A major target autoantigen in anti-neutrophil cytoplasmic antibody-associated vasculitis is myeloperoxidase (MPO). Although MPO-specific CD4+ Th cells seem to orchestrate renal injury, the role of the Th17 subset is unknown. We hypothesized that Th17 cells direct injurious anti-MPO autoimmunity in experimental murine anti-MPO-induced glomerulonephritis (GN). We immunized mice with MPO to establish autoimmunity, resulting in systemic IL-17A production with MPO-specific dermal delayed-type hypersensitivity. We triggered disease using antibodies to the glomerular basement membrane to induce glomerular deposition of MPO by neutrophils. Wild-type mice developed necrotizing GN with an influx of glomerular leukocytes and albuminuria. In contrast, mice deficient in the key Th17 effector cytokine IL-17A were nearly completely protected. The protective effects resulted partly from reduced neutrophil recruitment, which led to less disposition of glomerular MPO. To test whether IL-17A also drives autoimmune delayed-type hypersensitivity in the kidney, we injected MPO into the kidneys of MPO-sensitized mice. IL-17A deficiency reduced accumulation of renal macrophages and renal CCL5 mRNA expression. In conclusion, IL-17A contributes to the pathophysiology of autoimmune anti-MPO GN, suggesting that it may be a viable therapeutic target for this disease.

OBJECTIVE

To assess the characteristics of airway inflammation in patients with COPD.

METHODS

We measured the sputum concentration of interleukin-8 (IL-8), a chemokine involved in the migration and activation of neutrophils and eosinophils. We also measured myeloperoxidase (MPO) as a parameter of neutrophil activity and eosinophil cationic protein (ECP) as a parameter of eosinophil activity. Spontaneous sputum samples were obtained from 33 patients with stable COPD and 30 patients with asthma. Induced sputum samples were obtained from 12 normal control subjects.

RESULTS

The sputum concentration of IL-8 was significantly higher in the patients with COPD than in the patients with asthma or in the control subjects (p<0.0001). Concentrations of MPO and ECP were significantly higher in the patients with COPD than in the control subjects but did not differ significantly between the patients with COPD and those with asthma. In the patients with COPD, the sputum concentration of IL-8 was significantly correlated with the concentration of MPO (r=0.55, p<0.001) and of ECP (r=0.53, p<0.01). The sputum concentration of IL-8 was negatively correlated with FEV1/FVC (r=-0.78, p<0.0001) in the COPD group.

CONCLUSIONS

Results suggest the activation of both neutrophils and eosinophils in the airways of patients with COPD. It appears that IL-8 plays a primary role in this activation. The sputum concentration of IL-8 appeared to be closely associated with the degree of airflow obstruction in patients with COPD and may serve as a marker in evaluating the severity of airway inflammation, which is a risk factor for COPD.

Using data obtained from cDNA representational difference analysis to identify genes induced during neutrophilic differentiation of the 32D clone 3G (32Dcl3G) cells, we isolated cDNA clones for murine and human leucine-rich alpha2-glycoprotein (hLRG), a protein with unknown function purified 25 years ago. Expression of LRG during differentiation of 32Dcl3G cells preceded the expression of lactoferrin and gelatinase but followed myeloperoxidase. LRG transcripts were also detected in human neutrophils and progenitor cells but not in peripheral blood mononuclear cells. Notably, LRG expression was up-regulated during neutrophilic differentiation of human MPD and HL-60 cells but down-regulated during monocytic differentiation of HL-60 cells. The hLRG gene was localized to chromosome 19p13.3, a region to which the genes for several neutrophil granule enzymes also map. The putative promoter region of LRG was found to contain consensus-binding sites for PU.1, C/EBP, STAT, and MZF1. These results suggest that LRG is a novel marker for early neutrophilic granulocyte differentiation.

BACKGROUND

Activation of cannabinoid (CB)(1) receptors results in attenuation of experimental colitis. Our aim was to examine the role of CB(2) receptors in experimental colitis using agonists (JWH133, AM1241) and an antagonist (AM630) in trinitrobenzene sulfonic acid (TNBS)-induced colitis in wildtype and CB(2) receptor-deficient (CB(2) (-/-)) mice.

METHODS

Mice were treated with TNBS to induce colitis and then given intraperitoneal injections of the CB(2) receptor agonists JWH133, AM1241, or the CB(2) receptor antagonist AM630. Additionally, CB(2) (-/-) mice were treated with TNBS and injected with JWH133 or AM1241. Animals were examined 3 days after the induction of colitis. The colons were removed for macroscopic and microscopic evaluation, as well as the determination of myeloperoxidase activity. Quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) for CB(2) receptor was also performed in animals with TNBS and dextran sodium sulfate colitis.

RESULTS

Intracolonic installation of TNBS caused severe colitis. CB(2) mRNA expression was significantly increased during the course of experimental colitis. Three-day treatment with JWH133 or AM1241 significantly reduced colitis; AM630 exacerbated colitis. The effect of JWH133 was abolished when animals were pretreated with AM630. Neither JWH133 nor AM1241 had effects in CB(2) (-/-) mice.

CONCLUSIONS

We show that activation of the CB(2) receptor protects against experimental colitis in mice. Increased expression of CB(2) receptor mRNA and aggravation of colitis by AM630 suggests a role for this receptor in normally limiting the development of colitis. These results support the idea that the CB(2) receptor may be a possible novel therapeutic target in inflammatory bowel disease.

Human alveolar macrophages were lavaged from surgically resected lungs and from lungs of normal subjects. Macrophages that had been purified by glass adherence were maintained in tissue culture for as long as 54 days. After 3-4 wk in vitro they underwent transformation into multinucleated giant cells. These aged cells had more than 30 times the phagocytic capacity that the same group of cells had had after 1 day in vitro. Phagocytosis of heat-killed Candida albicans was inhibited by iodoacetate, sodium fluoride, potassium cyanide, and low partial pressures of oxygen, suggesting that these cells require both oxidative and glycolytic energy sources for maximal particle ingestion. Alveolar macrophages and monocyte-derived macrophages killed Listeria monocytogenes with similar efficiency, but neutrophils were more efficient than either of the other cell types. Bacterial killing is probably not dependent upon myeloperoxidase in the monocyte-derived macrophage or in the alveolar macrophage since histochemical stains for peroxidase do not stain either cell type. C. albicans blastospores, which are killed by neutrophils and monocytes that contain myeloperoxidase, were not killed by human alveolar macrophages during the 4 hr of observation. Large cells with supernormal phagocytic capacity were recovered from patients with postobstructive pheumonia and from one patient with recurrent bacterial pneumonia, indicating that macrophage function can be altered in certain disease states. Human alveolar macrophages are unique human phagocytes in their dependence on an oxygen tension greater than 25 mm HG for maximal phagocytosis. Carbon dioxide tensions as high as 70 mm Hg did not alter phagocytosis when the pH of the medium was held constant. These data suggest that the increased susceptibility to pneumonia of patients with chronic bronchitis or atelectasis may be in part related to suboptimal phagocytosis by macrophages in areas of the lung with depressed oxygen tension.

We measured the cyanide-insensitive pyridine nucleotide oxidase activity of fractionated resting and phagocytic neutrophils from 11 normal donors, 1 patient with hereditary deficiency of myeloperoxidase, and 7 patients with X-linked chronic granulomatous disease (CGD). When measured under optimal conditions (at pH 5.5 and in the presence of 0.5 mM Mn++), NADPH oxidase activity increased fourfold with phagocytosis and was six-fold higher than with NADH. Phagocytic neutrophils from patients with CGD were markedly deficient in NADPH oxidase activity.

ANCA have been demonstrated to induce the respiratory burst in primed neutrophils. In this study we have extended the investigations on neutrophil activation by ANCA directed against proteinase 3 (PR3), myeloperoxidase (MPO) and lactoferrin (LF), and we have analysed the underlying mechanisms. All three ANCA antigens were expressed on the cell surface of primed neutrophils. Superoxide production assayed by both cytochrome c reduction and oxidation of dihydrorhodamine 123, was induced by heterologous polyclonal anti-MPO and anti-LF antibodies, and ANCA-positive plasma samples. Induction of superoxide production was dose-dependent. F(ab')2 fragments did not induce the respiratory burst. Blockade of Fc receptors by specific MoAbs showed that anti-Fc gamma RII antibodies were able to turn off the ANCA-induced respiratory burst, whereas anti-Fc gamma RIII antibodies did not. Plasma samples that induced the respiratory burst did not differ from samples that did not induce superoxide production with respect to ANCA titre, but had higher levels of the IgG3 subclass of ANCA. Levels of the other subclasses of ANCA were comparable between those samples. We conclude that ANCA-induced activation of primed neutrophils is Fc gamma RII-dependent, and appears to be facilitated by antibodies of the IgG3 subclass.

OBJECTIVE

Overproduction of nitric oxide by inducible nitric oxide synthase (iNOS) has been proposed as a pathogenic factor in colitis. The objective of this study was to examine the role of iNOS using iNOS-deficient mice in experimental colitis.

METHODS

Colitis was induced by intrarectal instillation of 3% acetic acid and assessed for neutrophilic infiltration and intestinal injury over 7 days. iNOS messenger RNA expression was also measured.

RESULTS

At 24 hours, acetic acid induced a mild colitis in wild-type mice. An increase in neutrophil infiltration and tissue edema was also observed. In the iNOS-deficient mice, a twofold increase in macroscopic damage was observed. Neutrophil infiltration and tissue edema were similar to those in wild-type animals at this time point. Although inflammation in wild-type mice had resolved by 7 days, a sevenfold increase in damage score and elevated myeloperoxidase level were still evident in iNOS-deficient mice. A striking increase in the message for iNOS was observed in inflamed wild-type mice at 24 hours and was still present at 72 hours. No message was found in iNOS-deficient mice.

CONCLUSIONS

Induction of iNOS seems to be a critical protective response to injury in intestinal inflammation possibly by reducing leukocytic infiltration.

Thirty-five children <2 years of age mechanically ventilated for respiratory syncytial virus (RSV) infection were randomized to receive an intravenous infusion of 15 mg/kg MEDI-493 or placebo. RSV concentration was measured in tracheal secretions by plaque assay before and at 24-h intervals after treatment. The reduction in tracheal RSV concentration from day 0 to day 1 (-1.7+/-0.28 vs. -0. 6+/-0.21 log10 pfu/mL; P=.004) and from day 0 to day 2 (-2.5+/-0.26 vs. -1.0+/-0.41 log10 pfu/mL; P=.012) was significantly greater in the MEDI-493 group than in the placebo group. RSV concentration in nasal aspirates did not differ significantly between the groups. No significant differences were observed in the tracheal aspirate white blood cell count, or myeloperoxidase or eosinophilic cationic protein concentration, or in measures of disease severity between the groups. Thus, treatment with 15 mg/kg MEDI-493 intravenously was well-tolerated and significantly reduced RSV concentration in tracheal aspirates of children with respiratory failure due to RSV.

BACKGROUND

Mimetics of bacterial DNA, given orally or subcutaneously, protect mice from experimental colitis via a toll-like receptor (TLR)-9-dependent mechanism. The goal of the study was to define whether synthetic viral RNA, polyinosinic acid:cytidylic acid [poly(I:C)], which is also a potent immunomodulator, might also affect murine colitis and, if so, define whether such effects were mediated by TLR3, which is one of at least 4 known receptors for this viral RNA analog.

METHODS

Mice (C57BL6, IL-10KO, or TLR3 KO) were administered 1.5% dextran sodium sulfate (DSS) in drinking water for 7 days. Two hours before treatment with DSS, mice were given phosphate-buffered saline (PBS) or poly(I:C) 20 mug subcutaneously (s.c.), or 100 mug intragastrically (i.g.).

RESULTS

In wildtype mice s.c. administration of poly(I:C) dramatically protected against DSS-induced colitis as assessed by every parameter analyzed, which included body weight, rectal bleeding, colonic myeloperoxidase, histopathology, serum keratinocyte-derived chemokine, serum amyloid A, and lipocalin-2. In contrast, i.g. administration of poly(I:C) offered no protection in this colitis model nor did its administration activate the innate immune system as assessed by serologic parameters. Subcutaneous poly(I:C) protected against DSS-induced colitis equally well in C57BL6 and IL-10KO mice, indicating that this antiinflammatory cytokine is not required for such protection. Protection against colitis given by poly(I:C) treatment was ablated in TLR3 KO, indicating that the protective action of this viral RNA analog was mediated by this receptor.

CONCLUSIONS

Activation of TLR3 on cells that are accessible by systemic, but not oral, administration of synthetic viral RNA results in protection against the acute inflammation that can ensue upon damage of the gut epithelium. Thus, this viral RNA analog, which is under clinical trials for other inflammatory disorders (e.g., lupus), may also have therapeutic value for inflammatory bowel disease.

Wegener's granulomatosis and microscopic polyangiitis are idiopathic systemic vasculitides strongly associated with antineutrophil cytoplasmic autoantibodies (ANCA). In Wegener's granulomatosis, ANCA are mostly directed against proteinase 3 (PR3), whereas in microscopic polyangiitis ANCA are directed against myeloperoxidase; increases in levels of these autoantibodies precede or coincide with clinical relapses in many cases. In vitro, ANCA can further activate primed neutrophils to release reactive oxygen species and lytic enzymes, and, in conjunction with neutrophils, can damage and lyse endothelial cells. Patients with Wegener's granulomatosis or microscopic polyangiitis have an increased percentage of neutrophils that constitutively express PR3 on their membrane. These neutrophils can be stimulated by ANCA, without priming. In vivo, transfer of splenocytes from myeloperoxidase-deficient mice immunized with mouse myeloperoxidase into wild-type mice resulted in pauci-immune systemic vasculitis. A similar experiment in PR3-deficient mice did not cause significant vasculitic lesions. Together, clinical, in vitro and in vivo experimental data support a pathogenic role for ANCA in Wegener's granulomatosis and microscopic polyangiitis, although this role is more evident for myeloperoxidase-specific ANCA than for PR3-specific ANCA. Several controlled trials have led to an evidence-based approach for the treatment of ANCA-associated vasculitis, and further studies, based on new insights into pathogenesis, are in progress.

Allopurinol is a scavenger of the highly reactive hydroxyl radical (k2 approx. 10(9) M-1 X s-1). One product of attack of hydroxyl radical upon allopurinol is oxypurinol, which is a major metabolite of allopurinol. Oxypurinol is a better hydroxyl radical scavenger than is allopurinol (k2 approx. 4 X 10(9) M-1 X s-1) and it also reacts with the myeloperoxidase-derived oxidant hypochlorous acid. Hence the protective actions of allopurinol against reperfusion damage after hypoxia need not be entirely due to xanthine oxidase inhibition.

The release of "neutrophil extracellular traps" (NETs) has been identified as a novel immune response in innate immunity. Neutrophil extracellular traps are composed of neutrophil-derived circulating free DNA (cf-DNA), histones, and neutrophil cytoplasm-derived proteins such as proteases. Here, we studied the putative predictive value of plasma cf-DNA/NETs for the development of sepsis and mortality after multiple trauma. In a prospective pilot study with 45 multiple trauma (Injury Severity Score>16) patients, cf-DNA was directly quantified in plasma. Blood samples were sequentially obtained daily from admission to our Trauma Center until day 10. Because of limited intensive care unit (ICU) stay of less than 3 days, 8 patients have been excluded, resulting in 37 patients that were evaluated. Time kinetics of cf-DNA/NETs was compared with C-reactive protein (CRP), interleukin (IL) 6, leukocyte counts, and myeloperoxidase. The severity of the injury was calculated on the basis of the Injury Severity Score, as well as Multiple Organ Dysfunction Score, Sequential Organ Failure Assessment, and Simplified Acute Physiology Score II on ICU. Initially high cf-DNA/NETs values (>800 ng/mL) with recurrent increased values between days 5 to 9 were associated with subsequent sepsis, multiple organ failure, and death. In conjunction with cf-DNA/NETs, IL-6 was significantly elevated after admission. However, the development of a second hit was not indicated by IL-6. In contrast to cf-DNA/NETs, no difference in CRP kinetics was observed between patients with and without development of sepsis. Circulating free DNA/NETs kinetics rather followed kinetics of Multiple Organ Dysfunction Score, Sepsis-related Organ Failure Assessment, leukocyte counts, and partially of myeloperoxidase. Circulating free DNA/NETs seems to be a valuable additional marker for the calculation of injury severity and/or prediction of inflammatory second hit on ICU. However, a large clinical trial with severely injured patients should confirm the prognostic value of neutrophil-derived cf-DNA/NETs.

BACKGROUND

Patients have been described who have both anti-neutrophil cytoplasm antibodies (ANCA) and anti-glomerular basement membrane (GBM) antibodies. We have attempted to define the true prevalence of such "double positive" patients, and describe in detail their clinical features and outcome.

METHODS

We have reviewed all serologic assays performed between 1990 and 2000 in a single institution, and the case notes of patients having sera positive for both ANCA and anti-GBM antibodies. During this time 20,392 sera were initially tested for ANCA, and 4808 sera tested for anti-GBM antibodies.

RESULTS

Five percent of all ANCA-positive serum samples were also positive for anti-GBM antibodies, and 32% of all anti-GBM positive samples had detectable ANCA. Of 27 patients with both antibodies, 82% had anti-myeloperoxidase specific P-ANCA. Pulmonary hemorrhage occurred in 44%. Renal biopsy showed extensive glomerular cellular crescents in most patients. Patient and renal survival rates were 52% and 26%, respectively, at one year. Sixty-eight percent of patients were dialysis-dependent at presentation, and none of these recovered renal function, despite immunosuppression with or without plasma exchange.

CONCLUSIONS

Serologic evidence of double positivity for both ANCA and anti-GBM antibodies is common in patients with either antibody. In our study these patients have a poor prognosis when presenting with severe disease and initially behave more like anti-GBM disease than vasculitis. Recovery from severe renal failure is rare.

BACKGROUND

Strong evidence supports a role for CD40 ligand (CD40L) as marker and mediator of inflammatory diseases such as atherosclerosis. Despite extensive characterization of CD40, the classic receptor of CD40L, its role in immune defense against inflammatory diseases remains uncertain. The present study aimed to characterize the contribution of CD40 signaling to atherogenesis.

RESULTS

Surprisingly, mice deficient in both CD40 and the low-density lipoprotein receptor did not develop smaller lesions in the aortic arch, root, and thoracoabdominal aorta compared with mice deficient only in the low-density lipoprotein receptor that consumed an atherogenic diet for 8 and 16 weeks. By flow cytometry, radioactive binding assays, and immunoprecipitation, we demonstrate that CD40L interacts with the integrin Mac-1, which results in Mac-1-dependent adhesion and migration of inflammatory cells as well as myeloperoxidase release in vitro. Furthermore, mice deficient in CD40L show significantly reduced thioglycolate-elicited invasion of inflammatory cells into the peritoneal cavity compared with mice deficient in CD40 and wild-type controls. Inhibition of Mac-1 in low-density lipoprotein receptor-deficient mice attenuates lesion development and reduces lesional macrophage accumulation.

CONCLUSIONS

These observations identify the interaction of CD40L and Mac-1 as an alternative pathway for CD40L-mediated inflammation. This novel mechanism expands understanding of inflammatory signaling during atherogenesis.

OBJECTIVE

Mesalamine is the first-line therapy for colitis, but it lacks potency and is only effective for mild-to-moderate forms of this disease. Hydrogen sulphide has been shown to be a potent, endogenous anti-inflammatory substance, modulating leukocyte-endothelial adhesion and leukocyte migration. The purpose of this study was to determine if an H(2)S-releasing derivative of mesalamine (ATB-429) would exhibit increased potency and effectiveness in a mouse model of colitis.

METHODS

Colitis was induced in mice with trinitrobenzene sulphonic acid and the effects of ATB-429 and mesalamine were compared in several treatment regimens. The severity of colitis was determined using several indices, including a disease activity score (comprised of scores for diarrhea, weight loss and fecal blood), colonic myeloperoxidase activity and macroscopic/microscopic scoring of tissue injury.

RESULTS

Irrespective of the treatment regiment, ATB-429 was more effective than mesalamine in reducing the severity of colitis. ATB-429 was particularly effective in reducing granulocyte infiltration into the colonic tissue (by approximately 70%), as well as reducing the expression of mRNA for several key proinflammatory cytokines/chemokines (e.g., TNFalpha, IFNgamma). Treatment with ADT-OH, the H(2)S-releasing moiety of ATB-429, did not affect severity of colitis.

CONCLUSIONS

ATB-429 exhibits a marked increase in anti-inflammatory activity and potency in a murine model of colitis, as compared to mesalamine. These results are consistent with recently described anti-inflammatory effects of H(2)S. ATB-429 may represent an attractive alternative to mesalamine for the treatment of inflammatory bowel disease.

In serum and sputum samples from 15 patients with cystic fibrosis (CF) suffering from chronic Pseudomonas aeruginosa lung infections, concentrations and/or activities of elastase derived from polymorphonuclear leukocytes (PMN), cathepsin G, myeloperoxidase (MPO), and superoxide dismutase (SOD), as well as concentrations of the proteinase inhibitors alpha 1-proteinase inhibitor (alpha 1-PI) and alpha 2-macroglobulin (alpha 2-M), were determined. High enzyme concentrations compared with those in normal control subjects were found for PMN elastase (mean, 96.1 +/- 91.7 micrograms/ml), cathepsin G (mean, 5.9 +/- 6.0 micrograms/ml), and MPO (mean, 138.0 +/- 177 micrograms/ml) in patients' sputum samples. Superoxide dismutase was not detectable in any of the sputum specimens (below 1 ng/ml). Proteinase inhibitor concentrations were elevated in serum samples (alpha 1-PI: mean, 3,457 +/- 1,084 micrograms/ml; alpha 2-M: mean, 4,835 +/- 1,334 micrograms/ml). Means of 61 +/- 38 micrograms/ml alpha 1-PI and 29 +/- 31 micrograms/ml alpha 2-M were present in the sputum specimens. Both proteinase inhibitors were functional in the serum samples. However, sputum alpha 1-PI was proteolytically degraded, as shown by western blot technique, and was not able to bind 125I-labeled PMN elastase, as shown by autoradiography. Only 10.9 +/- 8.5% of the total alpha 1-PI in the sputum samples was complexed to PMN elastase and 3.6 +/- 3.2% to cathepsin G. On the other hand, 96.2 +/- 96.8% of the total PMN elastase and 78.0 +/- 100% of cathepsin G were unbound in the sputum samples. The study suggests that the imbalance between PMN proteinases and their inhibitors is due to inactivation of alpha 1-PI in the sputum caused by proteolytic or oxidative attack from PMN enzymes.

OBJECTIVE

Nonalcoholic fatty liver disease (NAFLD) is linked to obesity and diabetes, suggesting an important role of adipose tissue in the pathogenesis of NAFLD. Here, we aimed to investigate the interaction between adipose tissue and liver in NAFLD and identify potential early plasma markers that predict nonalcoholic steatohepatitis (NASH).

METHODS

C57Bl/6 mice were chronically fed a high-fat diet to induce NAFLD and compared with mice fed a low-fat diet. Extensive histological and phenotypical analyses coupled with a time course study of plasma proteins using multiplex assay were performed.

RESULTS

Mice exhibited pronounced heterogeneity in liver histological scoring, leading to classification into four subgroups: low-fat low (LFL) responders displaying normal liver morphology, low-fat high (LFH) responders showing benign hepatic steatosis, high-fat low (HFL) responders displaying pre-NASH with macrovesicular lipid droplets, and high fat high (HFH) responders exhibiting overt NASH characterized by ballooning of hepatocytes, presence of Mallory bodies, and activated inflammatory cells. Compared with HFL responders, HFH mice gained weight more rapidly and exhibited adipose tissue dysfunction characterized by decreased final fat mass, enhanced macrophage infiltration and inflammation, and adipose tissue remodeling. Plasma haptoglobin, IL-1β, TIMP-1, adiponectin, and leptin were significantly changed in HFH mice. Multivariate analysis indicated that in addition to leptin, plasma CRP, haptoglobin, eotaxin, and MIP-1α early in the intervention were positively associated with liver triglycerides. Intermediate prognostic markers of liver triglycerides included IL-18, IL-1β, MIP-1γ, and MIP-2, whereas insulin, TIMP-1, granulocyte chemotactic protein 2, and myeloperoxidase emerged as late markers.

CONCLUSIONS

Our data support the existence of a tight relationship between adipose tissue dysfunction and NASH pathogenesis and point to several novel potential predictive biomarkers for NASH.

Previous work in our laboratory and others using the weight drop (WD) model of traumatic brain injury (TBI) has shown that neutrophils accumulate in brain tissue during the initial 24 h posttrauma as measured by myeloperoxidase (MPO) activity and immunohistochemistry. This study compares the acute inflammatory response to TBI over time, as measured by MPO activity, in the WD and controlled cortical impact (CCI) models. Anesthetized adult Sprague-Dawley rats were traumatized using WD (10-g weight dropped 5 cm) or CCI (4 m/sec, 2.5 mm depth). At 2, 24, 48, or 168 h after trauma, rats (n = 4-5/group at each time) were anesthetized and killed, the brains were removed, and 6-mm coronal slices from traumatized and contralateral hemispheres were assayed for MPO activity. Nontraumatized rats (n = 4) served as controls. Three additional rats underwent a more severe CCI (3 mm depth) with MPO activity assayed at 24 h. A separate group of rats (n = 6) was subjected to WD trauma and killed at 2 weeks after injury for analysis of lesion volume. MPO activity in the traumatized hemisphere was demonstrated at 24 and 48 h in both the WD (0.3152 +/- 0.0472 and 0.3017 +/- 0.0228 U/g, respectively, p < 0.05 vs controls) and CCI (0.1866 +/- 0.0225 and 0.1937 +/- 0.0772 U/g, respectively, p < 0.05 vs controls) models. MPO activity was below the sensitivity of the assay in the control, 2 h, and 168 h groups in both models.(ABSTRACT TRUNCATED AT 250 WORDS)

The proteinase-activated receptor 2 (PAR-2) is a member of a family of G protein-coupled receptors for proteases. Proteases cleave PARs within the extracellular N-terminal domains to expose tethered ligands that bind to and activate the cleaved receptors. PAR-2 is highly expressed in colon in epithelial and neuronal elements. In this study we show that PAR-2 activation prevents the development and induces healing of T helper cell type 1-mediated experimental colitis induced by intrarectal administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) in mice. A role for PAR-2 in the protection against colon inflammation was explored by the use of SLIGRL-NH(2), a synthetic peptide that corresponds to the mouse tethered ligand exposed after PAR-2 cleavage. TNBS-induced colitis was dose-dependently reduced by the administration of SLIGRL-NH(2), whereas the scramble control peptide, LSIGRL-NH(2), was uneffective. This beneficial effect was reflected by increased survival rates, improvement of macroscopic and histologic scores, decrease in mucosal content of T helper cell type 1 cytokines, protein, and mRNA, and a diminished myeloperoxidase activity. SLIGRL-NH(2), but not the scramble peptide, directly inhibited IFN-gamma secretion and CD44 expression on lamina propria T lymphocytes. Protection exerted by PAR-2 in TNBS-treated mice was reverted by injecting mice with a truncated form of calcitonin gene-related peptide and by sensory neurons ablation with the neurotoxin capsaicin. Collectively, these studies show that PAR-2 is an anti-inflammatory receptor in the colon and suggest that PAR-2 ligands might be effective in the treatment of inflammatory bowel diseases.

Myeloperoxidase (MPO), a heme protein abundantly expressed in polymorphonuclear neutrophils (PMN), has long been viewed to function primarily as a bactericidal enzyme centrally linked to innate host defense. Recent observations now extend this perspective and suggest that MPO is profoundly involved in the regulation of cellular homeostasis and may play a central role in initiation and propagation of acute and chronic vascular inflammatory disease. For example, low levels of MPO-derived hypochlorous acid (HOCl) interfere with intracellular signaling events, MPO-dependent oxidation of lipoproteins modulates their affinity to macrophages and the vessel wall, MPO-mediated depletion of endothelial-derived nitric oxide (NO) impairs endothelium-dependent vasodilatation, and nitrotyrosine (NO(2)Tyr) formation by MPO sequestered into the vessel wall may affect matrix protein structure and function. Future studies are needed to further elucidate the significance of MPO in the development of acute and chronic vascular disease and to evaluate MPO as a potential target for treatment.

To study local lung inflammation, 34 subjects had endotoxin (1-4 ng/kg) instilled into a lung segment and saline instilled into a contralateral segment followed by bronchoalveolar lavage (BAL) at 2 h, 6 h, 24 h, or 48 h. Endotoxin instillation resulted in a focal inflammatory response with a distinct time course. An early phase (2 h to 6 h) revealed an increase in neutrophils (p = 0.0001) with elevated cytokines (tumor necrosis factor [TNF]-alpha, TNF receptors [TNFR], interleukin [IL]-1beta, IL-1 receptor antagonist, IL-6, granulocyte-colony-stimulating factor [G-CSF], all p < or = 0.002, but no change in IL-10) and chemokines (IL-8, epithelial neutrophil activating protein-78, monocyte chemotactic protein-1, macrophage inflammatory protein [MIP]-1alpha, MIP-1beta, all p < or = 0.001, but no change in growth-regulated peptide-alpha). A later phase (24 h to 48 h) showed increased neutrophils, macrophages, monocytes, and lymphocytes (all p < or = 0.02), and a return to basal levels of most mediators. Elevated levels of inflammatory markers (TNFR(1), TNFR(2), L-selectin, lactoferrin, and myeloperoxidase) persisted in the BAL at 48 h (p < or = 0.001). Increased permeability to albumin occurred throughout both phases (p = 0.001). Blood C-reactive protein, serum amyloid A, IL-6, IL-1ra, G-CSF, but not TNF-alpha increased by 8 h (all p < or = 0.008). The local pulmonary inflammatory response to endotoxin has a unique qualitative and temporal profile of inflammation compared with previous reports of intravenous endotoxin challenges. This model provides a means to investigate factors that initiate, amplify, and resolve local lung inflammation.

Histiocytic sarcoma is a rare malignant neoplasm that occurs in lymph nodes, skin, and the gastrointestinal tract. Many previously published cases were likely misdiagnosed examples of non-Hodgkin lymphoma. Only small numbers of bona fide examples exist in the world literature; cases arising primarily at extranodal sites are not well described and often seem to go unrecognized. To characterize these tumors further, 14 extranodal histiocytic sarcomas were analyzed. Hematoxylin and eosin sections were reexamined, immunohistochemistry was performed, and clinical details were obtained from referring hospitals. Eight patients were female and 6 male (median age, 55 years; range, 15-89 years). All patients presented with a solitary mass, ranging in size from 1.8 to 12 cm (median 6.8 cm). Seven tumors arose in soft tissue (6 lower limb; 1 upper limb), 5 in the gastrointestinal tract (1 involving both stomach and colon, 1 ileum, 2 rectum, 1 anus), 1 in the nasal cavity, and 1 in the lung. Three gastrointestinal tract tumors also involved regional lymph nodes, and 1 involved the liver. Most cases had infiltrative margins. The tumors were generally composed of sheets of large epithelioid cells with abundant eosinophilic cytoplasm, oval to irregular nuclei, vesicular chromatin, and large nucleoli. Binucleated cells were common, and 6 cases contained tumor giant cells. Mitoses ranged from 1 to 64 per 10 HPF (median 11 per 10 HPF). Necrosis was present in 8 cases. Nearly all tumors showed a striking inflammatory infiltrate, most often of neutrophils or lymphocytes. All cases were reactive for LCA, CD45RO, and CD68 (KP1 and PG-M1); 13 of 14 (93%) expressed CD4, 12 of 14 (86%) lysozyme, 8 of 10 (80%) CD31, 7 of 14 (50%) S-100 protein, and 5 of 14 (36%) focal CD1a. Two tumors showed weak, focal cytoplasmic positivity for CD30, and 1 for epithelial membrane antigen. The tumors were negative for ALK-1, CD21, CD35, CD3, CD20, CD34, myeloperoxidase, HMB-45, and keratins. Gastrointestinal tract cases were negative for c-kit and desmin. Six patients were treated with postoperative radiation and 7 with chemotherapy (CHOP or ProMACE-MOPP). Follow-up was available for 10 patients (median, 24 months; range, 4 months to 11 years). Two tumors recurred locally, and 5 patients developed distant spread: 3 to lymph nodes, 1 to lung, and 1 to bone. At the last follow-up, 2 patients have died of disseminated disease, 4 and 5 months following initial diagnosis. The patients who died thus far had the largest primary tumors. Histiocytic sarcoma may arise primarily in soft tissue and shows reproducible histologic features, including abundant eosinophilic cytoplasm and a prominent inflammatory infiltrate. Metastatic carcinoma, metastatic melanoma, and large cell non-Hodgkin lymphomas should be excluded by immunohistochemistry. Histiocytic sarcoma has the potential for an aggressive clinical course, most often with lymph node involvement. However, a subset of cases presenting with clinically localized disease have a favorable long-term outcome. Tumor size may be a prognostic factor.

BACKGROUND

The efflux capacity of high-density lipoprotein (HDL) with cultured macrophages associates strongly and negatively with coronary artery disease status, indicating that impaired sterol efflux capacity might be a marker-and perhaps mediator-of atherosclerotic burden. However, the mechanisms that contribute to impaired sterol efflux capacity remain poorly understood.

OBJECTIVE

Our aim was to determine the relationship between myeloperoxidase-mediated oxidative damage to apolipoprotein A-I, the major HDL protein, and the ability of HDL to remove cellular cholesterol by the ATP-binding cassette transporter A1 (ABCA1) pathway.

RESULTS

We quantified both site-specific oxidation of apolipoprotein A-I and HDL's ABCA1 cholesterol efflux capacity in control subjects and subjects with stable coronary artery disease or acute coronary syndrome. Subjects with coronary artery disease and acute coronary syndrome had higher levels of chlorinated tyrosine 192 and oxidized methionine 148 compared with control subjects. In contrast, plasma levels of myeloperoxidase did not differ between the groups. HDL from the subjects with coronary artery disease and acute coronary syndrome was less able to accept cholesterol from cells expressing ABCA1 compared with HDL from control subjects. Levels of chlorinated tyrosine and oxidized methionine associated inversely with ABCA1 efflux capacity and positively with atherosclerotic disease status. These differences remained significant after adjusting for HDL-cholesterol levels.

CONCLUSIONS

Our observations indicate that myeloperoxidase may contribute to the generation of dysfunctional HDL with impaired ABCA1 efflux capacity in humans with atherosclerosis. Quantification of chlorotyrosine and oxidized methionine in circulating HDL might be useful indicators of the risk of cardiovascular disease that are independent of HDL-cholesterol.