The atheroprotective effects of HDL are mediated by several mechanisms, including its role in reverse cholesterol transport and via its antiinflammatory properties. However, not all HDL is functionally similar. HDL and apolipoprotein A-I may become dysfunctional or even proinflammatory and thus promote atherosclerosis. ApoAI posttranslational modification can have a large impact on its function. Myeloperoxidase modification of apoAI impairs its function as a cholesterol acceptor, and the molecular changes induced by myeloperoxidase have been studied in detail. These studies provide the basis for the development of an oxidant-resistant form of apoAI and clinical measures of HDL modification and dysfunction, which may be useful as a treatment criterion.

Apolipoprotein A-I (apoAI), the major protein of high density lipoprotein, plays an important role in reverse cholesterol transport via its activity as an ABCA1-dependent acceptor of cellular cholesterol. We reported recently that myeloperoxidase (MPO) modification of apoAI inhibits its ABCA1-dependent cholesterol acceptor activity (Zheng, L., Nukuna, B., Brennan, M. L., Sun, M., Goormastic, M., Settle, M., Schmitt, D., Fu, X., Thomson, L., Fox, P. L., Ischiropoulos, H., Smith, J. D., Kinter, M., and Hazen, S. L. (2004) J. Clin. Invest. 114, 529-541). We also reported that MPO-mediated chlorination preferentially modifies two of the seven tyrosines in apoAI, and loss of parent peptides containing these residues dose-dependently correlates with loss in ABCA1-mediated cholesterol acceptor activity (Zheng, L., Settle, M., Brubaker, G., Schmitt, D., Hazen, S. L., Smith, J. D., and Kinter, M. (2005) J. Biol. Chem. 280, 38-47). To determine whether oxidative modification of apoA-I tyrosine residues was responsible for the MPO-mediated inactivation of cholesterol acceptor activity, we made recombinant apoAI with site-specific substitutions of all seven tyrosine residues to phenylalanine. ApoAI and the tyrosine-free apoAI were equally susceptible to dose-dependent MPO-mediated loss of ABCA1-dependent cholesterol acceptor activity, as well as lipid binding activity. MPO modification altered the migration of apoAI on SDS gels and decreased its alpha-helix content. MPO-induced modification also targeted apoAI tryptophan and lysine residues. Specifically, we detected apoAI tryptophan oxidation to mono- and dihydroxytryptophan and apoAI lysine modification to chlorolysine and 2-aminoadipic acid. Thus, tyrosine modification of apoAI is not required for its MPO-mediated inhibition of cholesterol acceptor activity.

OBJECTIVE

Preclinical and clinical studies have shown beneficial effects of infusions of apolipoprotein A-I (ApoA-I) on atherosclerosis. ApoA-I is also a target for myeloperoxidase-mediated oxidation, leading in vitro to a loss of its ability to promote ATP-binding cassette transporter A1-dependent macrophage cholesterol efflux. Therefore, we hypothesized that myeloperoxidase-mediated ApoA-I oxidation would impair its promotion of reverse cholesterol transport in vivo and the beneficial effects on atherosclerotic plaques.

RESULTS

ApoA-I(-/-) or apolipoprotein E-deficient mice were subcutaneously injected with native human ApoA-I, oxidized human ApoA-I (myeloperoxidase/hydrogen peroxide/chloride treated), or carrier. Although early postinjection (8 hours) levels of total ApoA-I in plasma were similar for native versus oxidized human ApoA-I, native ApoA-I primarily resided within the high-density lipoprotein fraction, whereas the majority of oxidized human ApoA-I was highly cross-linked and not high-density lipoprotein particle associated, consistent with impaired ATP-binding cassette transporter A1 interaction. In ApoA-I(-/-) mice, ApoA-I oxidation significantly impaired reverse cholesterol transport in vivo. In advanced aortic root atherosclerotic plaques of apolipoprotein E-deficient mice, native ApoA-I injections led to significant decreases in lipid content, macrophage number, and an increase in collagen content; in contrast, oxidized human ApoA-I failed to mediate these changes. The decrease in plaque macrophages with native ApoA-I was accompanied by significant induction of their chemokine receptor CCR7. Furthermore, only native ApoA-I injections led to a significant reduction of inflammatory M1 and increase in anti-inflammatory M2 macrophage markers in the plaques.

CONCLUSIONS

Myeloperoxidase-mediated oxidation renders ApoA-I dysfunctional and unable to (1) promote reverse cholesterol transport, (2) mediate beneficial changes in the composition of atherosclerotic plaques, and (3) pacify the inflammatory status of plaque macrophages.

Saffron has been proposed as a promising candidate for cancer chemoprevention. The purpose of this investigation was to investigate the chemopreventive action and the possible mechanisms of saffron against diethylnitrosamine (DEN)-induced liver cancer in rats. Administration of saffron at doses of 75, 150, and 300 mg/kg/day was started 2 weeks prior to the DEN injection and was continued for 22 weeks. Saffron significantly reduced the DEN-induced increase in the number and the incidence of hepatic dyschromatic nodules. Saffron also decreased the number and the area of placental glutathione S-transferase-positive foci in livers of DEN-treated rats. Furthermore, saffron counteracted DEN-induced oxidative stress in rats as assessed by restoration of superoxide dismutase, catalase, and glutathione-S-transferase levels and diminishing of myeloperoxidase activity, malondialdehyde and protein carbonyl formation in liver. The results of immunohistochemical staining of rat liver showed that saffron inhibited the DEN-mediated elevations in numbers of cells positive for Ki-67, cyclooxygenase 2, inducible nitric oxide synthase, nuclear factor-kappa B p-65, and phosphorylated tumor necrosis factor receptor. Saffron also blocked the depletion in the number of cells positive for TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling) and M30 CytoDeath in liver tissues of DEN-treated rats. In vitro experiments carried out using HepG2 cells also confirmed these findings and showed inhibition of nuclear factor-kappa B activation, increased cleavage of caspase-3, as well as DNA damage and cell cycle arrest upon saffron treatment.

CONCLUSIONS

This study provides evidence that saffron exerts a significant chemopreventive effect against liver cancer through inhibition of cell proliferation and induction of apoptosis. This report also shows some evidence that saffron protects rat liver from cancer via modulating oxidative damage and suppressing inflammatory response.

BACKGROUND

Helicobacter pylori (H. pylori) is the major cause of chronic active gastritis and peptic ulcer disease. Recent studies have shown that H. pylori produces various cytokines that are related to neutrophil or mononuclear cell accumulation. Interleukin-17 (IL-17) is the founding member of an emerging family of inflammatory cytokines whose biological activities remain incompletely defined. In this study, the contributions of IL-17 to the induction of gastric inflammation and to the protection from H. pylori infection were investigated using IL-17 gene-knockout (IL-17(-/-)) mice.

METHODS

IL-17(-/-)and wild-type C57BL/6 mice were challenged with H. pylori CPY2052 (2 x 10(8) CFU/mL) and the histological and microbiological evaluation were carried out at specified times. IL-17 and myeloperoxidase (MPO) protein levels in tissues were assayed in duplicate using ELISA kits.

RESULTS

In wild-type mice, IL-17 was undetected at baseline; however, the protein expression of IL-17 was induced after infection with H. pylori. A severe infiltration of neutrophils appeared in the submucosa and the lamina propria in wild-type mice. In contrast, the degree of neutrophil infiltration in IL-17(-/-) mice was significantly lower than that in wild-type mice. Although wild-type mice infected with H. pylori showed drastically higher MPO activity compared with uninfected wild-type mice, any significant increase in the enzyme activity was not revealed in infected IL-17(-/-) mice. The number of H. pylori colonized in the stomach of IL-17(-/-) mice was significantly lower than that of wild-type mice from 1 to 6 months after infection.

CONCLUSIONS

These results suggest that IL-17 may play an important role in the inflammatory response to the H. pylori infection and ultimately influence the outcome of the H. pylori-associated disease.

We tested the hypothesis that targeted transgenic overexpression of human extracellular superoxide dismutase (EC-SOD) would preserve alveolar development in hyperoxia-exposed newborn mice. We exposed newborn transgenic and wild-type mice to 95% oxygen (O2) or air x 7 days and measured bronchoalveolar lavage cell counts, and lung homogenate EC-SOD, oxidized and reduced glutathione, and myeloperoxidase. We found that total EC-SOD activity in transgenic newborn mice was approximately 2.5x the wild-type activity. Hyperoxia-exposed transgenic mice had less pulmonary neutrophil influx and oxidized glutathione than wild-type littermates at 7 days. We measured alveolar surface and volume density in animals exposed to 14 days more of air or 60% O2. Hyperoxia-exposed transgenic EC-SOD mice had significant preservation of alveolar surface and volume density compared with wild-type littermates. After 7 days 95% O2 + 14 days 60% O2, lung inflammation measured as myeloperoxidase activity was reduced to control levels in all treatment groups.

Nonalcoholic fatty liver disease (NAFLD) is currently the most common liver disease in the world. It encompasses a histological spectrum, ranging from simple, nonprogressive steatosis to nonalcoholic steatohepatitis (NASH), which may progress to cirrhosis and hepatocellular carcinoma. While liver-related complications are confined to NASH, emerging evidence suggests both simple steatosis and NASH predispose to type 2 diabetes and cardiovascular disease. The pathogenesis of NAFLD is currently unknown, but accumulating data suggest that oxidative stress and altered redox balance play a crucial role in the pathogenesis of steatosis, steatohepatitis, and fibrosis. We will examine intracellular mechanisms, including mitochondrial dysfunction and impaired oxidative free fatty acid metabolism, leading to reactive oxygen species generation; additionally, the potential pathogenetic role of extracellular sources of reactive oxygen species in NAFLD, including increased myeloperoxidase activity and oxidized low density lipoprotein accumulation, will be reviewed. We will discuss how these mechanisms converge to determine the whole pathophysiological spectrum of NAFLD, including hepatocyte triglyceride accumulation, hepatocyte apoptosis, hepatic inflammation, hepatic stellate cell activation, and fibrogenesis. Finally, available animal and human data on treatment opportunities with older and newer antioxidant will be presented.

BACKGROUND

The objective of this study was to characterize the acute and chronic inflammation induced by the intramural injection of peptidoglycan-polysaccharide (PG-PS) into the distal colon of genetically susceptible rats.

METHODS

Blood-to-lumen clearance of 51Cr-ethylenediaminetetraacetic acid, colonic myeloperoxidase activity, colon weight, and plasma nitrite and nitrate levels were determined to quantitate colonic mucosal injury, inflammation, and nitric oxide (NO) production, respectively.

RESULTS

Intramural injection of PG-PS into the distal colon produced a local biphasic inflammatory response composed of an acute episode 3 days after injection; this was followed by a spontaneous reactivation of chronic granulomatous colitis manifested by colonic thickening, adhesions, and infiltration of the submucosa and muscularis propria with macrophages, neutrophils, and lymphocytes at 3-4 weeks. Mucosal ulcers were evident only at 3 weeks, but hepatic nodules, splenic necrosis, and arthritis were evident at both 3 and 4 weeks after PG-PS injection. PG-PS produced significant increases in colonic mucosal permeability, myeloperoxidase activity, and plasma nitrite and nitrate levels at 3 weeks postinjection compared with controls. PG-PS stimulated the production of nitrite by elicited peritoneal macrophages and neutrophils in vitro.

CONCLUSIONS

PG-PS produces a chronic granulomatous colitis in rats; this colitis is characterized by enhanced NO production.

Chronic mucosal inflammation is considered a risk factor for colorectal cancer. Neutrophils are a major source of oxidants, whereas cyclooxygenase 2 (COX-2) and Hypoxia Inducible Factor-1alpha (HIF-1alpha) protein expression levels are increased in inflammatory and malignant lesions. The main purpose of the present study was to evaluate myeloperoxidase (MPO) positive cell infiltration, COX-2 and HIF-1alpha protein expression in colorectal carcinogenesis, especially in its early phases, using immunohistochemistry and immunofluorescence confocal microscopy techniques. MPO, COX-2 and HIF-1alpha proteins were expressed at higher rates in the normal colorectal mucosa of patients with inflammatory bowel diseases and colorectal tumours than in patients with normal colonoscopy. A gradual increase in COX-2 and HIF-1alpha protein expression was observed in dysplastic aberrant crypt foci, adenomas and carcinomas, showing a strong relation to dysplasia. In conclusion, the present study supports the hypothesis of a key role of inflammation in malignant transformation of colorectal mucosa. The evaluation of some early markers related to inflammation in the mucosa of the large bowel may serve as potential tool for prognosis and therapeutic strategies.

An appreciable percentage of patients with serum anti-glomerular basement membrane (anti-GBM) antibodies also have antineutrophil cytoplasmic antibodies (ANCA), against either myeloperoxidase (MPO-ANCA), or proteinase 3 (PR3-ANCA). In sera without ANCA, the anti-GBM antibodies have been shown to react mainly with the noncollagenous domain (NC1) of Type IV collagen, and especially with its alpha 3 chain, alpha 3(IV)NC1. In most sera, the antibodies can be partially blocked by a monoclonal antibody (Mab17) against alpha 3(IV)NC1, suggesting that a limited region is recognized. Although there is evidence that some anti-GBM antibodies that coexist with ANCA react with alpha 3(IV)NC1, extensive analysis of the specificity of such anti-GBM antibodies has not been reported. In the study presented here, sera were analyzed from 332 patients tested both for anti-GBM antibodies and ANCA (MPO or PR3-ANCA) and found to have one or more positive tests. Of the 100 sera with anti-GBM antibodies, 38 also had ANCA-25 with MPO-ANCA (66%), 12 with PR3-ANCA (32%), and one with both (2%). Of the 232 sera with ANCA only, 153 had MPO-ANCA (66%), 75 had PR3-ANCA (32%), and four had both (2%). Sera was also analyzed from 259 other patients who had positive ANCA tests and were not tested for anti-GBM antibodies: 138 had MPO-ANCA (54%), and 121 had PR3-ANCA (46%). The relative frequencies of MPO or PR3-ANCA in patients with coexisting anti-GBM antibodies did not differ significantly from those in all patients with ANCA (P = 0.35). Seventeen sera with anti-GBM antibodies only and 16 sera with anti-GBM antibodies plus ANCA were selected for further studies to compare the specificity of anti-GBM antibodies in sera with or without ANCA. Using enzyme-linked immunosorbent assays (ELISA), all sera in both groups were found to react with the NC1 domain (as a hexamer) of bovine Type IV collagen and with alpha 3 (IV)NC1 monomers. Furthermore, all but six sera also reacted with one or more of the alpha 1, 2, and 4 (IV)NC1 monomers, generally with considerably lower titers. Reactivity to alpha 3(IV)NC1 was partially blocked by Mab17, with comparable degrees of inhibition in both groups. Western blot analysis with the human NC1 domains revealed no differences in reactivity between the two groups. Thus, differences in antigen specificities of anti-GBM antibodies in sera with or without ANCA were not detected. The anti-GBM response in both situations is hypothesized to be driven by the same immunogen, which is probably derived from NC1 domains of endogenous Type IV collagen.

OBJECTIVE

To determine the occurrence of antineutrophil cytoplasmic antibodies (ANCA) and the specificity of these antibodies (Ab) in serum from patients with rheumatoid arthritis (RA) and patients with rheumatoid arthritis complicated by vasculitis (rheumatoid vasculitis [RV]).

METHODS

ANCA was detected with an indirect immunofluorescence test on ethanol-fixed granulocytes. Ab against the cytoplasmic antigens proteinase-3, elastase, lactoferrin (LF), and myeloperoxidase were measured by enzyme-linked immunosorbent assay.

RESULTS

ANCA were found in the serum of 43% of 49 patients with RV and in 36% of 50 patients with RA. Anti-LF Ab occurred more frequently in RV patients (45%) than in RA patients (4%), whereas reactivity against the other cytoplasmic antigens did not differe significantly between these groups.

CONCLUSIONS

Anti-LF Ab in serum of patients with RA may be useful in the diagnosis of vasculitis in RA.

Differentiation and maturation of myeloid cells is characterized by the sequential acquisition of two distinct cytoplasmic granule subsets, azurophil granules and specific granules. We recently showed the existence of a third granule subset, gelatinase granules. To investigate whether appearance of gelatinase granules marks a further step in maturation of myeloid cells beyond the appearance of specific granules, we sorted normal human bone marrow cells into one of three groups according to maturity by centrifugation on Percoll density gradients. The biosynthesis of myeloperoxidase (MPO) (an azurophil granule marker), lactoferrin and neutrophil gelatinase-associated lipocalin NGAL (specific granules markers) and gelatinase was then studied in each of these groups. We found that gelatinase was synthesized mainly in the group containing band cells and segmented cells. This contrasted with lactoferrin and NGAL, which were synthesized almost exclusively in the group containing myelocytes and metamyelocytes, and with MPO, which was mainly synthesized in the group containing myeloblasts and promyelocytes. Immunocytochemistry was in full agreement with the biosynthesis data, and showed that gelatinase appears in band cells, whereas NGAL and lactoferrin both appear in myelocytes. Thus, acquisition of gelatinase granules marks a step in neutrophil differentiation beyond the appearance of specific granules.

Hypochlorous acid is the most powerful oxidant generated by neutrophils and is likely to contribute to the damage mediated by these inflammatory cells. The haem enzyme myeloperoxidase catalyses its production from hydrogen peroxide and chloride. 4-Aminobenzoic acid hydrazide (ABAH) is a potent inhibitor of hypochlorous acid production. In this investigation we show that, in the presence of hydrogen peroxide, ABAH irreversibly inactivates myeloperoxidase. ABAH was oxidized by myeloperoxidase, and kinetic analysis of the inactivation conformed to that for a mechanism-based inhibitor. Inactivation was exacerbated by concentrations of hydrogen peroxide greater than 50 microM and by the absence of oxygen. Hydrogen peroxide alone caused minimal inactivation. Reduced glutathione inhibited the oxidation of ABAH as well as the irreversible inhibition of myeloperoxidase. In the presence of oxygen, ABAH and hydrogen peroxide initially converted myeloperoxidase into compound III, which subsequently lost haem absorbance. In the absence of oxygen, the enzyme was converted into ferrous myeloperoxidase and its haem groups were rapidly destroyed. We propose that myeloperoxidase oxidizes ABAH to a radical that reduces the enzyme to its ferrous intermediate. Ferrous myeloperoxidase reacts either with oxygen to allow enzyme turnover, or with hydrogen peroxide to give irreversible inactivation.

Defensins are a newly recognized class of small, cationic polypeptides that have in vitro microbicidal activity toward certain bacteria, fungi, and viruses. Human neutrophil granules were separated into 13 density fractions by using a high-resolution Percoll gradient centrifugation procedure, and the distribution of the three defensin polypeptides in these fractions was determined. Levels of defensins and several granule marker proteins were estimated in each fraction from relative staining intensities of bands following acid-urea and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of total acid-extractable proteins. These results were confirmed by enzyme immunoassay measurements of defensins and quantitative determinations of the typical azurophil granule components, myeloperoxidase, beta-glucuronidase, lysozyme, and elastase. The five higher density granule fractions (H1 through H5) contained fourfold higher relative amounts of defensins as compared with the eight lower density fractions (L1 through L8), accounting for approximately 50% of the total protein. In particular, fraction H5 was especially enriched in defensins but was relatively deficient in myeloperoxidase, beta-glucuronidase, lysozyme, and elastase. Ultrastructural morphology showed that fraction H5 contained the largest granules. Seventy percent of these granules exhibited electron-dense rims and electron-lucent central regions when stained with methanolic uranyl acetate-lead citrate, and 70% showed this same characteristic rim-staining pattern after limited reaction (30 minutes) for peroxidase with diaminobenzidine. These distinctively large, rim-stained granules were identified in intact, mature peripheral blood neutrophils as well as in human bone marrow promyelocytes, indicating that their synthesis occurs during early myeloid development. This unusual granule type may play a specialized role in the microbicidal functions of the neutrophil, distinct from that of typical azurophil granules.

Cardiovascular disease is common in patients with chronic kidney disease (CKD). As renal function fails, many patients become progressively malnourished, as evidenced by reduced levels of albumin, prealbumin, and transferrin. Malnourished patients have increased levels of C reactive protein (CRP), interleukin-6 (IL-6), and concomitant cardiovascular disease when they reach end stage. Many diseases that cause CKD, diabetes, and hypertension are also associated with cardiovascular disease. Thus the direct effect of renal failure per se directly contributing to the inflammation-malnutrition-atherosclerosis paradigm is not completely established in early stages of CKD. Some aspects of progressive renal failure, however, cause changes in plasma composition and endothelial structure and function that favor vascular injury. As renal function fails, hepatic apo A-I synthesis decreases and HDL levels fall. HDL is an important antioxidant and defends the endothelium from the effects of cytokines. Inflammation causes further structural and functional abnormalities in HDL. Apolipoprotein C III (apo C III), a competitive inhibitor of lipoprotein lipase is increased in CKD. Serum triglyceride levels increase as a result of accumulation of intermediate-density lipoprotein (IDL) comprising VLDL and chylomicron remnants. These impede vascular relaxation and are associated with cardiovascular disease. Activation of the renin angiotensin axis is a component of many renal diseases and adaptation to loss of renal mass. Angiotensin II (AngII) activates NADPH oxidases, leading to production of the superoxide anion and decreased availability of nitric oxide (NO), further impairing vascular function. H(2)O(2), produced as a consequence of superoxide dismutation, stimulates vascular cell proliferation and hypertrophy. Leukocyte-derived myeloperoxidase functions as an "NO Oxidase" in the inflamed vasculature and contributes to decreased NO bioavailability and compromised vascular reactivity. The changes in lipoprotein composition and structure as well as AngII-mediated alterations in endothelial function amplify the effect of subsequent inflammatory events.

OBJECTIVE

Receptor for advanced glycation end products (AGEs) (RAGE) plays a central role in the process of plaque rupture in diabetic patients. Recently, it has been reported that RAGE may be downregulated by improving glycemic control. In contrast, despite being well known that RAGE may be induced in human vessels in a glucose-independent fashion, also by myeloperoxidase (MPO)-dependent AGE generation, no data exist regarding the possibility of a pharmacological modulation of glucose-independent RAGE generation. Thus, the aim of this study was to characterize the effect of simvastatin on the expression of RAGE and RAGE-dependent plaque-destabilizing genes in human atherosclerotic plaques.

RESULTS

Seventy type 2 diabetic patients with asymptomatic carotid artery stenosis (>70%) were randomized to American Heart Association (AHA) step 1 diet plus simvastatin (40 mg/d) or AHA step 1 diet alone for 4 months before endarterectomy. Plaque expression of MPO, AGEs, RAGE, NF-kappaB, COX-2, mPGES-1, matrix metalloproteinase (MMP)-2 and MMP-9, lipid and oxidized LDL (oxLDL) content, procollagen 1, and interstitial collagen was analyzed by immunohistochemistry and Western blot; zymography was used to detect MMP activity. Plaques from the simvastatin group had less (P<0.0001) immunoreactivity for MPO, AGEs, RAGE, p65, COX-2, mPGES-1, MMP-2, and MMP-9, lipids and oxLDL; reduced (P<0.0001) gelatinolytic activity; increased (P<0.0001) procollagen 1 and collagen content; and fewer (P<0.0001) macrophages, T-lymphocytes, and HLA-DR+ cells. Of interest, RAGE inhibition by simvastatin, observed not only in plaque sections but also in plaque-derived macrophages, was reverted by addition of AGEs in vitro.

CONCLUSIONS

This study supports the hypothesis that simvastatin inhibits plaque RAGE expression by decreasing MPO-dependent AGE generation. This effect in turn might contribute to plaque stabilization by inhibiting the biosynthesis of PGE2-dependent MMPs, responsible for plaque rupture.

We assessed the effects of primary afferent nerve ablation (systemic treatment with capsaicin during adult or neonatal periods), primary afferent nerve activation (intracolonic capsaicin), and sympathectomy [6-hydroxydopamine (6-OHDA)] on the development of colitis induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS) in rats. We also examined whether lidocaine was effective after ablation of primary afferent nerves or sympathectomy. Colitis was assessed by macroscopic scoring, measurement of myeloperoxidase (MPO) activity, and histology. Systemic capsaicin treatment in adults increased the macroscopic damage score. Capsaicin treatment of neonates did not significantly increase damage score or MPO activity compared with vehicle-treated controls. However, all capsaicin-treated groups had a higher mortality. Intracolonic capsaicin treatment did not alter the severity of colitis. Chemical sympathectomy resulted in a decreased damage score and improved histology compared with controls. In 6-OHDA pretreated rats, lidocaine administration reduced the macroscopic and histological scores and MPO activity almost to control levels. However, lidocaine administration in capsaicin-treated rats attenuated the macroscopic damage but did not improve MPO activity or histology. These data suggest that capsaicin-sensitive nerves play a protective role in experimental colitis and sympathetic nerves contribute to the development of colitis. The beneficial effects of lidocaine appear to be due primarily to its action on enteric nerves.

BACKGROUND

The anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides are a group of heterogeneous diseases. This study was undertaken to investigate the outcome of Wegener's granulomatosis (WG), microscopic polyangiitis (MPA) and renal-limited vasculitis (RLV). Furthermore, we analysed the differences in patients with proteinase 3-ANCA (PR3-ANCA) and those with myeloperoxidase-ANCA (MPO-ANCA), which have not been assessed in a homogeneously treated group of patients with renal involvement.

METHODS

In this retrospective analysis, 80 patients with a new diagnosis of WG, MPA or RLV with biopsy-proven renal involvement were followed over a median of 46.7 months (range: 0.8-181.9 months). All patients had induction treatment with cyclophosphamide and oral corticosteroids.

RESULTS

At the end of follow-up, 23% were dependent on dialysis. Renal survival was significantly worse in patients with WG compared with patients with MPA or RLV (P = 0.04). A higher rate of end-stage renal disease (ESRD) was noticed in PR3-ANCA- vs MPO-ANCA-positive patients. A total of 21 patients (26%) died. Predictors of patient mortality were development of ESRD, older age and the maximum creatinine in the first month. Mortality was found to be higher in patients with WG and was significantly higher in PR3-ANCA-positive cases (P = 0.02). The relative risk of death was 9.32 times higher in PR3-ANCA- vs MPO-ANCA-positive patients.

CONCLUSIONS

Our data underscore the pathogenetic potential of ANCA by demonstrating a more aggressive disease state and a poorer outcome in patients with PR3-ANCA.

Administration of dextran sulphate sodium to animals induces acute colitis characterized by infiltration of large numbers of neutrophils into the colonic mucosa, which histologically resembles human active ulcerative colitis. It has been reported that neutrophils and the reactive oxygen metabolites produced by them are involved in the progress of ulcerative colitis. This study was intended to clarify their roles by using this animal model. First, possible sources and species of reactive oxygen metabolites were determined using luminol-dependent chemiluminescence with addition of enzyme inhibitors and reactive oxygen metabolite scavengers. Next, to examine whether neutrophils and hypochlorous acid derived from them contribute to tissue injury, we administered RP-3, a monoclonal antibody capable of selectively depleting neutrophils, and taurine, a hypochlorous acid scavenger, to rats treated with dextran sulphate sodium. Addition of azide, taurine, catalase, superoxide dismutase and dimethyl sulphoxide into colonic mucosal scrapings significantly inhibited chemiluminescence production, but allopurinol and indomethacin had no effects. These results suggest that excessive hypochlorous acid, hydrogen peroxide, superoxide anion and hydroxyl radical are generated by the inflamed colonic mucosa. Intraperitoneal injections of RP-3 significantly suppressed bleeding, tissue myeloperoxidase activity, chemiluminescence production and erosion formation. On the other hand, administration of taurine tended to inhibit bleeding and erosion formation to some extent, although it could not significantly suppress them. These data suggest that neutrophils play an important role in the development of this colitis and that hypochlorous acid might be one of the causes of tissue injury induced by neutrophils.

OBJECTIVE

We recently showed that patients with inflammatory bowel disease (IBD) report significantly more sleep disturbances. To determine whether disrupted sleep can affect the severity of inflammation and the course of IBD, we used an animal model of colonic inflammation to determine the effects of acute and chronic intermittent sleep deprivation on the severity of colonic inflammation and tissue damage in colitis and recovery from this damage.

METHODS

Acute sleep deprivation (ASD) consisted of 24h of forced locomotor activity in a mechanical wheel rotating at a constant speed. Chronic intermittent sleep deprivation (CISD) consisted of an acute sleep deprivation episode, followed by additional sleep deprivation periods in the wheel for 6h every other day throughout the 10day study period. To induce colitis, mice were given 2% dextran sodium sulfate (DSS) in their daily drinking water for 7days. The development and severity of colitis were monitored by measuring weight loss and tissue myeloperoxidase (MPO) activity daily and colon histology scores 10days after initiation of colitis.

RESULTS

ASD or CISD did not cause colonic inflammation in vehicle-treated mice. Changes in daily body weight, tissue MPO levels and colon histopathology score were similar between mice that were sleep deprived and controls. Daily DSS ingestion caused colitis in mice. ASD worsened colonic inflammation: tissue MPO levels in ASD/DSS-treated mice were significantly higher than in DSS-treated mice that were not sleep deprived. However, the worsening of colonic inflammation by ASD was not enough to exacerbate clinical manifestations of colitis such as weight loss. In contrast, the deleterious effects of CISD were severe enough to cause worsening of histological and clinical manifestations of colitis. The deleterious effects of sleep deprivation on severity of colitis appeared to be due to both increased colonic inflammation and a decrease in the ability of mice to recover from DSS-induced colonic injury.

CONCLUSIONS

Both acute and chronic intermittent sleep deprivation exacerbate colonic inflammation. Thus, sleep deprivation could be an environmental trigger that predisposes IBD patients to develop flare ups and a more severe disease course. These results provide a scientific rationale to conduct an interventional trial to determine whether improvement in sleep patterns will prevent IBD flare ups, modify the disease course, and improve quality of life.

Perilla frutescens extract showed marked reduction on tumorigenesis in a murine, two-stage skin carcinogenesis model. In this model, cancer is initiated by application of 7,12-dimethylbenz[a]anthracene (DMBA) and promoted by application of 12-tetradecanoylphorbol 13-acetate (TPA). Following tumor initiation with DMBA, topical application of a perilla-derived fraction (PF) at doses of 2 mg/mouse/application resulted in significant inhibition of tumorigenesis. The efficacy of each fraction was correlated with rosmarinic acid (RA) and luteolin concentration. Topical application of perilla extract (PE) that contained 68% RA or an equivalent amount of commercially available RA showed nearly identical antiinflammatory activity 5 h after TPA treatment. Application of luteolin had less anti-inflammatory activity. Marked neutrophil infiltration was observed in TPA-challenged skin by histological examination using hematoxylin-eosin. This change was greatly reduced by pre-treatment with PE or RA. Myeloperoxidase activity, a marker of neutrophil recruitment, was also increased in TPA-challenged skin and was significantly decreased in the PE and RA treated groups. Intercellular adhesion molecule 1 and vascular cell adhesion molecule-1 mRNA expression levels were reduced by pre-treatment with PE or RA. TPA-induced increases in synthesis of the chemokines KC and macrophage inflammatory protein-2 were significantly decreased by pre-treatment with PE or RA. Prostaglandin E2 and leukotriene B4 levels were slightly increased 5 h after TPA treatment. These levels were only numerically decreased in the PE and RA treated groups. However, induction of cyclooxygenase-2 mRNA expression was obviously reduced by pre-treatment with PE or RA. Reactive oxygen radical production, detected as thiobarbituric acid reactive substance and lipid peroxide, by double treatment of TPA was reduced by pre-treatment with PE or RA. Production of 8-hydroxy-2'deoxyguanosine, which was detected immunohistochemically, was also induced by double treatment with TPA. This adduct was barely visible in PE or RA treated mice. Thus, we conclude that part of the anticarcinogenic effects of P.frutescens extract is due to RA via two independent mechanisms: inhibition of the inflammatory response and scavenging of reactive oxygen radicals.

Neutrophil elastase, a potent serine protease carried and released by activated neutrophils, is not synthesized by neutrophils, but by their bone marrow precursor cells. Using in situ hybridization with 35S-labeled antisense and sense neutrophil elastase cRNA probes, the present study demonstrates that expression of the neutrophil elastase gene is tightly controlled in bone marrow precursors and occurs during a very limited stage of differentiation of the neutrophil myeloid series, almost entirely at the promyelocyte stage. Neutrophil elastase mRNA transcript levels are detectable to a limited extent in blasts, increase markedly in the promyelocyte stage, and then disappear as promyelocytes further differentiate. Control probes specific for myeloperoxidase, lactoferrin, and beta-globin mRNA transcripts, respectively, demonstrated contrasting gene expression. Myeloperoxidase mRNA transcripts were also found almost exclusively at the promyelocyte stage, but myeloperoxidase mRNA levels disappeared earlier than do neutrophil elastase mRNA levels, suggesting that expression of these genes may be differently controlled. In comparison, lactoferrin mRNA transcripts were detected late in the neutrophil lineage, while beta-globin mRNA was detected only in cells of the erythroid lineage. Together these observations suggest that the expression of the neutrophil elastase gene is likely under very tight control, and is likely different than that for other constituents of the neutrophil granules.

A well-established model of bowel inflammation is the HLA-B27 transgenic rat that exhibits a spontaneous disease phenotype resulting in chronic diarrhea caused by immune cell activation. Estrogens have previously been shown to modulate the immune system, and both estrogen receptors (ERalpha and ERbeta) are present in the intestine and cells of the immune system. Therefore, the ability of estrogen to ameliorate disease progression in the HLA-B27 transgenic rat was determined. HLA-B27 transgenic rats with chronic diarrhea were treated with 17alpha-ethynyl-17beta-estradiol (EE) for 5 days. EE treatment dramatically improved stool scores after only 3 days. Histological scores of the degree of ulceration, inflammatory cell infiltration, fibrosis, and lesion depth of the colon were also improved by EE treatment. Because neutrophil infiltration into the colon is involved in the development and propagation of disease, myeloperoxidase (MPO) activity was measured. MPO levels were reduced by 80% by EE treatment. Cotreatment with the pure ER antagonist ICI-182780 (ICI) blocked the effects of EE on stool character, MPO activity, and histology scores, strongly suggesting that the activity of EE is mediated through ER. Mast cell proteases can promote neutrophil infiltration, and gene expression analysis demonstrated that mast cell protease 1, 3, and 4 mRNA were all decreased in colons from estrogen-treated rats. In addition, a direct effect of estrogen on bone marrow-derived mast cell activity was demonstrated, suggesting that ER-mediated inactivation of mast cells may contribute to the improvement in the clinical sign and histological scores in this model.

CONCLUSIONS

Beta-arrestins 1 and 2 are ubiquitously expressed proteins that alter signalling by G-protein-coupled receptors. beta-arrestin 2 plays an important role as a signalling adaptor and scaffold in regulating cellular inflammatory responses. We hypothesized that beta-arrestin 2 is a critical modulator of inflammatory response in experimental sepsis. beta-arrestin 2(-/-) and wild-type (WT) mice were subjected to caecal ligation and puncture (CLP). The survival rate was significantly decreased (P < 0.05) in beta-arrestin 2(-/-) mice (13% survival) compared with WT mice (53% survival). A second group of mice were killed 18 hr after CLP for blood, peritoneal lavage and tissue sample collection. CLP-induced plasma interleukin (IL)-6 was significantly increased 25 +/- 12 fold and caecal myeloperoxidase (MPO) activity was increased 2.4 +/- 0.3 fold in beta-arrestin 2(-/-) compared with WT mice. beta-arrestin 2(-/-) mice exhibited more severe lung damage and higher bacterial loads compared with WT mice post CLP challenge as measured by histopathology and colony-forming unit count. In subsequent experiments, splenocytes, peritoneal macrophages and bone marrow-derived macrophages (BMDMs) were isolated and cultured from beta-arrestin 2(-/-) and WT mice and stimulated in vitro with lipopolysaccharide (LPS). Tumour necrosis factor (TNF)-alpha, IL-6 and IL-10 production induced by LPS was significantly augmented (2.2 +/- 0.2 fold, 1.8 +/- 0.1 fold, and 2.2 +/- 0.4 fold, respectively; P < 0.05) in splenocytes from beta-arrestin 2(-/-) mice compared with WT mice. The splenocyte response was different from that of peritoneal macrophages or BMDMs, which exhibited no difference in TNF-alpha and IL-6 production upon LPS stimulation between WT and beta-arrestin 2(-/-) mice. Our data demonstrate that beta-arrestin 2 functions to negatively regulate the inflammatory response in polymicrobial sepsis.