Despite recent advances in antibiotic therapy and intensive care, sepsis is still considered to be the most common cause of death in intensive care units. Excessive production of reactive oxygen species plays an important role in the pathogenesis of sepsis. Recently, it has been suggested that molecular hydrogen (H2) exerts a therapeutic antioxidant activity by selectively reducing hydroxyl radicals (*OH, the most cytotoxic reactive oxygen species) and effectively protects against organ damage induced by I/R. Therefore, we hypothesized that H2 treatment had a beneficial effect on sepsis. In the present study, we found that H2 inhalation starting at 1 and 6 h after cecal ligation and puncture (CLP) or sham operation significantly improved the survival rate of septic mice with moderate or severe CLP in a concentration- and time-dependent manner. Furthermore, moderate or severe CLP mice showed significant multiple organ damage characterized by the increases of lung myeloperoxidase activity, wet-to-dry weight ratio, protein concentration in bronchoalveolar lavage, serum biochemical parameters, and organ histopathologic scores at 24 h after CLP operation, which was significantly attenuated by 2% H2 treatment. In addition, we found that the beneficial effects of H2 treatment on sepsis and sepsis-associated organ damage were associated with the decreased levels of oxidative product, increased activities of antioxidant enzymes, and reduced levels of high-mobility group box 1 in serum and tissue. Thus, H2 inhalation may be an effective therapeutic strategy for patients with sepsis.

Gallic acid was found to possess antiinflammatory activity towards zymosan-induced acute food pad swelling in mice. In vitro studies on the mode of action of gallic acid revealed that this compound interferes with the functioning of polymorphonuclear leukocytes (PMNs). Scavenging of superoxide anions, inhibition of myeloperoxidase release and activity as well as a possible interference with the assembly of active NADPH-oxidase may account for the inhibition of inflammatory process by gallic acid. Structure-activity relationship analysis showed that the o-dihydroxy group of gallic acid is important for the inhibitory activity in vitro.

Much like other autoantibodies (eg, anti-double stranded DNA in systemic lupus erythematosus or antiglomerular basement membrane antibodies in Goodpasture's syndrome), antineutrophil cytoplasmic antibodies (ANCA) have provided doctors with a useful serological test to assist in diagnosis of small-vessel vasculitides, including Wegener's granulomatosis, microscopic polyangiitis, Churg-Strauss syndrome, and their localised forms (eg, pauci-immune necrotising and crescentic glomerulonephritis). 85-95% of patients with Wegener's granulomatosis, microscopic polyangiitis, and pauci-immune necrotising and crescentic glomerulonephritis have serum ANCA. ANCA directed to either proteinase 3 or myeloperoxidase are clinically relevant, yet the relevance of other ANCA remains unknown. Besides their diagnostic potential, ANCA might be valuable in disease monitoring. In addition, data seem to confirm the long-disputed pathogenic role of these antibodies. Present treatments for ANCA-associated vasculitis are not free from side-effects and as many as 50% of patients relapse within 5 years. Accurate understanding of the key pathogenic points of ANCA-associated vasculitis can undoubtedly provide a more rational therapeutic approach.

Reactive oxygen species (ROS) produced during exercise may be involved in delayed-onset muscle damage related to inflammation. To investigate this hypothesis, we studied whether oxidative stress increases nuclear translocation of nuclear factor-kappaB and chemokine expression in skeletal muscle using myotube L6 cells. We also assessed whether prolonged acute exercise could increase these parameters in rats. In L6 cells, H(2)O(2) induced nuclear translocation of p65 and increased the expression of cytokine-induced neutrophil chemoattractant-1 (CINC-1) and monocyte chemoattractant protein-1 (MCP-1), whereas preincubation with alpha-tocopherol limited the increase in these proteins. Sprague Dawley rats were divided into the following groups: rested control, exercised, rested with a high alpha-tocopherol diet, and exercised with a high alpha-tocopherol diet. After 3 weeks of acclimation, both exercise groups ran on a treadmill at 25 m/min for 60 min. Exercise increased nuclear p65, CINC-1, and MCP-1 in gastrocnemius muscle cells, but these changes were ameliorated by the high alpha-tocopherol diet. Increases in myeloperoxidase and thiobarbituric acid-reactive substrates were ameliorated by a high alpha-tocopherol diet, as were the histological changes. Neutrophil activity was not altered by either exercise or a high alpha-tocopherol diet. These results indicate that delayed-onset muscle damage induced by prolonged exercise is partly related to inflammation via phagocyte infiltration caused by ROS and that alpha-tocopherol (an antioxidant) can attenuate such inflammatory changes.

When polymorphonuclear leukocytes (PMNL) and soluble or particulate matter interact, the cells produce chemiluminescence. Luminol-dependent light emission from PMNL is linked to the myeloperoxidase (MPO)-H2O2 system. Light emission from a cell-free MPO-H2O2 system was found to be totally inhibited by human serum albumin (HSA), and since HSA is a large molecular protein that does not readily gain access to intracellular sites of PMNL, it could be used to determine the importance of extra- and intracellular events in PMNL chemiluminescence. In studies with cells from an MPO-deficient patient, we found that HSA inhibited more than 90% of extracellularly produced chemiluminescence. The chemotactic peptide formylmethionyl-leucyl-phenylalanine induced a two-peak chemiluminescence response in normal PMNL, and addition of HSA reduced the first peak, whereas the second peak was unaffected. This result indicated that the first peak was a result of extracellular reactions and the second peak was a result of intracellular reactions of the MPO-H2O2 system. Most of the phorbol myristate acetate-induced response in normal PMNL was due to intracellular events. Furthermore, chemiluminescence of intracellular origin seems to be limited not by generation of oxidative metabolites but by diffusion of luminol into the cells.

Sera of 64 patients with chronic inflammatory bowel disease (IBD) were screened for antibodies against neutrophil cytoplasmic antigens (ANCA) using an indirect immunofluorescence technique on ethanol-fixed human neutrophil granulocytes. 20 of 34 sera (59%) from patients with ulcerative colitis (UC) produced a fine-granular and perinuclear ANCA staining pattern (p-ANCA) clearly different from the typical diffuse and granular cytoplasmic ANCA fluorescence (c-ANCA, synonym ACPA) seen in active Wegener's granulomatosis (WG). The majority of the 20 p-ANCA positive UC patients had a high inflammatory disease activity. Among the 14 p-ANCA negative UC patients nine were without steroids; five of them had active disease, two were inactive and two had previously undergone colectomy. The remaining five patients still had active disease but received steroids for more than 4 weeks. Only 3 of the 30 sera from patients with Crohn's disease (CD) showed positive p-ANCA reactions. To narrow the specificity of the p-ANCA reaction all 64 sera were tested by ELISA for antibodies against anti-proteinase-3 (WG specific) and on HEp-2 cells for antinuclear (ANA) and anticytoplasmic antibodies. Ten p-ANCA positive UC sera were also tested in a myeloperoxidase ELISA. Only one UC serum reacted positively in the proteinase-3-ELISA and another one produced a weakly positive anti-nucleolar ANA fluorescence on HEp-2 cells. None of the tested sera reacted with myeloperoxidase suggesting that the p-ANCA staining pattern of granulocytes is not restricted to anti-myeloperoxidase antibodies as reported in the literature.(ABSTRACT TRUNCATED AT 250 WORDS)

The transient receptor potential vanilloid 1 receptor (TRPV1) is an important nociceptor involved in neurogenic inflammation. We aimed to examine the role of TRPV1 in experimental colitis and in the development of visceral hypersensitivity to mechanical and chemical stimulation. Male Sprague-Dawley rats received a single dose of trinitrobenzenesulfonic acid (TNBS) in the distal colon. In the preemptive group, rats received the TRPV1 receptor antagonist JYL1421 (10 mumol/kg, i.v.) or vehicle 15 min prior to TNBS followed by daily doses for 7 days. In the post-inflammation group, rats received JYL1421 daily for 7 days starting on day 7 following TNBS. The visceromotor response (VMR) to colorectal distension (CRD), intraluminal capsaicin, capsaicin vehicle (pH 6.7) or acidic saline (pH 5.0) was assessed in all groups and compared with controls and naïve rats. Colon inflammation was evaluated with H&E staining and myeloperoxidase (MPO) activity. TRPV1 immunoreactivity was assessed in the thoraco-lumbar (TL) and lumbo-sacral (LS) dorsal root ganglia (DRG) neurons. In the preemptive vehicle group, TNBS resulted in a significant increase in the VMR to CRD, intraluminal capsaicin and acidic saline compared the JYL1421-treated group (P<0.05). Absence of microscopic colitis and significantly reduced MPO activity was also evident compared with vehicle-treated rats (P<0.05). TRPV1 immunoreactivity in the TL (69.1+/-4.6%) and LS (66.4+/-4.2%) DRG in vehicle-treated rats was increased following TNBS but significantly lower in the preemptive JYL1421-treated group (28.6+/-3.9 and 32.3+/-2.3 respectively, P<0.05). JYL1421 in the post-inflammation group improved microscopic colitis and significantly decreased the VMR to CRD compared with vehicle (P<0.05,>>/=30 mm Hg) but had no effect on the VMR to chemical stimulation. TRPV1 immunoreactivity in the TL and LS DRG was no different from vehicle or naïve controls. These results suggest an important role for TRPV1 channel in the development of inflammation and subsequent mechanical and chemical visceral hyperalgesia.

BACKGROUND

Inflammation and oxidative stress are associated with atherosclerosis. Myeloperoxidase (MPO) is linked to both inflammation and oxidative stress by its location in leukocytes and its role in catalyzing the formation of oxidizing agents. Recent evidence suggests that MPO activity precipitates atherogenesis. Measurement of MPO in plasma may therefore contribute to cardiovascular disease (CVD) risk stratification.

BACKGROUND

Cross-sectional studies, case-control studies, and prospective-cohort studies investigating the relation between MPO and CVD have been evaluated. Differences in study populations, sample materials, sample handling, and assays were ascertained. Potential causal mechanisms linking MPO to accelerated atherosclerosis are discussed here. A majority of studies indicate that measurement of MPO in plasma was associated with improved CVD risk stratification above and beyond risk stratification results obtained with markers used in routine clinical practice. However, comparison of these epidemiological studies with regard to MPO and outcome is hampered because the reported MPO concentration depends on the assay method, sampling material, and preanalytical and analytical procedures. The link between MPO and CVD can, at least partly, be explained by MPO-dependent oxidation of LDL and HDL, subsequently leading to cholesterol accumulation in the arterial wall. Furthermore, MPO may reduce the bioavailability of nitric oxide, resulting in endothelial dysfunction. Finally, MPO destabilizes atherosclerotic plaques.

CONCLUSIONS

Increasing evidence suggests that MPO is causally linked to atherosclerosis and its measurement may improve CVD risk estimation. Before MPO can be used in routine clinical practice, however, standardization of sampling and laboratory procedures is needed.

BACKGROUND

Milk fat globule epidermal growth factor 8 (MFG-E8) is a potent opsonin for the clearance of apoptotic cells and is produced by mononuclear cells of immune competent organs including the spleen and lungs. It attenuates chronic and acute inflammation such as autoimmune glomerulonephritis and bacterial sepsis by enhancing apoptotic cell clearance. Ischemia-reperfusion (I/R) injury of the gut results in severe inflammation, apoptosis, and remote organ damage, including acute lung injury (ALI).

OBJECTIVE

To determine whether MFG-E8 attenuates intestinal and pulmonary inflammation after gut I/R.

METHODS

Wild-type (WT) and MFG-E8(-/-) mice underwent superior mesenteric artery occlusion for 90 minutes, followed by reperfusion for 4 hours. A group of WT mice was treated with 0.4 microg/20 g recombinant murine MFG-E8 (rmMFG-E8) at the beginning of reperfusion. Four hours after reperfusion, MFG-E8, cytokines, myeloperoxidase activity, apoptosis, and histopathology were assessed. A 24-hour survival study was conducted in rmMFG-E8- and vehicle-treated WT mice.

RESULTS

Mesenteric I/R caused severe widespread injury and inflammation of the small intestines and remote organs, including the lungs. MFG-E8 levels decreased in the spleen and lungs by 50 to 60%, suggesting impaired apoptotic cell clearance. Treatment with rmMFG-E8 significantly suppressed inflammation (TNF-alpha, IL-6, IL-1beta, and myeloperoxidase) and injury of the lungs, liver, and kidneys. MFG-E8-deficient mice suffered from greatly increased inflammation and potentiated ALI, whereas treatment with rmMFG-E8 significantly improved the survival in WT mice.

CONCLUSIONS

MFG-E8 attenuates inflammation and ALI after gut I/R and may represent a novel therapeutic agent.

OBJECTIVE

The role of sensory neurones in colitis was studied by chemical denervation of primary sensory neurones as well as antagonism of the vanilloid receptor-1 (VR-1) in rats prior to administration of dextran sulphate sodium (DSS) to induce colitis.

METHODS

Neonatal rats were chemically denervated by subcutaneous administration of capsaicin; controls received capsaicin vehicle only. When animals reached maturity, colitis was induced by administration of 5% DSS in drinking water for seven days. Additionally, normal adult rats were treated with a VR-1 antagonist capsazepine (CPZ) or vehicle twice daily via an enema from day 0 to day 6 of the DSS regimen. Control rats were treated with an enema infusion of vehicle and 5% DSS, or without either an enema infusion or DSS in drinking water. For both groups of rats, severity of inflammation was quantitated by disease activity index (DAI), myeloperoxidase (MPO) activity, and histological examination.

RESULTS

DSS induced active colitis in all control rats with resultant epithelial ulceration, crypt shortening, and neutrophil infiltration. Both neonatal capsaicinised rats and normal adult rats treated with CPZ enemas exhibited significantly lower levels of DAI, MPO, and histological damage compared with vehicle treated rats (p< 0.05).

CONCLUSIONS

Neonatal capsaicinisation and local administration of CPZ prevents intestinal inflammation in a well established model of colitis indicating that primary sensory neurones possessing VR-1 receptors are required in the propagation of colonic inflammation.

The object of this study was to investigate the impact of cigarette smoke on bacterial clearance and immune inflammatory parameters after infection with Pseudomonas aeruginosa in mice. We observed a delayed rate of bacterial clearance in smoke-exposed compared with sham-exposed mice. This was associated with increased inflammation characterized by greater numbers of neutrophils and mononuclear cells in the bronchoalveolar lavage. After infection, we observed increased levels of proinflammatory cytokines (tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6) and chemokines (monocyte chemoattractant protein-1 [MCP-1] and macrophage inflammatory protein-2 [MIP-2]) as well as myeloperoxidase and proteolytic activity in the lungs of smoke-exposed compared with sham-exposed animals. Delayed clearance was associated with increased morbidity and greater weight loss of smoke-exposed mice. After delivery of inactivated bacteria, we observed a similar inflammatory response, clinical score, and tumor necrosis factor-alpha expression in smoke- and sham-exposed animals, suggesting that increased inflammation and altered clinical presentation are due to the delayed rate of bacterial clearance. Our findings suggest that cigarette smoke affects respiratory immune-inflammatory responses elicited by bacteria. We postulate that altered respiratory host defense may be implicated in smoking-related diseases such as chronic obstructive pulmonary disease.

Phagocyte activation is accompanied by assembly of an NADPH oxidase that reduces oxygen to form a number of reactive species. These oxygen radicals can eradicate invading microorganisms, regulate the function of other immune reactive cells, and cause damage to "innocent bystander" cells. It is generally assumed that the NADPH oxidase is activated exclusively in the plasma membrane. In neutrophils, this assumption does not fit with the subcellular localization of the membrane component of the oxidase, which is stored in granule compartments. It has now become increasingly evident that oxidants are also produced in an intracellular compartment that we identify as the specific granules. Myeloperoxidase is stored in another granule subset, the azurophil granules, and participates in the processing of the oxidative metabolites. We suggest that neutrophil activation is accompanied by fusion between azurophil and specific granules, allowing these peroxidase-dependent reactions to take place. The presented data suggest a requisite role for neutrophil oxidants complementing their function as microbial killing agents. Signaling capabilities of the oxidants, affecting for example, the state of protein phosphorylation, regulation of transcription factors, and induction of apoptosis, are discussed.

Ultraviolet (UV) radiation-induced infiltrating leukocytes, depletion of antigen-presenting cells, and oxidative stress in the skin play an important role in the induction of immune suppression and photocarcinogenesis. Earlier we have shown that topical application of polyphenols from green tea or its major chemopreventive constituent (-)-epigallocatechin-3-gallate (EGCG) prevents UV-B-induced immunosuppression in mice. To define the mechanism of prevention, we found that topical application of EGCG (3 mg/mouse/3 cm(2) of skin area) to C3H/HeN mice before a single dose of UV-B (90 mJ/cm(2)) exposure inhibited UV-B-induced infiltration of leukocytes, specifically the CD11b+ cell type, and myeloperoxidase activity, a marker of tissue infiltration of leukocytes. EGCG treatment was also found to prevent UV-B-induced depletion in the number of antigen-presenting cells when immunohistochemically detected as class II MHC+ Ia+ cells. UV-B-induced infiltrating cell production of H2O2 and nitric oxide (NO) was determined as a marker of oxidative stress. We found that pretreatment of EGCG decreased the number of UV-B-induced increases in H2O2-producing cells and inducible nitric oxide synthase-expressing cells and the production of H2O2 and NO in both epidermis and dermis at a UV-B-irradiated site. Together, these data suggest that prevention of UV-B-induced infiltrating leukocytes, antigen-presenting cells, and oxidative stress by EGCG treatment of mouse skin may be associated with the prevention of UV-B-induced immunosuppression and photocarcinogenesis.

The terminal complement components play an important role in mediating tissue injury after ischemia and reperfusion (I/R) injury in rats and mice. However, the specific complement pathways involved in I/R injury are unknown. The role of the alternative pathway in I/R injury may be particularly important, as it amplifies complement activation and deposition. In this study, the role of the alternative pathway in I/R injury was evaluated using factor D-deficient (-/-) and heterozygote (+/-) mice. Gastrointestinal ischemia (GI) was induced by clamping the mesenteric artery for 20 minutes and then reperfused for 3 hours. Sham-operated control mice (+/- versus -/-) had similar baseline intestinal lactate dehydrogenase activity (P = ns). Intestinal lactate dehydrogenase activity was greater in -/- mice compared to +/- mice after GI/R (P = 0.02) thus demonstrating protection in the -/- mice. Intestinal myeloperoxidase activity in +/- mice was significantly greater than -/- mice after GI/R (P < 0.001). Pulmonary myeloperoxidase activity after GI/R was significantly higher in +/- than -/- mice (P = 0.03). Addition of human factor D to -/- animals restored GI/R injury and was prevented by a functionally inhibitory antibody against human factor D. These data suggest that the alternative complement pathway plays an important role in local and remote tissue injury after GI/R. Inhibition of factor D may represent an effective therapeutic approach for GI/R injury.

BACKGROUND

Interleukin-1 (IL-1) plays a role in mediating acute inflammation during ischemia-reperfusion (I/R) injury in the heart, which leads to both necrosis and apoptosis of cardiomyocytes. IL-1 receptor antagonist (IL-1ra) is known to inhibit the effects of IL-1alpha and IL-1beta, resulting in attenuated inflammatory injury, and to protect cells from IL-1beta-induced apoptosis in vitro. We hypothesized that IL-1ra overexpression would provide cardioprotection by reducing inflammation-mediated myocardial damage including apoptosis after I/R injury in vivo.

RESULTS

Rat hearts were transfected with human secreted-type IL-1ra gene by intracoronary infusion of Hemagglutinating Virus of Japan liposome and were heterotopically transplanted. IL-1ra overexpression in these hearts was confirmed by enzyme immunoassay and immunohistochemistry. Myocardial tolerance of the transplanted heart was evaluated with the use of a novel system in which the heart, existing within the recipient's abdomen, was given 30 minutes of ischemia by left coronary artery occlusion and 24 hours of reperfusion. Consequently, infarct size was decreased in IL-1ra-transfected hearts compared with control-transfected ones (26.9+/-3.2% versus 46.2+/-3.0%, P=0.001), corresponding to lower myocardial myeloperoxidase activity (2.20+/-0.69 versus 6.82+/-1.19 U/g wet wt, P<0.001) and decreased neutrophil infiltration in histological study. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling and DNA-laddering studies demonstrated that cardiomyocyte apoptosis was attenuated in IL-1ra-transfected hearts (21.4+/-3.3 versus 41.4+/-3.4%, P=0.002), correlating with reduced post I/R upregulation of Bax, Bak, and caspase-3.

CONCLUSIONS

IL-1ra introduced by gene transfection protected myocardium from I/R injury by attenuating the inflammatory response, which was associated with decreased apoptosis. This suggests a potentially important role of IL-1/IL-1ra in myocardial I/R injury and the value of IL-1ra-gene therapy for myocardial preservation.

There is increasing evidence that parasitic helminth infection has the ability to ameliorate other disease conditions. In this study the ability of the rat tapeworm, Hymenolepis diminuta, to modulate dinitrobenzene sulfonic acid (DNBS)-induced colitis in mice is assessed. Mice receiving DNBS (3 mg intrarectally) developed colitis by 72 h after treatment. Mice infected 8 days before DNBS with five H. diminuta larvae were significantly protected from the colitis, as gauged by reduced clinical disease, histological damage scores, and myeloperoxidase levels. This anticolitic effect was dependent on a viable infection and helminth rejection, because no benefit was observed in mice given killed larvae or in infected STAT6 knockout mice or rats, neither of which eliminate H. diminuta. The anticolitic effect of H. diminuta was associated with increased colonic IL-10 mRNA and stimulated splenocytes from H. diminuta- plus DNBS-treated mice produced more IL-10 than splenocytes from DNBS-only treated mice. Coadministration of an anti-IL-10 Ab blocked the anticolitic effect of prophylactic H. diminuta infection. Also, mice infected 48 h after DNBS treatment showed an enhanced recovery response. Finally, using a model of OVA hypersensitivity, we found no evidence of concomitant H. diminuta infection enhancing enteric responsiveness to subsequent ex vivo OVA challenge. The data show that a viable infection of H. diminuta in a nonpermissive system exerts a profound anticolitic effect (both prophylactically and as a treatment) that is mediated at least in part via IL-10 and does not predispose to enhanced sensitivity to bystander proteins.

Antiproteinase 3- and antimyeloperoxidase-associated vasculitis. Wegener's granulomatosis, microscopic polyangiitis, and idiopathic pauci-immune necrotizing crescentic glomerulonephritis (NCGN) are strongly associated with antineutrophil cytoplasmic autoantibodies (ANCAs) directed against either proteinase 3 (anti-PR3) or myeloperoxidase (anti-MPO). This has led some investigators to prefer combining these diseases under the common heading of ANCA-associated vasculitides. However, it is increasingly recognized that there are characteristic differences between patients with anti-PR3 and those with anti-MPO-associated vasculitis. This review focuses on the clinical, histopathologic, and possibly pathophysiologic differences between anti-PR3- and anti-MPO-associated vasculitis. Although there is considerable overlap, the anti-PR3- and anti-MPO-associated vasculitides are each characterized by particular clinical and histopathological findings. Extrarenal organ manifestations and respiratory tract granulomas occur more frequently in patients with anti-PR3 than in those with anti-MPO. Anti-PR3-positive patients with NCGN generally have a more dramatic deterioration of their renal function compared with anti-MPO-positive patients. The term "ANCA-associated vasculitis" is considered as a useful concept in the presence of systemic vasculitis. Likewise, in the presence of vasculitis, the terms "anti-PR3-associated vasculitis" and "anti-MPO-associated vasculitis" are useful concepts.

BACKGROUND

Research on autoantibody formation in patients treated with TNF alpha inhibitors has produced contradictory results.

OBJECTIVE

To study the prevalence of autoantibodies in patients with rheumatoid arthritis treated with the TNF alpha inhibitor infliximab.

METHODS

53 patients (48 female, 11 male) treated with infliximab for rheumatoid arthritis were followed for autoantibody production before treatment and after 14, 30, and 54 weeks. Six patients treated with etanercept were studied for comparison. The analyses included antibodies against nuclear antigens (ANA), extractable nuclear antigens, double stranded (ds)DNA (by ELISA, IIF on Crithidia luciliae for IgM and IgG, and Farr assay), nucleosomes, cardiolipin, smooth muscle, mitochondria, proteinase 3, and myeloperoxidase antigens.

RESULTS

The number of patients treated with infliximab who developed antibodies against dsDNA of both IgG and IgM class (tested by IIF) increased significantly. The prevalence of patients positive for IgG class increased to 66% at 30 weeks and 45% at 54 weeks, and of IgM class to 85% and 70%, respectively. The titre and number of patients expressing antibodies against nucleosomes and ANA also increased significantly. The number of rheumatoid factor or anticardiolipin positive patients was stable and there was no increase in antibodies against the other antigens. A lupus-like syndrome was seen in one patient. No patient treated with etanercept developed any of these autoantibodies.

CONCLUSIONS

Patients treated with infliximab may develop anti-dsDNA antibodies of both IgM and IgG class, anti-nucleosome antibodies, and ANA, with a gradual increase until 30 weeks.

The human granulocytic ehrlichiosis (HGE) agent resides and multiplies exclusively in cytoplasmic vacuoles of granulocytes. Double immunofluorescence labeling was used to characterize the nature of the HGE agent replicative inclusions and to compare them with inclusions containing the human monocytic ehrlichia, Ehrlichia chaffeensis, in HL-60 cells. Although both Ehrlichia spp. can coinfect HL-60 cells, they resided in separate inclusions. Inclusions of both Ehrlichia spp. were not labeled with either anti-lysosome-associated membrane protein 1 or anti-CD63. Accumulation of myeloperoxidase-positive granules were seen around HGE agent inclusions but not around E. chaffeensis inclusions. 3-(2, 4-Dinitroanilino)-3'-amino-N-methyldipropylamine and acridine orange were not localized to either inclusion type. Vacuolar-type H+-ATPase was not colocalized with HGE agent inclusions but was weakly colocalized with E. chaffeensis inclusions. E. chaffeensis inclusions were labeled with the transferrin receptor, early endosomal antigen 1, and rab5, but HGE agent inclusions were not. Some HGE agent and E. chaffeensis inclusions colocalized with major histocompatibility complex class I and II antigens. These two inclusions were not labeled for annexins I, II, IV, and VI; alpha-adaptin; clathrin heavy chain; or beta-coatomer protein. Vesicle-associated membrane protein 2 colocalized to both inclusions. The cation-independent mannose 6-phosphate receptor was not colocalized with either inclusion type. Endogenously synthesized sphingomyelin, from C6-NBD-ceramide, was not incorporated into either inclusion type. Brefeldin A did not affect the growth of either Ehrlichia sp. in HL-60 cells. These results suggest that the HGE agent resides in inclusions which are neither early nor late endosomes and does not fuse with lysosomes or Golgi-derived vesicles, while E. chaffeensis resides in an early endosomal compartment which accumulates the transferrin receptor.

There is considerable interest in the role that mammalian heme peroxidase enzymes, primarily myeloperoxidase, eosinophil peroxidase and lactoperoxidase, may play in a wide range of human pathologies. This has been sparked by rapid developments in our understanding of the basic biochemistry of these enzymes, a greater understanding of the basic chemistry and biochemistry of the oxidants formed by these species, the development of biomarkers that can be used damage induced by these oxidants in vivo, and the recent identification of a number of compounds that show promise as inhibitors of these enzymes. Such compounds offer the possibility of modulating damage in a number of human pathologies. This reviews recent developments in our understanding of the biochemistry of myeloperoxidase, the oxidants that this enzyme generates, and the use of inhibitors to inhibit such damage.

OBJECTIVE

Although most acute enteric infections in humans resolve, some herald the onset of chronic symptomatology and persistent gastrointestinal dysfunction--so-called postinfectious irritable bowel syndrome. This entity is poorly understood, and there are no animal models for testing hypotheses. The aim of this study was to investigate changes in intestinal neuromuscular function during and after recovery from acute intestinal inflammation due to primary Trichinella spiralis infections in NIH Swiss mice.

METHODS

Morphometric scores and myeloperoxidase activity were used to monitor mucosal inflammation. Neuromuscular function was assessed in vitro by pharmacological or electrical stimulation of longitudinal muscle.

RESULTS

Acute inflammation resulted in an approximately 50% reduction of villus height, an approximately 50% increase in crypt depth, and a threefold increase in myeloperoxidase activity. Carbachol- and KCl-induced contractions of longitudinal muscle were also increased threefold, whereas contraction induced by electrical field stimulation of intramural nerves was decreased by 60%. Mucosal morphology and myeloperoxidase activity rapidly returned to control values, but the increased muscle contractility and the decreased excitatory neurotransmission persisted as long as 42 and 28 days after infection, respectively.

CONCLUSIONS

These findings show that transient mucosal inflammation alters enteric neuromuscular function; this alteration persists after recovery from the infection and mucosal restitution.

In this study we examined the leukocytic oxidant species that induce oxidant damage of DNA in whole cells. H2O2 added extracellularly in micromolar concentrations (10-100 microM) induced DNA strand breaks in various target cells. The sensitivity of a specific target cell was inversely correlated to its catalase content and the rate of removal of H2O2 by the target cell. Oxidant species produced by xanthine oxidase/purine or phorbol myristate acetate-stimulated monocytes induced DNA breakage of target cells in proportion to the amount of H2O2 generated. These DNA strand breaks were prevented by extracellular catalase, but not by superoxide dismutase. Cytotoxic doses of HOCl, added to target cells, did not induce DNA strand breakage, and myeloperoxidase added extracellularly in the presence of an H2O2-generating system, prevented the formation of DNA strand breaks in proportion to its H2O2 degrading capacity. The studies also indicated that H2O2 formed hydroxyl radical (.OH) intracellularly, which appeared to be the most likely free radical responsible for DNA damage: .OH was detected in cells exposed to H2O2; the DNA base, deoxyguanosine, was hydroxylated in cells exposed to H2O2; and intracellular iron was essential for induction of DNA strand breaks.

OBJECTIVE

To synthesize novel inhibitors of the nuclear enzyme poly(adenosine 5'-diphosphate [ADP]-ribose) synthetase (PARS), also known as poly(ADP-ribose) polymerase (PARP), and to test them in in vitro models of oxidant-induced cytotoxicity and in endotoxin and splanchnic occlusion-reperfusion-induced shock.

METHODS

Randomized, prospective laboratory study.

METHODS

Research laboratory.

METHODS

Murine macrophages, thymocytes, and endothelial cells; Balb/c mice and Wistar rats.

METHODS

Macrophages and endothelial cells were treated with peroxynitrite and bleomycin to induce PARS activation, and thymocytes were treated with peroxynitrite to induce cell necrosis. Novel PARS inhibitors were synthesized and used to reduce PARS activation and to reverse cytotoxicity. Balb/c mice were subjected to splanchnic occlusion and reperfusion and were pretreated with various doses (1-10 mg/kg intraperitoneally) of PJ34, a selected, potent, water-soluble PARS inhibitor. The passage of fluorescein isothiocyanate-conjugated dextran (4 kDa) was analyzed in everted gut ileal sacs incubated ex vivo as an index of gut permeability. Wistar rats were subjected to Escherichia coli bacterial lipopolysaccharide (40 mg/kg intraperitoneally). PJ34 was also used at 10 mg/kg intraperitoneally, 1 hr before lipopolysaccharide or at 25 mg/kg intraperitoneally 1 hr after lipopolysaccharide treatment. Serum concentrations of indicators or multiple organ injury, concentrations of various proinflammatory mediators, and tissue concentrations of myeloperoxidase and malondialdehyde were measured. In addition, survival rates and vascular contractile and relaxant responses were recorded.

RESULTS

Appropriate modifications of the phenanthridinone core structure yielded significant increases in the potency of the compounds, both as PARS inhibitors and as cytoprotective agents. The compound N-(6-oxo-5,6-dihydro-phenanthridin-2-yl) -N,N-dimethylacetamide (designated as PJ34) was one of the potent PARS inhibitors of the series, and it dose-dependently protected against thymocyte necrosis, with a half-maximal restoration of cell viability of 35 nM and complete protection at 200 nM. PARS activation also was visualized by immunohistochemistry and was dose-dependently suppressed by PJ34. The effect of PJ34 was dose-dependently reversed by excess nicotinamide adenine dinucleotide (oxidized). The PARS inhibitors dose-dependently suppressed proinflammatory cytokine and chemokine production and restored viability in immunostimulated macrophages. PJ34 was selected for the subsequent in vivo studies. PJ34 significantly protected against splanchnic reperfusion-induced intestinal hyperpermeability in the mouse. PJ34 reduced peak plasma concentrations of tumor necrosis factor-alpha, interleukin-1beta, and nitrite/nitrate in the plasma of lipopolysaccharide-treated rats. PJ34 ameliorated the lipopolysaccharide-induced increases in indexes of liver and kidney failure and concentrations of myeloperoxidase and malondialdehyde in the lung and gut. Lipopolysaccharide elicited vascular dysfunction, which was normalized by PJ34. Lipopolysaccharide-induced mortality was reduced by PJ34 (both pre- and posttreatment).

CONCLUSIONS

The novel series of phenanthridinone PARS inhibitors have potent cytoprotective effects in vitro and significant protective effects in shock and reperfusion injury in rodent models in vivo.

OBJECTIVE

The aim of the present study is to determine whether hypochlorous acid (HOCl), the major oxidant of leukocyte-derived myeloperoxidase (MPO), oxidizes the zinc-thiolate center of endothelial nitric oxide synthase (eNOS) and uncouples the enzyme.

RESULTS

Exposure of purified recombinant eNOS to HOCl >> or = 100 micromol/L) released zinc and disrupted the enzyme-active eNOS dimers. In parallel with increased detections of both O2*- and ONOO-, clinically relevant concentrations of HOCl disrupted eNOS dimers in cultured human umbilical vein endothelial cells (HUVEC) at concentration 10- to 100-fold lower than those required for recombinant eNOS. In HUVEC, HOCl increased the translocation of both p67(phox) and p47(phox) of NAD(P)H oxidase and the phosphorylation of atypical protein kinase C-zeta. Further, genetic or pharmacological inhibition of either NAD(P)H oxidase-derived O2*- or PKC-zeta or NOS abolished the effects of HOCl on eNOS dimers. Consistently, HOCl increased both O2*- and ONOO- and eNOS dimer oxidation in isolated mouse aortas from C57BL/6 but less in those of gp91(phox) knock-out mice. Finally, in human carotid atherosclerotic arteries, eNOS predominantly existed as monomers in parallel with increased staining of both MPO and 3-nitrotyrosine.

CONCLUSIONS

We conclude that HOCl uncouples eNOS by ONOO- generated from PKC-zeta-dependent NAD(P)H oxidase.