovial cells and chondrocytes, plays a pivotal role in the pathogenesis of cartilage destruction in osteoarthritis (OA). We examined the specific expression and function of IL-1 receptor family-related genes in human joint tissues. Gene array analysis of human normal and OA-affected cartilage showed mRNA expression of IL-1 receptor accessory protein (IL-1RAcp) and IL-1 type I receptor (IL-1RI), but not IL-1 antagonist (IL-1ra) and IL-1 type II decoy receptor (IL-1RII). Similarly, human synovial and epithelial cells showed an absence of IL-1RII mRNA. Functional genomic analyses showed that soluble (s) IL-1RII, at picomolar concentrations, but not soluble TNF receptor:Fc, significantly inhibited IL-1β-induced nitric oxide (NO) and/or prostaglandin E2production in chondrocytes, synovial and epithelial cells. In OA-affected cartilage, the IC50for inhibition of NO production by sIL-1RII was 2 log orders lower than that for sIL-1RI. Human chondrocytes that overexpressed IL-1RII were resistant to IL-1-induced IL-1β mRNA accumulation and inhibition of proteoglycan synthesis. In osteoarthritis, deficient expression by chondrocytes of innate regulators or antagonists of IL-1 such as IL-1ra and IL-1RII (soluble or membrane form) may allow the catabolic effects of IL-1 to proceed unopposed. The sensitivity of IL-1 action to inhibition by sIL-1RII has therapeutic implications that could be directed toward correcting this unfavorable tissue(s) dependent imbalance.

Nitric oxide (NO) is induced by exposure of endothelial cells (EC) to acetylcholine, where it acts in a paracrine manner to relax smooth muscle and as a defensive molecule to inhibit the adhesion of leukocytes to EC. The mechanism(s) of the antiadhesive properties of constitutive NO are poorly understood. In these studies, we found that NO induced by acetylcholine exerts autocrine effects, which interfere with normal adhesion mechanisms. The function of the adhesion molecule intercellular adhesion molecule 1 (CD54) of EC was measured using latex beads coated with antibody to CD54 as a model for CD54 ligation by the leukocyte β2 integrin. Recruitment of filamentous actin (F-actin) and of the signaling molecule vasodilator-stimulated phosphoprotein (VASP) was measured by immunofluorescence microscopy. Exposure of EC to anti-CD54 beads induced the subplasmalemmal assembly of F-actin and VASP. Acetylcholine blocked the anti-CD54 bead–induced translocation of F-actin and VASP; this effect was reversed by inhibition of NO production. The NO action did not interfere with binding, but completely inhibited the assembly of the focal activation complex, which we believe is necessary for firm heterotypic adhesion between leukocyte and EC. Further studies indicated that the NO effect was due to its capacity to raise cGMP. Platelet endothelial cell adhesion molecule 1 (CD31, also implicated in leukocyte adhesion) did not mimic CD54 responses. These results indicate that the ligation of endothelial cell CD54 induces the assembly of subplasmalemmal F-actin and the recruitment of VASP. NO derived from constitutive nitric oxide synthase acts to disrupt these CD54-elicited endothelial cell responses. This action may protect vascular endothelium from leukocyte-mediated injury.

No abstract.

Pharmacogenomics is a revolution in molecular medicine, especially in view of the development of microarray technologies and proteomics to monitor gene and protein expression. The ability to monitor up to 60,000 potential parameters in a clinical setting gives a whole new meaning to 1) toxic effects, 2) side effects, 3) primary and secondary targets, and 4) drug resistance and nonresponders during a clinical trial. Pharmacogenomics may set a new standard to monitor the effects of drugs such as NSAIDS or disease‐modifying drugs in diseases such as arthritis. The present article outlines a pharmacogenomics clinical protocol that is in progress, a study that will address some of the above questions. Drug Dev. Res. 49:29–33, 2000. © 2000 Wiley‐Liss, Inc.

In these studies we provide conclusive evidence that (β/γ) actin present in human neutrophils is a substrate for nitric oxide (NO)‐dependent ADP ribosylation and that this modification is associated with the inhibition of actin polymerization. A 43‐kDa substrate for NO‐dependent ADP ribosylation was identified as actin by four methods: (1) comigration with the botulinum C2 toxin substrate by two‐dimensional gel electrophoresis (pI 5.2), (2) identity between the peptide map generated by V8 protease digestion of the NO and botulinum C2 substrates, (3) immunoprecipitation with antiactin antibodies, and (4) the ability of NO to ADP ribosylate purified neutrophil G‐actin in the presence of plasma membrane cofactors. Because the ADP ribosylation of actin by the botulinum C2 toxin is known to inhibit F‐actin polymerization, we examined the effect of NO on actin assembly. Flow cytometry revealed that NO inhibited formyl‐methionine‐leucine‐phenylalanine (fMLP)‐dependent (SO s at 37°) F‐actin formation (108 ± 8 vs. 89 ± 6 relative fluorescence units, P< .02). These results were confirmed by quantification of F‐actin formation by gel scanning (10% sodium dodecyl sulfate gel, Coomassie, and densitometry): pretreatment of polymorphonuclear leukocytes with NO resulted in a reduction of fMLP‐induced, cytoskeletal‐associated F‐actin, which was accompanied by an increase of Triton‐soluble G‐actin. NO also inhibited F‐actin formation, as observed by means of rhodamine phalloidin staining of neutrophils adherent to a fibronectin‐coated surface. This effect was accompanied by a dose‐dependent inhibition of neutrophil adherence in NO‐treated cells. The data indicate that NO inhibits cytoskeletal assembly and adherence in human neutrophils in association with the ADP ribosylation of actin. J. Leukoc. Biol.58: 196–202; 1995.

Neutrophil aggregation is mediated by the β2integrin CD11b/CD18, which has limited expression on the surface membrane of resting cells but is recruited from intracellular organelles after cell activation. We have previously found that CD11b/CD18 newly translocated to the plasma membrane does not contribute to adhesion but must be modified to be functional. Because neutrophil aggregation induced by phorbol myristate acetate (PMA) is accompanied by de novo phosphorylation of the CD18 cytoplasmic tail, we sought to determine whether CD11b/CD18 phosphorylation is separately regulated in the different cellular compartments. Accordingly, [32P]‐labeled CD11b/CD18 was immunoprecipitated from purified neutrophil‐specific granule or plasma membrane lysates. In plasma membrane fractions, as in whole cell lysates, CD18 became phosphorylated in cells exposed to PMA but not in untreated cells or cells treated with N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP). The a chain, CD11h, was phosphorylated under all conditions. In contrast, only marginal phosphorylation of specific granule‐associated CD18 or CD11b was observed. Calyculin A, an inhibitor of serine/threonine phosphatases (ppl > pp2a), induced strong phosphorylation of CD18 in the plasma membrane but not in the specific granules. Addition of intact specific granule membranes to the plasma membranes from PMA‐treated neutrophils markedly decreased phosphorylation in both CD11b and CD18 subunits. These data suggest that the phosphorylation of CD11b/CD18, which accompanies neutrophil activation, is limited to plasma membrane‐associated molecules. Phosphorylation, either constitutive or induced, is absent in the specific granule membranes. The difference may be due to a specific granule‐associated phosphatase, probably distinct from ppl. Therefore adhesion‐competent plasma membrane CD11b/CD18 and adhesion‐incompetent specific granule CD11b/CD18 differ in their state of phosphorylation. J. Leukoc. Biol. 61: 313–321; 1997.

ABSTRACT: To evaluate a flare of systemic lupus erythematosus (SLE) during pregnancy and to differentiate it from diseases of pregnancy, serological parameters are often utilized. However, there are conflicting reports regarding the merit of conventional measurements of complement and activation products. While in normal pregnancy the levels of serum C3, C4, and CH50gradually rise, a decline in these levels occurs during the course of pregnancy in selected SLE patients. There is controversy regarding whether such falls represent decreases in the overall synthesis of complement or activation, the former theory being supported by a report of normal levels of the C1s‐C1 inhibitor complex. During normal pregnancies, increases of complement split products, such as plasma C3a, may occur, and these correlate positively with elevations of C3. In pregnancies complicated by lupus, increases of C3a are often accompanied by a decline in total C3 and CH50. In a minority of non‐SLE patients, preeclampsia has been associated with elevations of a variety of complement split products. Ba, C3a, C4d, SC5b‐9, indicating activation of both the classical and alternative pathways. The CH50levels tend to remain normal in these patients. In contrast, elevations of complement split products frequently accompany disease flares in patients with SLE. A high ratio of CH50/Ba may differentiate patients with preeclampsia from those with active SLE. A decline in conventional measures of C3, C4, or CH50which is accompanied by elevations of complement split products appears to differentiate a lupus flare from non‐SLE diseases of pregnancy.

Nonsteroidal antiinflammatory drugs (NSAIDs) inhibit neutrophil functions via mechanisms separate from their capacity to inhibit prostaglandin synthesis. We have studied discrete events in the process of signal transduction: NSAIDs but not a related analgesic drug (acetaminophen), inhibited aggregation in response to the chemoattractants f-Met-Leu-Phe (FMLP), leukotriene B4, and C5a. NSAIDs, but not acetaminophen, inhibited binding of radiolabeled FMLP to purified neutrophil membranes. Gpp(NH)p, a GTPase insensitive analog of GTP, also inhibited the binding of FMLP but, paradoxically, enhanced superoxide anion generation and lysozyme release. The inhibition of ligand binding by NSAIDs did not correlate with their capacity to inhibit FMLP-induced increments in diacylglycerol (DG): piroxicam, but not salicylate effectively inhibited appearance of label ([3H]arachidonate, [14C] glycerol) in DG. Finally, NSAIDs exerted differential effects on the viscosity of neutrophil plasma membranes and multilamellar vesicles (liposomes): membrane viscosity was increased by piroxicam and indomethacin, decreased by salicylate, and unaffected by acetaminophen. Thus, the different effects of NSAIDs on discrete pathways are not due to their shared capacity to reduce ligand binding but rather to a capacity to uncouple postreceptor signaling events that depend upon the state of membrane fluidity.

Chemically modified tetracyclines [CMT‐3 (IC50∼6–13 μM = ∼2.5–5 μg/ml) and CMT‐8 (IC50∼26 μM = 10 μg/ml), but not CMT‐1, ‐2 or ‐5], which lack anti‐microbial activity, inhibited nitrite production in LPS‐stimulated macrophages. Unlike competitive inhibitors of l‐arginine which inhibited the specific activity of inducible nitric oxide synthase (iNOS) in cell‐free extracts, CMTs exerted no such direct effect on the enzyme. CMTs could, however, be shown to inhibit both iNOS mRNA accumulation and protein expression in LPS‐stimulated cells. Tetracyclines (doxycycline and CMT‐3) unlike hydrocortisone had no significant effect on murine macrophages transfected with iNOS promoter (tagged to a luciferase reporter gene) in the presence of LPS. However, doxycycline and CMT‐3 augmented iNOS mRNA degradation, in LPS‐stimulated murine macrophages. These studies show a novel mechanism of action of tetracyclines which harbours properties to increase iNOS mRNA degradation and decrease iNOS protein expression and nitric oxide production in macrophages. This property of tetracyclines may have beneficial effects in the treatment of various diseases where excess nitric oxide has been implicated in the pathophysiology of these diseases.

Objective. Neuropsychiatric manifestations are common in patients with systemic lupus erythematosus (SLE), but accurate diagnosis is often difficult. We conducted a prospective study to determine the utility of neurometric quantitative electroencephalography (QEEG) as an indicator of cerebral dysfunction in SLE patients.

Nitric oxide (NO), first identified as an endothelium-derived relaxation factor, is now recognized to regulate the functions of many mammalian cells and tissues. Nitric oxide is synthesized via the oxidation of arginine by a family of nitric oxide synthases (NOS), which are either constitutive and calcium dependent (cNOS) or inducible and calcium independent (iNOS). The endogenous production of nitric oxide plays a vital role in the regulation of physiological processes (e.g., blood vessel tone, neurotransmission) as well as in host defense and immunity. There is increasing evidence that nitric oxide also plays a complex role in the modulation of the inflammatory response. The induction of nitric oxide synthase (iNOS) in tissues can lead to the sustained production of high concentrations of nitric oxide which may exert pro-inflammatory effects including vasodilation, edema, cytotoxicity, and the mediation of cytokine dependent processes. Conversely, the production of NO by endothelial cell cNOS may serve a protective, or anti-inflammatory, function by preventing the adhesion and release of oxidants by activated neutrophils in the microvasculature. In this review we describe the multifaceted role of nitric oxide in inflammation, particularly focusing on the regulation of inflammatory cells and the repertoire of molecular processes targeted by NO that control cellular functions.Biosynthesis of Nitric OxideNitric oxide is synthesized via L-arginine oxidation by a family of nitric oxide synthases (NOS). Isomeric forms of nitric oxide synthase, representing at least three distinct gene products, have been cloned in bovine, rat, mice, and human tissues (1-5) (Table I). The three isoforms of the enzyme vary in calcium dependence, kinetics, and regulation. However, all nitric oxide synthases are flavoproteins which require NADPH and tetrahydrobiopterin as co-factors. The amino acid sequences deduced from the cDNA of the endothelial and neuronal cNOSs and iNOS have been reported (1-3, 6).

Nitric oxide (NO) has been implicated in both cartilage degradation and cell survival. Importantly, NO has been shown, in a cell-type-dependent manner, to directly cause cell death or indirectly promote cell death by compromising the ability of cells to detoxify intra- or extracellular oxidants. In this study we examined the role of NO in the survival of bovine chondrocytes exposed to catabolic cytokines (interleukin-1 (IL-1); tumor necrosis factor [TNF]) with or without the addition of an exogenous oxidant stress (e.g., H2O2, HOOCl, etc.). The exposure of chondrocytes to a mixture of IL-1 and TNF (IL-1/TNF) results in the release of NO but did not alter cell viability. However, there was evidence of NO-dependent oxidative responses in the IL-1/TNF group, as we observed an increased level of intracellular oxidants as well as the appearance of a 55 kD nitrated protein which reflects the formation of peroxynitrite. We next analyzed viability with H2O2. The LD50 for IL-1/TNF-treated cells was 0.1 mM (vs. 1 mM for control). The enhanced sensitivity was completely reversed when cells were incubated with the NO synthase inhibitor 1-n5-1-iminoethylornithine (NIO). To test whether cell death was caused by compromising the ability of cells to detoxify extracellular oxidants, we examined the hexose monophosphate shunt (HMPS) response in cells given H2O2. Treatment of control cells with H2O2 resulted in a fourfold increase in HMPS activity. In contrast, IL-1/TNF cells exhibited no increase in HMPS activity. The attenuation of stimulated HMPS activity was reversed by the coaddition of NIO. Thus, these data indicate that 1) endogenous NO mediates cytokine-dependent susceptibility to oxidant injury and 2) this effect is in part due to impaired activation of the HMPS. In inflamed joints replete with cytokines and oxidants, NO may contribute to chondrocyte death and progressive joint destruction. J. Cell. Physiol. 172:183–191, 1997. © 1997 Wiley-Liss, Inc.

Previous studies have shown that neutrophils in the circulation of patients with active systemic lupus erythematosus (SLE) are activated as judged by their increased surface expression of the β2-integrin CD11b/CD18. Since activation of neutrophils leads to altered expression of another adhesion molecule, L-selectin (LS), we examined neutrophils from patients with SLE for changes in the expression of CD11b/CD18 and LS by cytofluorographic analysis of immunofluorescent-labeled cells. Overall there was no difference between surface expression of CD11b/CD18 on neutrophils from SLE patients or controls [mean fluorescence 225 ± 26 vs 225 ± 13 relative fluorescence units (RFU), respectively]. However, as previously reported, neutrophils from patients with more active disease (activity score 3, UCH Middlesex activity score) expressed greater CD11b/CD18 than neutrophiIs from controls (319 ± 40 RFU, P &lt; 0.03, n = 9) or from patients with less active disease (193 ± 10 RFU, P &lt; 0.006). Indeed, CD11b/CD18 expression correlated directly with disease activity (r = 0.54, P &lt; 0.02). Stimulation of neutrophils ex vivo with the chemoattractant N-formyl-methionylleucyl-phenylalanine (100 nM) induced up-regulation of CD11b/CD18 in cells from both SLE patients and controls (205 ± 12% vs 239 ± 15% of basal, respectively), but neutrophiIs from the most active patients (score 3) increased CD11b/CD18 expression less than controls (175 ± 12% of basal, P &lt; 0.003, n = 9). The magnitude of the stimulated increment in expression of CD11b/CD18 on neutrophils correlated inversely with SLE activity (r = -0.64, P &lt; 0.003, n = 20). Surprisingly, we observed no change in LS expression on neutrophils from SLE patients compared to controls (143 ± 14 vs 141 ± 16 RFU, respectively) even in patients with the highest activity indices (154 ± 21 RFU). In contrast to CD11b/CD18, there was no correlation between LS expression and disease activity (r = 0.12, P = NS). Stimulation of neutrophils reduced the expression of LS similarly in both controls and SLE patients (67 ± 3% vs 58 ± 4% reduction, respectively) and did not correlate with disease activity (r = 0.07, P = NS, n = 20). These results show, for the first time, that changes in CD11b/CD18 expression do not correlate with LS expression on neutrophils from patients with active SLE. Moreover, since C5a and other chemoattractants (over a range of concentrations) stimulated changes of identical magnitude in surface expression of CD11b/CD18 and LS, our data suggest that the exposure of neutrophils to chemoattractants in the circulation of patients with active SLE is not sufficient to account for all of the alterations observed in neutrophil adhesion molecules. Copyright 1994, 1999 Academic Press

The object of this study was to determine the incidence of seropositivity to B. burgdorferiby the commonly available enzyme-linked immunosorbent assay (ELISA) in patients with SLE and other rheumatic diseases and to evaluate immunoblot analysis as a tool to differentiate true from false positive ELISA. Sera were obtained from patients with SLE (n= 35), rheumatoid arthritis (n= 26), seronegative arthritis (n = 28) and Lyme disease (n= 18). Reactivity to B. burgdorferi antigens was analysed by two available diagnostic techniques: ELISA and immunoblot. Correlations were made between seroreactivity to B. burgdorferiand standard serological tests of autoimmunity: antibodies to nuclear antigens, dsDNA, cardiolipin, SSA and SSB. Seroreactivity to B. burgdorferiantigens by the ELISA system was detected in 40% of patients with SLE, 8% of patients with rheumatoid arthritis and 4% with seronegative arthritis. Among patients seropositive by ELISA, immunoblots were negative in all cases. However, eight of 14 patients with rheumatoid arthritis (57%) showed cross-reactivity to multiple borrelial antigens. No significant correlations were found between Lyme seropositivity by ELISA and other autoantibodies except IgM rheumatoid factor (r = 0.61, P < 0.01 ) in patients with rheumatoid arthritis.In conclusion: a positive ELISA for Lyme disease was found in up to 40% of patients with established SLE and also in other rheumatic diseases. However, specific serum antibodies to Borrelia were not confirmed by the more specific immunoblot technique. We conclude that immunoblot analysis can help differentiate a false from true positive ELISA for Lyme disease. These findings should caution the clinician with regard to the limitations of current diagnostic testing for Lyme disease, particularly in patients with connective tissue disease.

Production of nitric oxide by solid tumors may have important ramifications regarding tumor growth and potential metastasis. This study demonstrated that the chondrosarcoma of the Swarm rat has upregulated mRNA for inducible nitric oxide synthase and produces nitric oxide. These results were confirmed by (a) the, presence of a 4.4‐kb band of mRNA detected by Northern blot using a probe for inducible nitric oxide synthase, (b) a 133‐kDa band of prothein that was detected with either a polyclonal or monoclonal antibody to the inducible nitric oxide synthase of the murine macrophage. and (c) the detection of nitrites from the culture medium of freshly cultured, isolated chondrosarcoma cells. This study showed that the expression of inducible nitric oxide synthase and the production of nitric oxide by the tumor can be increased by stimulation with endotoxin lipopolysaccharide and can be inhibited by inducible nitric oxide synthase inhibitors (L‐N(g)‐monomethyl arginine and aminoguanidine). Immunostaining confirmed the presence of inducible nitric oxide synthase within the tumor cells and appeared to localize the enzyme to the cytoplasm of the cells. A human chondrosarcoma was also shown to have an upregulated inducible nitric oxide synthase by both the detection of mRNA for inducible nitric oxide synthase and the presence of nitrites from the culture medium of the tumor in organ culture. Because the chondrosarcoma of the Swarm rat is a well differentiated solid tumor that rarely metas‐tasizes, nitric oxide may be produced by the tumor to promote local growth by effects on vascular supply.

The destructive capacity of the neutrophil has long been appreciated, and the presence of extraordinary numbers of neutrophils in the synovial fluid of patients with RA supports a role for these cells in the pathogenesis of joint destruction. In this article, we reviewed the current state of knowledge of neutrophil function in the inflammatory response, and emphasized the subjects of neutrophil/endothelial adhesion and the role of chemoattractants and cytokines in neutrophil mobilization. We also discussed the mechanisms of action of neutrophil destruction of cartilage and the interplay of signals between the neutrophil and the chondrocyte. The capacity of many of the drugs used to treat RA to interfere with one or several of these processes underscores the importance of the neutrophil in RA and suggests that future therapeutic strategies could target neutrophil activation within the synovial space.

Cyclooxygenase (COX), the key enzyme required for the conversion of arachidonic acid to prostaglandins was first identified over 20 years ago. Drugs, like aspirin, that inhibit cyclooxygenase activity have been available to the public for about 100 years. In the past decade, however, more progress has been made in understanding the role of cyclooxygenase enzymes in biology and disease than at any other time in history. Two cyclooxygenase isoforms have been identified and are referred to as COX‐1 and COX‐2. Under many circumstances the COX‐1 enzyme is produced constitutively (i.e., gastric mucosa) whereas COX‐2 is inducible (i.e., sites of inflammation). Here, we summarize the current understanding of the role of cyclooxygenase‐1 and ‐2 in different physiological situations and disease processes ranging from inflammation to cancer. We have attempted to include all of the most relevant material in the field, but due to the rapid progress in this area of research we apologize that certain recent findings may have been left out.—DuBois, R. N., Abramson, S. B., Crofford, L., Gupta, R. A., Simon, L. S., van de Putte, L. B. A., Lipsky, P. E. Cyclooxygenase in biology and disease. FASEB J.12, 1063–1073 (1998)

Objective: To examine the effects of non-steroidal anti-inflammatory drugs (NSAIDS) on nitric oxide (NO) and prostaglandin E2(PGE2) production in chondrocytes from three different species.

Activation of the membrane-associated NADPH oxidase of neutrophils requires several cytosolic factors including p47phox, p67phoxand p21roc2. We compared NADPH oxidase activity with the membrane translocation of p47phox, p67phox, and p21rac2. In a cell-free system, GTPγS stimulated translocation of p47phoxand p67phoxto the plasma membrane only in the presence of arachidonate, and this translocation correlated with NADPH oxidase activity of the reisolated plasma membranes (R= 0.94 and 0.97, respectively). In contrast, GTPγS-stimulated p21rac2translocation with or without arachidonate, and the extent of translocation did not correlate with oxidase activity (R= 0.17). Neutrophil cytoplasts were used to relate membrane translocation of p47phox, p67phoxand p21rac2to membrane oxidase activity in response to the inflammatory agonists. Whereas N-formyl-methionyl-leucyl-phenylalanine stimulated equimolar, transient membrane translocation of p47phoxand p67phoxwhich kinetically paralleled NADPH oxidase activity, relatively little p21rac2translocated (moles of p47phox/p21rac2= 16.6). Moreover, although phorbol 12-myristate 13-acetate stimulated both the stable translocation of p47phoxand p67phoxand sustained NADPH oxidase activity, it did not stimulate p21rac2translocation. From these data we conclude that membrane translocation of p21rac2does not regulate NADPH oxidase activity stoichiometrically.