Luminol-dependent Chemiluminescence Response Research Articles

Biomarkers of system inflammation in local and diffuse peritonitis

In cases of any acute surgical abdominal disease the progression of purulent inflammation can lead to local or diffuse peritonitis. The indicators of the degree and specificity of the inflammatory response in blood such as cytokine concentration, neutrophil activity, plasma antioxidant capacity (thiols concentration) could be considered as potential predictors of complications. The luminol-dependent chemiluminescence (CL) response of blood activated by the phorbol ester (PMA), and the concentration of cytokines IL-6, IL-8, IL-10, myeloperoxidase (MPO) and thiols in plasma were measured in patients with uncomplicated condition (group 1, n=8), local peritonitis (group 2, n=9) or diffuse peritonitis (group 3, n=9) at admission to surgery (before surgical operation, b/o), immediately after surgical operation (a/o) and a day after surgery (1 day) as well as in healthy volunteers (norm, n=12). In all time-points the cytokines and MPO concentrations measured by ELISA, in group 3 were higher than in healthy volunteers and in patients in groups 1 and 2. Blood CL demonstrated a more than 5-fold increase above the normal values in all patients, and was also higher in group 2 as compared to group 1 (b/o and a/o). Patients in group 3 had shown both maximum and minimum of CL values, which could be a consequence of neutrophil priming or exhaustion ("immune paralysis"), respectively. The same patients' plasma exhibited low thiol concentration (≤30% vs normal values). In patients with fatal outcomes (group 3, n=2) within a day after surgery, either a decrease of the CL to zero values concurrently with elevated IL-8 and IL-6 concentrations and low thiol levels was observed, or CL exceeded normal values more than 20 times with concurrent complete exhaustion of the plasma thiol pool. No clear dependency between the plasma parameters and neutrophil activity was found. Hence a parameter set for prognosis and/or early diagnosis of infectious complications in acute abdominal pathology should include different biomarkers of the inflammatory response: cytokine profile (IL-6, IL-8, IL-10), MPO and neutrophil activity, antioxidant plasma capacity (e.g., total thiols concentration).

The modulating effects of propofol and its lipid carrier on canine neutrophil functions.

Propofol (2,6-diisopropylphenol), being used as an intravenous sedative and anesthetic agent, influences not only upon nervous system but also for host inflammatory response through modulating neutrophil functions. This study is designed to evaluate the modulating effects of propofol and its lipid carrier administration at clinically relevant rate on canine neutrophil functions. Clinically healthy beagle dogs were received propofol (8.8 mg/kg) from cephalic vein and maintained with propofol dropping infusion (26.4 mg/kg/hr). Blood samples were collected from the dogs before infusion and 30 min after the start of propofol administration, and neutrophil functions were evaluated. The dogs were also administered lipid carrier, and neutrophil functions were evaluated in the same manner as propofol administration. Peripheral white blood cell and neutrophil counts decreased after the propofol or lipid carrier administration. The administration of propofol or lipid carrier significantly reduced neutrophil adherence ability. The superoxide production of neutrophils was measured by luminol-dependent chemiluminescence response using with opsonized zymosan. Peak height of neutrophil chemiluminescence curve was reduced by propofol and lipid carrier administration, on the contrary, peak time of neutrophil chemiluminescence curve was delayed. Administration of propofol or lipid carrier also reduced neutrophil adherence ability to nylon fibers. In the present study, we showed the modulating effects of propofol and its lipid carrier on canine neutrophil functions. However, there was no significant difference in the modulating effects between propofol group and lipid carrier group. Therefore, the modulating effects observed here were deeply concerned in lipid carrier administration.

Chemiluminescence Response of Murine Macrophages on Multilayer Microfluidic Chips

We have demonstrated an integrated platform for microfluidics and chemiluminescence (CL) detection that is capable of parallel cell culture, convenient liquid manipulation, and sensitive chemiluminescent detection. Luminol-dependent CL responses induced by three different stimuli, phytohemagglutinin (PHA), concanavalin A (ConA), and lipopolysaccharides (LPS), which can evoke a CL response in macrophages, were evaluated on this microfluidic chip. We studied the dose-dependent effect of these three stimuli on CL response in murine macrophages. PHA produced the highest CL response compared to LPS and ConA. The CL intensity produced by PHA was 6.85 and four times higher than that by LPS and ConA, respectively, at the low concentration of 100 μg/ml. We have found microfluidic based CL to be a very useful screening tool, which is less laborious and more sensitive. This microfluidic system is disposable and capable of rapid device prototyping; it may prove to be very useful in clinical and pharmaceutical applications.

Effect of smoking on neutrophil oxidative metabolism

Alterations in neutrophil function by tobacco products may play a central role in the pathogenesis of periodontal diseases and several smoking-related systemic diseases. The aim of the study was to evaluate the effect of smoking on neutrophil oxidative metabolism. The study included 17 smoking men free of systemic diseases who were referred for treatment of various odontological diseases to outpatient department of Kaunas University of Medicine Hospital. The age of subjects varied from 22 to 43 years. All subjects answered the questions about smoking habits. Clinical examination included assessment of oral hygiene status according to the OHI-s index and periodontal status according to Russell and Ramfjord indices. To evaluate the oxidative metabolism of neutrophils, luminol- and liucigenin-dependent chemiluminescence and nitroblue tetrazolium test were used. After smoking, extracellular liucigenin-dependent chemiluminescence response was higher as compared to the response before smoking, but total (intra- and extracellular) luminol-dependent chemiluminescence response was the same both before and after smoking. Exposure of neutrophils to smoking caused a significant increase in nitroblue tetrazolium reduction. The release of reactive oxygen species in neutrophils exposed to smoking may alter the pathogenic processes in periodontal diseases.

Neisseria gonorrhoeae Catalase Is Not Required for Experimental Genital Tract Infection despite the Induction of a Localized Neutrophil Response

Neisseria gonorrhoeae produces several antioxidant defenses, including high levels of catalase, which may facilitate the persistence during an inflammatory response via neutralization of H2O2 produced by phagocytes. In vivo testing of the role of catalase in gonococcal survival is critical since several physiological factors impact interactions between N. gonorrhoeae and polymorphonuclear leukocytes (PMNs). Here we assessed the importance of gonococcal catalase in a surrogate model of female genital tract infection. Female BALB/c mice were treated with 17-beta estradiol to promote susceptibility to N. gonorrhoeae and inoculated intravaginally with wild-type gonococci or a catalase (kat) deletion mutant. A localized PMN influx occurred in an average of 43 and 81% of mice infected with wild-type or kat mutant gonococci, respectively, and PMNs associated with numerous wild-type or catalase-deficient bacteria were observed in vaginal smears. The combined results of six experiments showed a significant difference in the number of days wild-type bacteria were recovered compared to the catalase-deficient gonococci. However, there was much variability between experiments, and we found no correlation between PMN influx, colonization load, and clearance of wild-type or kat mutant bacteria. Estradiol treatment did not impair bacterial uptake, the luminol-dependent chemiluminescence response, or the killing capacity of isolated murine PMNs against N. gonorrhoeae or Staphylococcus aureus. Our data suggest N. gonorrhoeae is not significantly challenged by H2O2 produced by PMNs in the murine lower genital tract; alternatively, redundant defense mechanisms may protect the gonococcus from reactive oxygen species during infection.

Effects of Maximal Exercise on Nonspecific Immunity in Athletes under Trained and Detrained Conditions

1755 Very few studies have examined the effect of maximal exercise on nonspecific immunity in athletes under trained and detrained conditions. PURPOSE: To investigate both the acute effects of maximal exercise and the chronic effects of training on nonspecific immunity in winter-sports athletes during different training periods. METHODS: In 15 winter-sports athletes, these acute and chronic effects were investigated during different training periods: (a) in summer, when the athletes were enhancing their aerobic capacity through extensive endurance training; (b) during the winter sports season, in early winter, when both endurance and athletic training were being undertaken; and (c) in spring, when the athletes were resting (detraining for a month). RESULTS: The mean value ± standard deviation) of the maximal oxygen uptake in each of the three training periods (a, b and c) were 65.4 ± 4.6 mL·kg−1·min−1, 63.1 ± 5.5 mL·kg−1·min−1, and 58.3 ± 5.8 mL·kg−1·min−1, respectively. Following maximal exercise, acute peripheral leukocytosis due to lymphocytosis and neutrophila was observed in all three periods. The capacity of isolated and stimulated neutrophils to produce reactive oxygen species as measured by luminol-dependent chemiluminescence (LDCL) was significantly enhanced after maximal exercise before and during the athletic season, but decreased during after-season detraining, suggesting that the conditioning state affected the exercise-induced changes in neutrophil functional status. Serum opsonic activity also showed a similar pattern. As for the chronic effects of training, a significant decline was particularly seen in the resting values of the segmented neutrophil count, the neutrophil LDCL response and the serum IgG level, in the pre-season training period. CONCLUSION: The nonspecific immune status of the subjects might have been partially compromised due to chronic overload while engaged in exhaustive endurance training under heat exposure.

Effects of Maximal Exercise on Nonspecific Immunity in Athletes under Trained and Detrained Conditions

1755 Very few studies have examined the effect of maximal exercise on nonspecific immunity in athletes under trained and detrained conditions. PURPOSE: To investigate both the acute effects of maximal exercise and the chronic effects of training on nonspecific immunity in winter-sports athletes during different training periods. METHODS: In 15 winter-sports athletes, these acute and chronic effects were investigated during different training periods: (a) in summer, when the athletes were enhancing their aerobic capacity through extensive endurance training; (b) during the winter sports season, in early winter, when both endurance and athletic training were being undertaken; and (c) in spring, when the athletes were resting (detraining for a month). RESULTS: The mean value ± standard deviation) of the maximal oxygen uptake in each of the three training periods (a, b and c) were 65.4 ± 4.6 mL·kg−1·min−1, 63.1 ± 5.5 mL·kg−1·min−1, and 58.3 ± 5.8 mL·kg−1·min−1, respectively. Following maximal exercise, acute peripheral leukocytosis due to lymphocytosis and neutrophila was observed in all three periods. The capacity of isolated and stimulated neutrophils to produce reactive oxygen species as measured by luminol-dependent chemiluminescence (LDCL) was significantly enhanced after maximal exercise before and during the athletic season, but decreased during after-season detraining, suggesting that the conditioning state affected the exercise-induced changes in neutrophil functional status. Serum opsonic activity also showed a similar pattern. As for the chronic effects of training, a significant decline was particularly seen in the resting values of the segmented neutrophil count, the neutrophil LDCL response and the serum IgG level, in the pre-season training period. CONCLUSION: The nonspecific immune status of the subjects might have been partially compromised due to chronic overload while engaged in exhaustive endurance training under heat exposure.

Zymosan-induced luminol-dependent chemiluminescence response of circulating and extravasated leukocytes in experimental sepsis.

This study examines a concurrent profiling of circulating and extravasated polymorphonuclear leukocytes (PMNs) in a rat model of experimental sepsis. Fecal peritonitis was induced in Wistar male rats by intraperitoneal instillation of a fecal suspension in saline (1:1 w/v). Blood and peritoneal fluid were collected 8 h following fecal inoculation for the evaluation of inflammatory response of PMNs using zymosan-induced luminol-dependent chemiluminescence. Fifty microliters of pre-diluted blood or peritoneal fluid samples were mixed with 150 microl of reaction mixture (4 x 10(-4) M luminol+50 microg opsonized zymosan+0.1% gelatin in Hank's balanced salt solution) and the chemiluminescence signal was measured in a luminometer at 37 degrees C. Fecal peritonitis caused a significant leukocytopenia (3540+/-297 mm(-3) versus control value of 7525+/-711 mm(-3), p < 0.001) accompanied by massive infiltration of PMNs in the peritoneal cavity (34700+/-4006 versus 7325+/-425 mm(-3), p < 0.001). The phagocytic activity of circulating blood PMNs was down-regulated whereas a significant up-regulation was observed in the activity of PMNs from peritoneal fluid. In conclusion, this study clearly demonstrates sepsis-induced alterations in both blood and peritoneal fluid PMNs and their quantitative assessment may be helpful in disease evaluation and designing effective therapies.

Ecabet sodium attenuates reactive oxygen species produced by neutrophils after priming with bacterial lipopolysaccharides.

The pathogenic roles of reactive oxygen species (ROS) have been implicated in ulcerative colitis (UC). The aim of this study was to examine the effects of ecabet sodium on ROS produced by human neutrophils, particularly after being primed by bacterial lipopolysaccharides (LPS). Neutrophils were isolated from six healthy volunteers. Each well of a 96-well microplate received neutrophil suspension (1.0 x 10(5) cells) and the plates were incubated at 37 degrees C for 30 min with or without E. coli LPS (f.c. 0.001 ng/ micro L). Ecabet sodium (f.c. 0-5.0 mg/mL) was added before starting or after finishing the incubation. Neutrophils were stimulated by opsonized zymosan (OZ; 1.0 mg/mL) or calcium ionophore (A21837; 0.3 micro mol/L) and luminol-dependent chemiluminescence response was measured using a Lumi Box H-1000. Ecabet sodium attenuated ROS production at a concentration of 5.0 mg/mL (p < 0.05) in LPS-primed neutrophils. However, attenuating effects were not significantly different when ecabet sodium was added before or after the incubation with E. coli LPS. Ecabet sodium may have some attenuating effects on ROS produced by human neutrophils even after neutrophils are primed by bacterial LPS. These results may explain, in part, the therapeutic effects of ecabet sodium for UC.

Helicobacter pylori water soluble surface proteins prime human neutrophils for enhanced production of reactive oxygen species and stimulate chemokine production

Backgrounds/Aims: Chronic gastritis induced by Helicobacter pylori is characterised by considerable neutrophil infiltration into the gastric mucosa without mucosal invasion of bacteria. Bacteria have different characteristics with respect to their...

Impact of a competitive marathon race on systemic cytokine and neutrophil responses.

To investigate whether cytokines and neutrophils mediate exercise-related pathogenesis, we examined their responses and possible association after exhaustive exercise. Plasma and urine samples were obtained from 10 male runners before and after a 42.195-km marathon race. Major cytokines and neutrophil activation markers [myeloperoxidase (MPO) and lactoferrin (LTF)] were measured by enzyme-linked immunosorbent assays. Functional modulation of standard neutrophils and monocytes by plasma was determined on their luminol-dependent chemiluminescence responses. The race induced peripheral neutrophilia accompanied by an increase in band neutrophils and monocytosis. Plasma MPO and LTF concentrations increased significantly by 1.8 and 1.4 times after the race. There was a greater increase in urine concentrations of MPO and LTF, 12.3 and 3.5 times after exercise, respectively, suggesting that neutrophil activation occurred and that renal clearance exceeded the increase in plasma concentrations. Plasma interleukin (IL)-6, IL-8, IL-10, granulocyte colony-stimulating factor (G-CSF), macrophage CSF (M-CSF), and monocyte chemotactic protein 1 (MCP-1) increased significantly after the race, and urine IL-1beta, IL-6, G-CSF, M-CSF, and MCP-1 increased significantly. The plasma IL-6 responses correlated with the increases of band neutrophil count (r = 0.860, P < 0.01), suggesting IL-6-mediated bone marrow release of neutrophils. Furthermore, the increases in urine MPO concentration were correlated with increases in urine IL-6 (r = 0.868, P < 0.01) and G-CSF (r = 0.875, P < 0.01), suggesting that these cytokines promoted neutrophil activation. However, preincubation of neutrophils and monocytes with postexercise plasma could not cause priming responses, possibly because of the exercise-induced enhancement of plasma antioxidant activity. Although many cytokines recruiting and priming neutrophils and monocytes were secreted and functional after exhaustive exercise, overwhelming antioxidant and antiinflammatory defenses were induced, preventing exercise-induced oxidative stress.

Cytotoxic activity of Mannheimia haemolytica strains in relation to diversity of the leukotoxin structural gene lktA

The leukotoxin of Mannheimia haemolytica has a very high degree of amino acid diversity because the lktA gene has a complex mosaic structure that has been derived by horizontal DNA transfer and intragenic recombination. The objective of the present study was to determine the effect of this amino acid diversity on leukotoxin cytotoxicity against bovine and ovine cell types. This was done by comparing the luminol-dependent chemiluminescence response of bovine and ovine neutrophils after pre-incubation with the various leukotoxin types. The LktA1.1-type leukotoxin associated with bovine serotype A1 and A6 strains differs from the LktA1.2-type leukotoxin produced by ovine serotype A1 and A6 strains at a single amino acid position and has enhanced activity against bovine but reduced activity against ovine neutrophils. These findings, together with the exclusive association of the LktA1.1-type leukotoxin with bovine strains, suggest that this leukotoxin type has an adaptive advantage in the bovine host. Leukotoxins LktA6–LktA10 are associated with ovine strains and have complex mosaic structures and diverse amino acid sequences but similar levels of cytotoxic activity against bovine and ovine neutrophils, respectively. However, ovine neutrophils were more sensitive to the cytotoxic activities of these leukotoxins than were bovine neutrophils. LktA8- and LktA10-type leukotoxins are associated with serotype A2 and A7 strains that are responsible for the majority of ovine disease cases, but LktA6-, LktA7- and LktA9-type leukotoxins are associated with less common serotypes. These findings contribute to the growing body of evidence suggesting that factors other than leukotoxin cytotoxicity are responsible for the full expression of virulence in M. haemolytica. Overall, the extensive recombinational exchanges within the lktA gene of M. haemolytica have had little effect on leukotoxin function which is highly conserved.

N-Acetylcysteine Effect on the Luminol-dependent Chemiluminescence Pattern of Reactive Oxygen Species Generation by Human Polymorphonuclear Leukocytes

The evidence of the effect of N-acetylcysteine on reactive oxygen species produced by human polymorphonuclear leukocytes (PMNs) is often contradictory, as thiol compounds may react with not only reactive oxygen and nitrogen species but also they may influence intracellular glutathione levels. The effect of 20, 100 and 200μM N-aceylcysteine (NAC) on luminol dependent chemiluminescence (LDCL) of human PMNs (106 cells/ml phospate buffered saline (PBS)) and whole blood to N-formyl-methionyl-leucyl-phenylalanine (fMLP) and phorbol-12-myristate-13-acetate (PMA) was studied. Further, the ability of NAC to increase PMNs intracellular thiols and affect subsequent PMNs, stimulation was assessed. NAC 100 and 200μM, but not 20μM, was found to attenuate the kinetic parameters of initial phase of fMLP-stimulated PMNs oxidative burst. NAC at the concentration of 100 and 200μM decreased the rate, maximum and integrated value of PMNs response to fMLP. The integrated value of PMA-induced PMNs and fMLP-induced whole blood LDCL response was also decreased by 100 and 200μM NAC. Furthermore, all tested NAC concentrations decreased LDCL of resting PMNs suspension.The chemiluminescence pattern of reactive oxygen species (ROS) generation by PMNs stimulated with fMLP or PMA did not differ significantly from those preincubated with either 20, 100, or 200μM NAC. Similarly, NAC did not increase the concentration of intracellular thiols in resting PMNs. However, addition of 200μM NAC to PMA-stimulated PMNs prevented the decline in intracellular thiols pool. Both PMA- and fMLP-activated PMNs oxidized extracellular NAC. These results indicate that NAC decreases the intensity of PMNs oxidative burst by direct scavenger activity.

Production of chemokines and reactive oxygen species by human neutrophils stimulated by Helicobacter pylori.

Bacteria have different characteristics in stimulation of human neutrophils to produce reactive oxygen species (ROS) and chemokines. This study examined the ability of Helicobacter pylori to induce production of ROS and chemokines by human neutrophils. H. pylori strains (1.5 x 108 CFU/ml) were cocultured with 5 x 104 neutrophils isolated from healthy subjects. Samples were incubated with human serum with or without IgG antibodies to H. pylori. ROS production was measured using luminol-dependent chemiluminescence (LmCL), and the concentrations of chemokines (IL-8, RANTES, MIP-1alpha and MCP-1) were measured by ELISA. The mean of the highest LmCL (peak height; PH) value stimulated by H. pylori was 3318 in the absence of serum. PH increased to 4687 when incubated with anti-H. pylori antibody-positive sera (p <.001) but antibody-negative sera did not affect LmCL response. The mean final concentration of IL-8 produced in the absence of serum was 142.6 pg/ml. Increased IL-8 production was seen by addition of antibody positive serum (p <.01). IL-8 production was not significantly correlated with production of ROS. On the other hand, H. pylori stimulation did not induce neutrophil production of RANTES, MIP-1alpha or MCP-1. H. pylori was capable of inducing IL-8 production by human neutrophils, but not C-C chemokines. Production of C-X-C dominant chemokine by neutrophils is consistent with the pathological characteristics of H. pylori-induced gastritis, where persistent neutrophil infiltration is present.

Priming of dolphin neutrophil respiratory burst by recombinant tumor necrosis factor alpha

We studied the effects of recombinant dolphin tumor necrosis factor alpha (rdoTNFα) on the respiratory burst activity of dolphin neutrophils. rdoTNFα enhanced the luminol-dependent chemiluminescence response of dolphin neutrophils induced by concanavalin-A, opsonized zymosan, and heated plasma, but not that induced by phorbol myristate acetate. The TNF-associated priming activity was concentration- and preincubation time-dependent, and heat-instable. These data suggest that, as in human neutrophils, TNFα enhances the respiratory burst in dolphin neutrophils that follows short-term incubation with various receptor-mediated agonists.

Different metabolic effects of ganglioside GM1 in brain synaptosomes and phagocytic cells.

The metabolic effects of ganglioside GM1 were found to be quite different in brain synaptosomes and phagocytic cells. Incubation of rat brain cortex synaptosomes with GM1 was shown to decrease the production of reactive oxygen species induced by Fe2+-H2O2 system and measured by chemiluminometric method in the presence of luminol. Gangliosides GM1, GD1a, and GT1b significantly diminished the induced accumulation of lipid peroxidation product in brain synaptosomes, but protein kinase inhibitor (polymyxin B) abolished this effect. Incubation with antioxidants or GM1 significantly diminished the increase of 45Ca2+ influx and oxidative inactivation of Na+,K+-ATPase in brain synaptosomes exposed to glutamate, the effect of GM1 was concentration-dependent in the range 10(-11)-10(-8) M. But the incubation of human neutrophils and mouse peritoneal macrophages with 10(-11)-10(-10) M GM1, on the contrary, increased several times the luminol-dependent chemiluminescence response of these cells to activation by low concentrations of 12-myristate-13-acetate phorbol ester. The opposite effects of GM1 in the nerve endings and phagocytic cells seem to be protective in both cases as the inhibition of reactive oxygen species production in the nerve cells may enhance their viability in damaged brain, while the intensification of their production in phagocytic cells may promote the resistance of organism to infection.

Increased leukocyte activity as a predictor for flow-limiting coronary lesions in patients with angina pectoris

Myocardial ischemia initiates an acute inflammatory response, in which polymorphonuclear leukocytes (PMNs) are major participants. We investigated whether increased PMN activity in the peripheral blood is a marker for high-grade coronary artery stenosis in patients with angina pectoris. PMN activity was examined in 45 patients by measuring the luminol-dependent chemiluminescence (CL) response of the peripheral blood upon addition of phorbol myristate acetate (PMA). The CL of patients with angiographic filling delay was 1.6-fold higher than in patients without delay (480±54 vs. 302±33 counts/PMN, P<0.01). The CL of patients with a filling delay of the left anterior descending artery (LAD) was 1.7-fold higher than in patients without an LAD filling delay (537±79 vs. 317±27 counts/PMN, P<0.01). With the CL cut-off at 400 counts/PMN, the sensitivity and specificity for detecting an angiographic filling delay of the LAD were 69 and 75%, respectively. These data indicate that PMN activity in the peripheral blood is increased with the presence of a flow-limiting coronary lesion showing an angiographic filling delay. Measuring PMN activity may be a useful approach to assess indications for and timing of angiography and/or adjunctive invasive therapies in patients with angina pectoris.

Helicobacter pylori water extracts prime human neutrophils for enhanced reactive oxygen species production and stimulate production of CXC chemokine but not CC chemokine

BACKGROUND: Chronic gastritis induced by Helicobacter pylori (H. pylon) is charactsrisnd by considerable neutrophil infiltration into the gastric mucosa. Neutropbils are considered to play an important role in oxidative injury of gastric mucosa. The aim of this study was to examine the effects of H. pyloriwater extracts on oxidative burst and chemokine production of human neutrophils. METHODS: H. pylori cells were extracted by harvesting into distilled water and centr'itugation. Filtered water extracts were added to 5x10' neotrophils separated from volunteers without H. pylori infection. Neutrophil oxidative burst was measured using luminol-dependent chemiluminescence (LmCL) at 37 °C for 1 hour. Further incubation was performed at 37 °C for 12 hours and the production of IL-8 (CXC chemoldne) and MIP-la,(CC chemokine) was measured by ELISA. Neutrophils were also pre-incubated with H. pylor/water extracts (f.c. 0-2.0 %) for 30 min. After the pre-incubation, neutrophils were stimulated by H. pyloricells to examine the neutrophil-priming effects of H. pylor/water extracts. RESULTS: H. pyloriwater extracts induced neutrophil oxidative burst but the LmCL response was weak (<5000 cpm). On the other hand, pre-incubation with water extracts enhanced LmCL response of neutrophils stimulated by H. pylori cells in a concentration dependent manner (p<O.01). H. pylor/water extracts also stimulated oeutrophil IL-8 production coflcentration..(lepandentty (p<O.O01) while M IP-lo~vas not detectable. DISCUSSION: H. pylor/water extracts are capable of priming human neutrophils for enhanced production of reactive oxygen species. H. p/for/ water extracts stimulates neutrophil CXC chemokine production but not CC chemoldne. These results may suggest H. pylori products in water extract have a role in the activation and migration of neutrophils in H. pylori induced gastritis.

Effect of interferon-alpha on production of reactive oxygen species by human neutrophils.

In order to examine that the effects of interferon alpha (IFN-alpha) on the production of reactive oxygen species from human neutrophils, we investigated in vitro effects of IFN-alpha on neutrophil chemiluminescence responses. Human neutrophils were incubated with 0-1 x 10(5) IU/mL IFN-alpha for 10-60 min just before stimulation with opsonized zymosan and phorbol 12-myristate 13-acetate. Luminol-dependent (LmCL) and lucigenin-dependent (LgCL) chemiluminescence responses were measured using a Lumi Box H-1000. The LmCL response was inhibited by pre-incubation with high concentrations of IFN-alpha (more than 1 x 10(4) IU/mL). In contrast, LgCL response was increased when neutrophils were pre-incubated with high concentration of IFN-alpha. Effects of pre-incubation of IFN-alpha were similar between CL responses stimulated with OZ and PMA. The increase of LgCL might be due to the inhibitory effect of IFN-alpha on myeloperoxidase degranulation, in view of the decreased LmCL responses.

A Characterization of Amiodarone-Induced Pulmonary Toxicity in F344 Rats and Identification of Surfactant Protein-D as a Potential Biomarker for the Development of the Toxicity

Amiodarone (AD) is gaining support as a first-line antiarrhythmic drug despite its potentially fatal pulmonary toxicity involving inflammation and fibrosis. The goals of this study were to characterize a rat model of AD-induced pulmonary toxicity (AIPT) and identify a serum biomarker to aid in the diagnosis of the onset of pulmonary toxicity. Male F344 rats were instilled intratracheally with AD (6.25 mg/kg with a 3.125 mg/ml solution) in sterile water or the sterile water vehicle on days 0 and 2, a protocol that led to the development of pulmonary fibrosis on day 28 in the AD-treated animals. Animals were killed on days 3, 5, 6, 7, or 10 and bronchoalveolar lavage (BAL) was performed. Recovery of alveolar macrophages and eosinophils was increased on days 3 and 5, while neutrophil recovery and albumin levels in the first BAL fraction were significantly elevated only on day 3. BAL cells recovered from AD-treated rats at day 3 produced more phorbol myristate acetate-stimulated luminol-dependent chemiluminescence (LDCL) over 20 min than BAL cells from control rats. Experiments using specific inhibitors implicated superoxide and nitric oxide in at least part of the LDCL response. Serum levels of surfactant protein-D (SP-D), a surfactant-associated protein, were increased concurrently with the inflammatory response in the lungs. These findings indicate that this model exhibits transient pulmonary inflammation and damage, with the potential for elevated oxidant production in the lungs and subsequent pulmonary fibrosis. Also, SP-D is proposed as a specific biomarker to monitor the onset of AIPT in this model.