We examined the effect of intracolonic administration of anti-adhesion molecule antibodies on DSS-induced colitis in mice. Immunohistochemical staining in mice with colitis showed increased expression of ELAM-1 and ICAM-1 on endothelial cells of vessels in the lamina propria and submucosa at sites of inflamed lesions. Intracolonic administration of anti-ELAM-1 or anti-ICAM-1 antibody decreased bloody stools, anaemia, and histologically evident damage, as well as myeloperoxidase activity and IL-1beta content. We concluded that adhesion molecule expression is important in the development of DSS-induced colitis in mice and that intracolonic administration of anti-adhesion molecule antibodies, especially anti-ELAM-1 antibody, effectively inhibits the colonic inflammation. Intracolonic administration of anti-adhesion molecule antibodies may show therapeutic promise in ulcerative colitis.

The ability of nonsteroidal anti-inflammatory drugs and cyclooxygenase-2 inhibitors to exacerbate inflammatory bowel disease suggests that prostaglandins are important anti-inflammatory mediators in this context. Prostaglandin D(2) has been suggested to exert anti-inflammatory effects. We investigated the possibility that prostaglandin D(2) derived from cyclooxygenase-2 plays an important role in downregulating colonic inflammation in rats. Colitis was induced by intracolonic administration of trinitrobenzene sulfonic acid. At various times thereafter (from 1 h to 7 days), colonic prostaglandin synthesis and myeloperoxidase activity (index of granulocyte infiltration) were measured. Prostaglandin D(2) synthesis was elevated >4-fold above controls within 1-3 h of induction of colitis, preceding significant granulocyte infiltration. Treatment with a selective cyclooxygenase-2 inhibitor abolished the increase in prostaglandin D(2) synthesis and caused a doubling of granulocyte infiltration. Colonic granulocyte infiltration was significantly reduced by administration of prostaglandin D(2) or a DP receptor agonist (BW-245C). These results demonstrate that induction of colitis results in a rapid increase in prostaglandin D(2) synthesis via cyclooxygenase-2. Prostaglandin D(2) downregulates granulocyte infiltration into the colonic mucosa, probably through the DP receptor.

OBJECTIVE

The complement cascade is activated after experimental intracerebral hemorrhage (ICH). It remains unclear, however, whether depleting the complement system will improve injury resulting from ICH. This study investigated the effects of systemic complement depletion on brain edema formation after ICH.

METHODS

Fifty-six pentobarbital-anesthetized Sprague-Dawley rats were used. Treatment animals were complement-depleted with cobra venom factor (CVF) (intraperitoneally). Control rats received an equal volume of saline injection (intraperitoneally). In both treatment and control rats, autologous blood (100 microL) was infused stereotaxically into the right basal ganglia. Rats were killed 2, 24, or 72 hours later for brain water, ion, and tumor necrosis factor-alpha (TNF-alpha) measurements, for Western blot analysis, and for immunohistochemical studies. Brain edema was quantitated by wet/dry weight. TNF-alpha levels were measured by enzyme-linked immunosorbent assay. Western blot analysis was applied for C9 semiquantification. Immunohistochemistry was used to detect complement C3d, C5a, C9, and myeloperoxidase.

RESULTS

Perihematomal brain edema was reduced by systemic complement depletion at 24 hours (78.8+/-0.6% versus 81.5+/-0.8% in control, P:<0.01) and 72 hours (81.5+/-1.5% versus 83.6+/-0.9% in control, P:<0.05), while cerebellar water content was unaffected (78.2+/-0.3% versus 78.0+/-0. 1%). Complement depletion reduced TNF-alpha production 2 hours after ICH. Immunocytochemistry showed that complement depletion significantly reduced perihematomal C9 deposition, C3d production, and the number of C5a- and myeloperoxidase-positive cells.

CONCLUSIONS

Complement depletion by CVF attenuates brain edema in ICH, indicating that complement activation plays an important role in ICH-induced brain edema. Preventing complement activation may be effective in the treatment of ICH.

Human respiratory syncytial virus (RSV) is the most important cause of severe lower respiratory tract disease (LRTD) in young children worldwide. Extensive neutrophil accumulation in the lungs and occlusion of small airways by DNA-rich mucus plugs are characteristic features of severe RSV-LRTD. Activated neutrophils can release neutrophil extracellular traps (NETs), extracellular networks of DNA covered with antimicrobial proteins, as part of the first-line defence against pathogens. NETs can trap and eliminate microbes; however, abundant NET formation may also contribute to airway occlusion. In this study, we investigated whether NETs are induced by RSV and explored their potential anti-viral effect in vitro. Second, we studied NET formation in vivo during severe RSV-LRTD in infants and bovine RSV-LRTD in calves, by examining bronchoalveolar lavage fluid and lung tissue sections, respectively. NETs were visualized in lung cytology and tissue samples by DNA and immunostaining, using antibodies against citrullinated histone H3, elastase and myeloperoxidase. RSV was able to induce NET formation by human neutrophils in vitro. Furthermore, NETs were able to capture RSV, thereby precluding binding of viral particles to target cells and preventing infection. Evidence for the formation of NETs in the airways and lungs was confirmed in children with severe RSV-LRTD. Detailed histopathological examination of calves with RSV-LRTD showed extensive NET formation in dense plugs occluding the airways, either with or without captured viral antigen. Together, these results suggest that, although NETs trap viral particles, their exaggerated formation during severe RSV-LRTD contributes to airway obstruction.

The oxidation of lipoproteins is considered to play a key role in atherogenesis, and tyrosyl radicals have been implicated in the oxidation reaction. Tyrosyl radicals are generated in a system containing myeloperoxidase, H2O2, and tyrosine, but details of this enzyme-catalyzed reaction have not been explored. We have performed transient spectral and kinetic measurements to study the oxidation of tyrosine by the myeloperoxidase intermediates, compounds I and II, using both sequential mixing and single-mixing stopped-flow techniques. The one-electron reduction of compound I to compound II by tyrosine has a second order rate constant of (7.7 +/- 0.1) x 10(5) M-1 s-1. Compound II is then reduced by tyrosine to native enzyme with a second order rate constant of (1.57 +/- 0.06) x 10(4) M-1 s-1. Our study further revealed that, compared with horseradish peroxidase, thyroid peroxidase, and lactoperoxidase, myeloperoxidase is the most efficient catalyst of tyrosine oxidation at physiological pH. The second order rate constant for the myeloperoxidase compound I reaction with tyrosine is comparable with that of its compound I reaction with chloride: (4.7 +/- 0.1) x 10(6) M-1 s-1. Thus, although chloride is considered the major myeloperoxidase substrate, tyrosine is able to compete effectively for compound I. Steady state inhibition studies demonstrate that chloride binds very weakly to the tyrosine binding site of the enzyme. Coupling of tyrosyl radicals yields dityrosine, a highly fluorescent stable compound that had been identified as a possible marker for lipoprotein oxidation. We present spectral and kinetic data showing that dityrosine is further oxidized by both myeloperoxidase compounds I and II. The second order rate constants we determined for dityrosine oxidation are (1.12 +/- 0.01) x 10(5) M-1 s-1 for compound I and (7.5 +/- 0.3) x 10(2) M-1 s-1 for compound II. Therefore, caution must be exercised when using dityrosine as a quantitative index of lipoprotein oxidation, particularly in the presence of myeloperoxidase and H2O2.

Endotoxin-induced microvascular lung injury in mice is a commonly used experimental model of the acute respiratory distress syndrome (ARDS). The present paper aimed to characterize this popular model in a comprehensive and systematic fashion. Male C57bl/6 mice (n = 5) were administered an LD55 dose of E. coli endotoxin (15 mg/kg, i.p.), and lungs were harvested at several time points and evaluated for injury as well as for expression of a variety of inflammatory mediators. Endotoxin induced many features characteristic of acute microvascular lung injury. These included early (1-2 h) expression of inflammatory mediators (IL-1alpha, IL-1beta, IL-4, IL-6, IL-10, TNF-alpha, interferon-alpha, interferon gamma, and MCP-1) and leukocyte accumulation in lung tissue (lung myeloperoxidase activity 18.5 +/- 7.8 U/g tissue, P < 0.05), followed by pulmonary edema (lung water content index 17.4% +/- 2.5%, P < 0.05) and mortality. Histopathological evaluation of lung tissue was compatible with these findings. The characterization of this murine model of endotoxin-induced microvascular injury will facilitate its utilization in ARDS research.

Spinal cord injury (SCI) triggers a well characterized, acute, local inflammation leading to secondary damage at the lesion site. Another little recognized problem may be the activation of circulating inflammatory cells that potentially damage tissues outside the cord. We investigated this problem using severe clip-compression SCI in rats. We studied systemic inflammation after SCI and its effects on lungs and kidneys, as dysfunction of these organs is a frequent, early complication after SCI. From 2-24 h after SCI, the number of circulating neutrophils (especially immature cells) significantly increased by 3-10 fold. Flow cytometry experiments revealed that SCI transiently activates these neutrophils, causing increased oxidative responses to phorbolmyristic acid at 2 h after SCI; then, from 4-24 h, the neutrophils were less responsive. Neutrophil longevity was increased (30-50% decrease in apoptosis) at 2-8 h after SCI. Immunohistochemical analyses demonstrated the invasion of neutrophils into lungs and kidneys (2 h-7 d after SCI) and more phagocytic macrophages in lungs (12 h, 3 d after SCI). Myeloperoxidase and matrix metalloproteinase-9 activity in lung and kidney homogenates increased (12 h-7 d after SCI). Expression of COX-2 increased and lipid peroxidation also occurred within this time. Control experiments inducing local cord damage by excitotoxic quisqualate injection verified that SCI per se is sufficient to trigger systemic inflammation and organ damage. In summary, SCI mobilizes and activates neutrophils that then migrate into visceral organs, a phenomenon occurring in parallel with their well-known entry into the cord injury site. The systemic inflammatory response to SCI should be targeted in the development of new therapeutic strategies to treat SCI.

Granulocyte colony-stimulating factor (G-CSF) is a cytokine that regulates the proliferation and differentiation of neutrophils. The G-CSF receptor (G-CSFR) is a member of the hemopoietic growth factor receptor family. A G-CSFR expression plasmid was introduced into interleukin-3 (IL-3)-dependent mouse myeloid precursor FDC-P1 cells that normally do not respond to G-CSF. G-CSF stimulated proliferation of the transformants, down-regulated Thy-1 and F4/80 antigens on the cell surface, and induced expression of neutrophil-specific genes such as myeloperoxidase (MPO) and leukocyte elastase. On the other hand, neither granulocyte/macrophage colony-stimulating factor (GM-CSF) nor IL-3 induced MPO gene expression, but they inhibited G-CSFR-mediated MPO gene expression. These results suggested that the G-CSFR, but not the IL-3/GM-CSF receptors, transduced the neutrophilic differentiation signal into cells. Mutational analysis of the G-CSFR indicated that the N-terminal region of its cytoplasmic domain is sufficient to transduce the proliferation signal into cells, while the C-terminal region plays an essential role in transducing the differentiation signal.

We investigated the effect of activated protein C (APC) on lipopolysaccharide (LPS)-induced pulmonary vascular injury in rats to investigate the possible usefulness of APC as a treatment for adult respiratory distress syndrome. Intravenously administered LPS (5 mg/kg) significantly increased pulmonary vascular permeability. APC prevented the LPS-induced increase in pulmonary vascular permeability observed at 6 hours. Heparin plus antithrombin III (ATIII) and active site-blocked factor Xa (DEGR-Xa), a selective inhibitor of thrombin generation, inhibited LPS-induced coagulopathy but did not prevent LPS-induced pulmonary vascular injury. LPS-induced pulmonary vascular injury was significantly attenuated in rats with nitrogen mustard-induced leukocytopenia and in rats treated with ONO-5046, a potent granulocyte elastase inhibitor. Administration of LPS also increased pulmonary accumulation of leukocytes, as evaluated by measurement of myeloperoxidase activity in the lungs. APC significantly reduced LPS-induced increases in pulmonary accumulation of leukocytes at 1 hour. Neither ATIII plus heparin nor DEGR-Xa inhibited leukocyte accumulation. Active site-blocked APC (DIP-APC) prevented neither the LPS-induced pulmonary accumulation of leukocytes nor the LPS-induced increase in pulmonary vascular permeability. These results suggest that the mechanism of APC inhibition of LPS-induced pulmonary vascular injury was independent of its anticoagulant activity and was related to its ability to inhibit accumulation of leukocytes. In addition, these findings suggest that the serine protease activity of APC may be essential to its inhibitory effect on LPS-induced pulmonary accumulation of leukocytes and subsequent pulmonary vascular injury.

OBJECTIVE

Hypoxic preconditioning (PC) confers robust neuroprotection against neonatal hypoxic-ischemic brain injury (H-I), yet the underlying mechanism is poorly understood. In the adult brain, neuronal survival after ischemia is associated with the activation of the phosphatidylinositol 3-kinase (PI3-K)/Akt signaling pathway. Suppression of inflammation is a newly identified direct consequence of PI3-K/Akt signaling. We therefore investigated whether PI3-K/Akt suppresses inflammation and contributes to PC-induced neuroprotection.

METHODS

Postnatal day 7 rats were exposed for 3 hours to either ambient air or 8% oxygen, which induces hypoxic PC. H-I was produced 24 hours later by unilateral carotid artery ligation followed by 2.5 hours of hypoxia. Animals were euthanized 0 to 24 hours later for detecting Akt and glycogen synthetase kinase-3beta phosphorylation (p-Akt, p-GSK-3beta), 24 hours later for assessing cytokine expression and inflammatory markers, and 7 days later for measuring brain tissue loss. In addition, LY294002 was injected intracerebroventricularly to inhibit PI3-K/Akt.

RESULTS

Brains with H-I without PC showed delayed but sustained reduction in p-Akt. PC restored the levels of p-Akt and the Akt substrate GSK-3beta, reduced proinflammatory markers (NF-kappaB, COX-2, CD68, myeloperoxidase, and microglial activation), and markedly ameliorated H-I-induced brain tissue loss. Inhibition of PI3-K/Akt using LY294002 attenuated PC neuroprotection and promoted the expression of NF-kappaB, COX-2, and CD68. Proteomic microarray analysis revealed that PC inhibited expression of proinflammatory cytokines induced by H-I or a dose of lipopolysaccharide that resulted in minimal tissue damage.

CONCLUSIONS

Suppression of inflammatory responses may contribute to PC neuroprotection against neonatal H-I brain injury. This effect is mediated in part via upregulating PI3-K/Akt activity.

Antineutrophil cytoplasmic autoantibodies with specificity for myeloperoxidase (MPO) were found in 53 patient sera that were routinely submitted for antineutrophil cytoplasmic antibody determination. Based on clinical and histologic criteria, 15 of these 53 patients were classified as having systemic necrotizing vasculitis of the polyarteritis group, 11 patients were classified as having Wegener's granulomatosis (WG), and 14 were classified as having idiopathic crescentic glomerulonephritis. The remaining 13 patients did not fulfill the diagnostic criteria for these disorders, although most of these patients had clinical symptoms compatible with these disorders. While all patients with WG had renal involvement, only 4 of the 15 patients with systemic necrotizing vasculitis of the polyarteritis group had glomerulonephritis. The sensitivity of autoantibodies to MPO for either systemic necrotizing vasculitis of the polyarteritis group, WG, or idiopathic crescentic glomerulonephritis was further tested in all our patients with these disorders (n = 104). Twenty-seven of 104 patients had autoantibodies to MPO. Furthermore, 69 of the remaining 77 patients had autoantibodies specific for the 29-kd serine protease, which has been reported to be specifically associated with WG. Sera from 8 patients were negative for either of these antibodies (92% sensitivity of autoantibodies to MPO and/or the 29-kd serine protease). The specificity of autoantibodies to MPO for either systemic necrotizing vasculitis of the polyarteritis group, WG, or idiopathic crescentic glomerulonephritis was also tested in selected groups of patients who had closely related diseases. Two of 144 patients had autoantibodies to MPO (specificity 99%).(ABSTRACT TRUNCATED AT 250 WORDS)

Streptococcus pneumoniae is the most frequent cause of community-acquired pneumonia. We sought to determine the role of tumor necrosis factor-alpha (TNF) in the pathogenesis of pneumococcal pneumonia. Induction of pneumonia in C57B1/6 mice by intranasal inoculation with 10(6) colony-forming units (cfu) S. pneumoniae resulted in a sustained increase in TNF activity in lung homogenates reaching a plateau between 12 and 72 h (72 h: 185.49 +/- 54.41 ng/g), while plasma TNF activity remained low or undetectable. Treatment with a neutralizing anti-TNF monoclonal antibody 2 h before inoculation strongly reduced lung TNF activity, but only modestly diminished lung interleukin (IL)-1beta levels, and did not significantly influence lung IL-6, IL-10, and interferon-gamma concentrations. Anti-TNF-treated mice had fourfold more S. pneumoniae cfu isolated from lungs than control mice 40 h after inoculation (p < 0.05), although lung myeloperoxidase activities were similar in both treatment groups. Anti-TNF-treated mice died significantly earlier from pneumococcal pneumonia than control mice (p < 0.05). Endogenously produced TNF is important for host defense during pneumococcal pneumonia.

We have previously described protective effects of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) on pulmonary endothelial cell (EC) barrier function and demonstrated the critical role of cyclopentenone-containing modifications of arachidonoyl moiety in OxPAPC protective effects. In this study we used oxidized phosphocholine (OxPAPC), phosphoserine (OxPAPS), and glycerophosphate (OxPAPA) to investigate the role of polar head groups in EC barrier-protective responses to oxidized phospholipids (OxPLs). OxPAPC and OxPAPS induced sustained barrier enhancement in pulmonary EC, whereas OxPAPA caused a transient protective response as judged by measurements of transendothelial electrical resistance (TER). Non-OxPLs showed no effects on TER levels. All three OxPLs caused enhancement of peripheral EC actin cytoskeleton. OxPAPC and OxPAPS completely abolished LPS-induced EC hyperpermeability in vitro, whereas OxPAPA showed only a partial protective effect. In vivo, intravenous injection of OxPAPS or OxPAPC (1.5 mg/kg) markedly attenuated increases in the protein content, cell counts, and myeloperoxidase activities detected in bronchoalveolar lavage fluid upon intratracheal LPS instillation in mice, although OxPAPC showed less potency. All three OxPLs partially attenuated EC barrier dysfunction induced by IL-6 and thrombin. Their protective effects against thrombin-induced EC barrier dysfunction were linked to the attenuation of the thrombin-induced Rho pathway of EC hyperpermeability and stimulation of Rac-mediated mechanisms of EC barrier recovery. These results demonstrate for the first time the essential role of polar OxPL groups in blunting the LPS-induced EC dysfunction in vitro and in vivo and suggest the mechanism of agonist-induced hyperpermeability attenuation by OxPLs via reduction of Rho and stimulation of Rac signaling.

OBJECTIVE

We investigated the prognostic performance of myeloperoxidase (MPO), and soluble CD40 ligand (sCD40L) along with B-type natriuretic peptide (BNP), high-sensitivity C-reactive protein (hsCRP), and cardiac troponin I (cTnI) for non-fatal recurrent ischaemic events in non-ST elevation acute coronary syndrome (ACS).

RESULTS

We measured plasma MPO and sCD40L in 1524 patients with ACS treated with tirofiban and randomized to early invasive vs. conservative management in the TACTICS-TIMI 18 trial who survived to 180 days. Patients with elevated baseline MPO (>884 pM) were at higher risk of non-fatal myocardial infarction or rehospitalization for ACS at 30 days (9.3 vs. 4.6%, P < 0.001). In contrast, no difference was observed with higher sCD40L (>989 pg/mL, 7.6 vs. 6.3%, P = 0.31). MPO remained associated with recurrent ischaemic events after adjustment for age, ST-deviation, diabetes, prior coronary artery disease, heart failure, cTnI, hsCRP, and sCD40L (OR 2.10; 95% CI 1.36-3.23, P = 0.001). This association was attenuated by 180 days (OR 1.26; 0.95-1.68). Stratification using baseline MPO, BNP, and cTnI identified a >3-fold gradient of risk.

CONCLUSIONS

MPO adds to BNP and cTnI for short-term risk assessment for recurrent ischaemic events in non-ST elevation ACS. sCD40L was not associated with risk in this population treated with a platelet GPIIb/IIIa receptor antagonist.

BACKGROUND

Inflammation and nociceptive sensitization are hallmarks of tissue surrounding surgical incisions. Recent studies demonstrate that several cytokines may participate in the enhancement of nociception near these wounds. Since opioids like morphine interact with neutrophils and other immunocytes, it is possible that morphine exerts some of its antinociceptive action after surgical incision by altering the vigor of the inflammatory response. On the other hand, keratinocytes also express opioid receptors and have the capacity to produce cytokines after injury. Our studies were directed towards determining if opioids alter cytokine production near incisions and to identify cell populations responsible for producing these cytokines.

RESULTS

A murine incisional model was used to measure the effects of acute morphine administration (0.1-10 mg/kg) on nociceptive thresholds, neutrophil infiltration and cytokine production in hind paw skin 30 minutes and 2 hours after incision. Incised hind paws displayed profound allodynia which was reduced by morphine (0.1-10 mg/kg) in the 2 hours following incision. Skin samples harvested from these mice showed enhanced levels of 5 cytokines: IL-1 beta, IL-6, tumor necrosis factor alpha (TNFalpha), granulocyte colony stimulating factor (G-CSF) and keratinocyte-derived cytokine (KC). Morphine reduced these incision-stimulated levels. Separate analyses measuring myeloperoxidase (MPO) and using immunohistochemistry demonstrated that morphine dose-dependently reduced the infiltration of neutrophils into the peri-incisional tissue. The dose of morphine required for reduction of cytokine accumulation, however, was below that required for inhibition of peri-incisional neutrophil infiltration. Additional immunohistochemical studies revealed wound edge keratinocytes as being an important source of cytokines in the acute phase after incision.

CONCLUSIONS

Acute morphine administration of doses as low as 0.1 mg/kg reduces peri-incisional cytokine expression. A reduction in neutrophil infiltration does not provide a complete explanation for this effect, and keratinocytes may be responsible for some incision area cytokine production. These studies suggest that morphine may alter the inflammatory milieu of incisional wounds, but these alterations do not likely contribute significantly to analgesia in the acute setting.

BACKGROUND

Sepsis is defined as a systemic inflammatory response to infection, which in its severe form is associated with multiple organ dysfunction syndrome (MODS). The precise mechanisms by which MODS develops remain unclear. Neutrophils have a pivotal role in the defense against infections; however, overwhelming activation of neutrophils is known to elicit tissue damage.

OBJECTIVE

We investigated the role of the chemokine receptor CCR2 in driving neutrophil infiltration and eliciting tissue damage in remote organs during sepsis.

METHODS

Sepsis was induced in wild-type mice treated with CCR2 antagonist (RS504393) or CCR2(-/-) mice by cecal ligation and puncture (CLP) model. Neutrophil infiltration into the organs was measured by myeloperoxidase activity and fluorescence-activated cell sorter. CCR2 expression and chemotaxis were determined in neutrophils stimulated with Toll-like receptor agonists or isolated from septic mice and patients.

RESULTS

CCR2 expression and responsiveness to its ligands was induced in circulating neutrophils during CLP-induced sepsis by a mechanism dependent on Toll-like receptor/nuclear factor-κB pathway. Genetic or pharmacologic inhibition of CCR2 protected mice from CLP-induced mortality. This protection was associated with lower infiltration of neutrophils into the lungs, heart, and kidneys and reduced serum biochemical indicators of organ injury and dysfunction. Importantly, neutrophils from septic patients express high levels of CCR2, and the severity of patient illness correlated positively with increasing neutrophil chemotaxis to CCR2 ligands.

CONCLUSIONS

Collectively, these data identify CCR2 as a key receptor that drives the inappropriate infiltration of neutrophils into remote organs during sepsis. Therefore, CCR2 blockade is a novel potential therapeutic target for treatment of sepsis-induced MODS.

BACKGROUND

The development of colitis in interleukin 10 (IL-10) deficient mice, together with the known anti-inflammatory and immunomodulatory properties of this cytokine have prompted consideration of IL-10 as a treatment for inflammatory bowel disease (IBD). However, studies using hrIL-10 in IBD models have yielded inconsistent results.

OBJECTIVE

To examine the therapeutic potential of overexpressing the IL-10 gene before and after the induction of experimental colitis in rats.

METHODS

Gene transfer was achieved by intraperitoneal injection of non-replicating human type 5 adenovirus bearing the IL-10 gene, either 24 hours before or one hour after intrarectal administration of dinitrobenzene sulphonic acid in rats. Colonic damage and inflammation was assessed macroscopically and by measuring myeloperoxidase activity and leukotriene B4 concentrations.

RESULTS

Gene transfer increased IL-10 protein in serum for up to six days. IL-10 gene transfer prior to colitis improved colitis macroscopically and histologically, and significantly reduced colonic myeloperoxidase activity and leukotriene B4 concentrations. In contrast, IL-10 gene transfer after the onset of colitis had no beneficial effect.

CONCLUSIONS

Gene therapy using an adenovirus-IL-10 construct was successful in preventing but not in reversing experimental colitis in the rat.

Both Angelica sinensis (Oliv.) Diels (AS) and Ligusticum chuanxiong Hort. (LC) have been used to treat stroke in traditional Chinese medicine for centuries. Ferulic acid (FA), a component in both AS and LC, plays a role in neuroprotection. The purpose of this study was to investigate the effects of FA on cerebral infarct and the involvement of neuroprotective pathway. Rats underwent 2 hours and 24 hours of reperfusion after 90 min middle cerebral artery occlusion (MCAo). The cerebral infarct and neurological deficits were measured after 24 hours of reperfusion. Furthermore, the expression of superoxide radicals, intercellular adhesion molecule-1 (ICAM-1), myeloperoxidase (MPO), nuclear factor-kappaB (NF-kappaB) immunoreactive cells were assessed after 2 hours and 24 hours of reperfusion. Administration of 80 and 100 mg/kg of FA at the beginning of MCAo significantly reduced cerebral infarct and neurological deficit-score, similar results were obtained by 100 mg/kg of FA administered 30 min after MCAo. FA treatment (100 mg/kg i.v.) effectively suppressed superoxide radicals in the parenchyma lesion, and ICAM-1 immunoreactive vessels in the ischemic striatum after 2 hours of reperfusion. FA (100 mg/kg i.v.) reduced the expression of ICAM-1 and NF-kappaB in the ischemic cortex and striatum, also down-regulated MPO immunoreactive cells in the ischemic cortex after 24 hours of reperfusion. These results showed that the effect of FA on reducing cerebral infarct area and neurological deficit-score were at least partially attributed to the inhibition of superoxide radicals, ICAM-1 and NF-kappaB expression in transient MCAo rats.

Peptidylarginine deiminase, registered as PAD V in the DDBJ/GenBank/EMBL data banks, is expressed in HL-60 cells differentiated into granulocytes or monocytes. We analyzed PAD activities in density-fractionated human peripheral blood cell fractions. PAD activity with similar substrate specificity to that of PAD V was found in the eosinophil and neutrophil fractions, which showed single bands comigrating with authentic PAD V on immunoblotting with an anti-PAD V antibody. Both the biochemical and immunoblotting analyses showed marked enrichment of PAD V in the eosinophil fraction. Its immunoreactivity appeared to localize in eosinophilic granules at high density and in myeloperoxidase-negative cytoplasmic granules of neutrophils at low density, as determined by confocal laser-scanning microscopy. Possible roles of PAD V in myeloid differentiation and granulocyte function are discussed. In addition, we present evidence for the presence of PAD(s) that are antigenically different from PAD V in monocytes and lymphocytes.

OBJECTIVE

We sought to investigate the hypothesis that smoking is accompanied by systemic inflammation.

RESULTS

We examined the relation of smoking to 11 systemic inflammatory markers in Framingham Study participants (n=2944, mean age 60 years, 55% women, 12% ethnic minorities) examined from 1998-2001. The cohort was divided into never (n=1149), former (n=1424), and current smokers with last cigarette >6h (n=134) or < or =6h (n=237) prior to phlebotomy. In multivariable-adjusted models there were significant overall between-smoking group differences (defined as p<0.0045 to account for multiple testing) for every inflammatory marker tested, except for serum CD40 ligand (CD40L), myeloperoxidase (MPO) and tumor necrosis factor receptor-2 (TNFR2). With multivariable-adjustment, pair-wise comparisons with never smokers revealed that former smokers had significantly lower concentrations of plasma CD40L (p<0.0001) and higher concentrations of (CRP) C-reactive protein (p=0.002).

CONCLUSIONS

As opposed to never smokers, those with acute cigarette smoke exposure (< or =6h) had significantly higher concentrations of all markers (p<0.0001) except serum CD40L, MPO, and TNFR2; plasma CD40L were significantly lower. Compared with never smokers, cigarette smokers have significantly elevated concentrations of most circulating inflammatory markers, consistent with the hypothesis that smoking is associated with a systemic inflammatory state.

Regarding the mechanisms of methotrexate (MTX) hepatotoxicity and nephrotoxicity, several hypotheses have been put forward, among which oxidative stress (including depletion of glutathione) is likely. This investigation elucidates the role of free radicals in MTX-induced toxicity and the protection by melatonin. Wistar albino rats were injected with MTX intraperitoneally. Following a single dose of MTX (20 mg/kg), either saline (MTX group) or melatonin (10 mg/kg, MTX + Mel group) was administered for 5 days. In other rats, physiologic saline (control group) or melatonin (10 mg/kg, Mel group) was injected for 5 days, following a single injection of saline. On the sixth day, rats were killed to obtain blood, liver, and kidney tissue samples. Malondialdehyde (MDA), an end product of lipid peroxidation, and glutathione (GSH), a key antioxidant, levels were evaluated in blood and tissue homogenates. Reactive oxygen metabolite-induced inflammatory changes in kidney and liver tissues were evaluated by measuring myeloperoxidase (MPO) activity, an index of neutrophil infiltration. MTX administration resulted in increased MDA levels and MPO activity and decreased GSH levels in the blood, liver, and kidney whereas melatonin reversed these effects. When melatonin was administered alone, no significant changes in biochemical parameters were noted. In conclusion, the present study suggests that melatonin may be of therapeutic benefit when used with MTX.

To evaluate the bronchial inflammatory response and its relationship to bacterial colonization in bronchiectasis, we performed a bronchoalveolar lavage (BAL) in 49 patients in stable clinical condition and in nine control subjects. BAL was processed for differential cell count, quantitative bacteriologic cultures, and measurement of inflammatory mediators. An increase was observed in the percentage of neutrophils (37 [0 to 98]) (median[range]) versus 1[0 to 4]%, p = 0.01), in the concentration of elastase (90.5 [8 to 2,930] versus 34 [9 to 44], p = 0.03), myeloperoxidase (9.1 [0 to 376] versus 0.3 [0.1 to 1.4], p = 0.01), and in the levels of TNF-alpha (4 [0 to 186] versus 0 [0 to 7], p = 0.03), IL-8 (195 [0 to 5,520] versus 3 [0 to 31], p = 0.001), and IL-6 (6 [0 to 115] versus 0 [0 to 3], p = 0.001) in patients with bronchiectasis compared with control subjects. Noncolonized patients showed a more intense bronchial inflammatory reaction than did control subjects. This inflammatory reaction was exaggerated in patients colonized by microorganisms with potential pathogenicity (MPP), with a clear relationship with the bronchial bacterial load. Patients with bronchiectasis showed a slight systemic inflammatory response, with poor correlations between systemic and bronchial inflammatory mediators, suggesting that the inflammatory process was mostly compartmentalized. We conclude that patients with bronchiectasis in a stable clinical condition present an active neutrophilic inflammation in the airways that is exaggerated by the presence of MPP, and the higher the bacterial load the more intense the inflammation.