Zymosan-induced Chemiluminescence Research Articles

Effect of periimplantitis treatment on the chemiluminescent activity of neutrophilic granulocytes <i>in vitro</i>

BACKGROUND: Periimplantitis causes implant loss, which reduces the quality of dental treatment. The effect of periimplantitis treatment on the immune response, particularly the chemiluminescent activity of neutrophilic granulocytes, is unclear. The paper addresses this issue, as well as improvements in periimplantitis treatment approaches. AIM: To assess the biocompatibility of implants removed from the inflammation site and treated with Air Flow and laser. MATERIALS AND METHODS: Three types of implant surface were assessed: anodized titanium dioxide (TiO2); sand-blasted, large grit, acid-etched (SLA); and resorbable blast media (RBM). Implants were removed in patients with confirmed periimplantitis, followed by an air-powder abrasive surface treatment with Air Flow and chlorhexidine, using a YSGG laser with a wave length of 2,780 nm. New (out of the box) implants were used as a control. Biocompatibility was assessed by the synthesis of primary and secondary reactive oxygen species (ROS) by neutrophils; the intensity and kinetics of synthesis were examined using chemiluminescence analysis. RESULTS: Lucigenin- and luminol-dependent chemiluminescence of neutrophils was assessed following in vitro incubation with SLA, RMB, and TiO2 implants removed in patients with confirmed periimplantitis and treated with Air Flow and chlorhexidine. The study found a decrease in the time to maximum and an increase in the maximum intensity and area under the curve of spontaneous and zymosan-induced chemiluminescence of neutrophils, regardless of the studied implant type. Changes in the zymosan-induced chemiluminescence of neutrophils following incubation with implants were greater than changes in spontaneous chemiluminescence, resulting in a higher activation index. No significant changes in neutrophil chemiluminescence were observed after in vitro incubation with laser-treated SLA, RMB, and TiO2 implants. CONCLUSION: SLA, RMB, and TiO2 implants removed in periimplantitis patients and treated with Air Flow and chlorhexidine have low biocompatibility. However, Air Flow-treated RBM implants show relatively superior biocompatibility than SLA and TiO2 implants, which is attributed to the decreased synthesis of primary and secondary ROS by neutrophils during in vitro incubation. The degree of ROS synthesis by neutrophils during incubation with laser-treated implants corresponds to that of the control, indicating increased biocompatibility of laser-treated implants. Laser-treated TiO2 implants had the lowest neutrophil activation during incubation, determining their maximum biocompatibility among the studied implants.

The level of blood neutrophils primary reactive oxygen species production in Graves’ disease patients undergoing conservative therapy with thiamazole.

Background. Conservative therapy with thiamazole in Graves’ disease helps to reduce the level of oxidative stress by restoring euthyroidism and the total reactive antioxidant potential of plasma. However, until now, in patients with Graves’ disease, the radical-producing ability of neutrophils remains unexplored at the initial stage of free radical conversion at different durations of drug-induced euthyroidism, which seems significant in revealing the mechanisms of disease recurrence. The aim is to study the level of production of primary reactive oxygen species in peripheral blood neutrophils depending on the duration of drug — induced euthyroidism in patients with Graves’ disease. Materials and methods. A single-center, prospective, controlled study was conducted with the participation of patients with laboratory-confirmed Graves’ disease. Subgroups of patients with Graves’ disease were formed depending on the duration of achieved drug-induced euthyroidism with continuous conservative therapy with thiamazole. The level of ROS production in peripheral blood neutrophils was determined by chemiluminescence. The assessment of spontaneous and zymosan-induced chemiluminescence was carried out for 90 minutes on a 36-channel chemiluminescence analyzer BLM-3607 (Medbiotech LLC, Krasnoyarsk). Results. The study included 102 Graves’ disease women with an average age of 47.54 ± 15.47 years. Of these, 75 (73.5 %) with relapse and 27 (26.47 %) with newly diagnosed disease. The total synthesis of (S) primary ROS increased statistically significantly relative to the control values in the examined patients with a duration of the euthyroid state of 5–8 and 9–12 months. It was found that in patients with Graves’ disease with stable drug-induced euthyroidism for 5–8 and 9–12 months, the S index of zymosan-induced lucigenin-dependent chemiluminescence of neutrophils increased statistically significantly, respectively, by 10.7 and 7.4 times relative to the control values. Conclusion. The restoration and maintenance of drug-induced euthyroidism for more than 12 months against the background of conservative thiamazole therapy in patients with Graves’ disease reduces the load of systemic oxidative stress on the immune system. However, the high total production of primary ROS in neutrophils of patients with Graves’ disease in a state of drug-induced euthyroidism with conservative thiamazole therapy may play a significant role in disconnecting the functioning of the NADPH oxidase complex and determine the functional capabilities of neutrophils in case of disease recurrence.

Phagocytic and chemiluminescent activity of blood neutrophils in patients with bladder cancer

BACKGROUND: Tumor microenvironment modulates (including with the help of metabolites) the functional activity of the neutrophils that contribute to the reprogramming of the antitumor activity into a protumor one.
 AIMS: To study the phagocytic and chemiluminescent activity of neutrophils in patients with bladder cancer (BC) under the influence of metabolites of the tumor microenvironment in vitro.
 MATERIALS AND METHODS: We examined 37 patients with superficial BC (T1,а,isN0M0) and considered 32 healthy individuals as a control group. Neutrophils isolated from their blood were incubated in vitro with lactate, ADP, and glutamate. Phagocytic activity was examined using flow cytometry, and the intensity of the respiratory burst of neutrophils was evaluated via chemiluminescent analysis.
 RESULTS: In patients with BC, the phagocytic index (PhI) values are reduced compared to the control sample (without in vitro metabolite exposure) and when exposed to glutamate, while the effect of lactate on cells causes an increase in the phagocytic number and PhI. Moreover, under the influence of lactate in vitro, the activity of spontaneous and zymosan-induced chemiluminescence of neutrophils decreases. ADP causes a decrease in spontaneous chemiluminescence only. Finally, under the influence of glutamate, the indicators of spontaneous and induced chemiluminescence decrease.
 CONCLUSIONS: Under the influence of lactate and ADP (products of tumor cells), the phagocytic activity of a population of immature neutrophils is stimulated, which leads to myeloid suppressor cells that inhibit antitumor immunity. Thus, metabolites of the tumor microenvironment modulate the activity of the respiratory burst of neutrophils in patients with BC.

The chemiluminescent and enzymatic activity of blood neutrophils in patients with Graves' disease depending on hyperthyroidism compensation

BACKGROUND: Oxidative stress in Graves’ disease can potentiate the cytotoxicity of neutrophils and support autoimmune inflammation. Elimination of hyperthyroidism with conservative therapy with thiamazole only in some cases leads to remission, which determines the necessity to search an immunological markers for etiotropic therapeutic approaches in treatment of the disease.AIM: To study the chemiluminescent and enzymatic activity of peripheral blood neutrophils in patients with Graves’ disease depending on hyperthyroidism compensation to determine the intracellular targets of immunotropic treatment.MATERIALS AND METHODS: Spontaneous and zymosan-induced chemiluminescence was evaluated using a 36-channel chemiluminescence analyzer «BLM-3607» (MedBioTech, Krasnoyarsk). The reactivity of peripheral blood neutrophils was characterized by: Tmax — the rate of development of the chemiluminescent reaction, Imax — the maximum level of reactive oxygen species (ROS) synthesis and the area under the chemiluminescence curve (S — total synthesis of ROS for 90 minutes of measurement). The activity of NAD(P)-dependent dehydrogenases in neutrophils was determined using the bioluminescent method.RESULTS: The prospective study included 126 women with Graves’ disease, aged 18 to 65 years, 93 (73.81%) with compensated and 33 (26.19%) with uncompensated hyperthyroidism. In uncompensated hyperthyroidism, the indicator S of spontaneous ROS and zymosan-induced lucigenin-dependent chemiluminescence increases significantly, both relative to the control and to the values of compensated hyperthyroidism patients. Antigenic neutrophils stimulation during luminol-dependent chemiluminescence in patients with recurrent hyperthyroidism demonstrated more than tenfold increase in the total synthesis of ROS relative to the control, but no statistically significant differences with compensated hyperthyroidism patients. A high level of NADH-GDG was established in patients with recurrent hyperthyroidism, both relative to the control range and to the patients with euthyroidism.CONCLUSION: Violation of oxygen peripheral blood neutrophils metabolism in patients with euthyroidism mainly affects the production of ROS, which is associated with hyperthyroidism compensation and the immunosuppressive effect of thiamazole. In patients with recurrent hyperthyroidism, there are more changes in the production of high-energy oxidants not only at initial oxidative reactions stage, but also at the level of secondary ROS, indicating the activation of cellular response immunological mechanisms.

Monocyte Chemiluminescence Traits in Gastric Cancer

Aim. A study of monocyte chemiluminescent activity at variant stages of gastric cancer.Materials and methods. The study enrolled 90 gastric cancer patients and 70 healthy donors. Spontaneous and induced chemiluminescence in monocytes was assessed for 90 min with a “BLM 3607” 36-channel chemiluminescence analyser (Russia). Opsonized zymosan-induced chemiluminescence enhancement was measured as a ratio of the areas under the induced vs. spontaneous chemiluminescence curves, the activation index. Statistical significance was estimated with the Mann—Whitney criterion (p < 0.05).Results. The maximal spontaneous monocyte chemiluminescence intensity significantly decreased in stage IV gastric cancer patients compared to the control cohort (p = 0.035). Time to maximum in spontaneous chemiluminescence increased in all gastric cancer patients vs. control (p = 0.001), and in stage IV gastric cancer vs. stage I patients (p = 0.043). The areas under a curve in spontaneous and induced monocyte chemiluminescence increased in all gastric cancer patients vs. control (p = 0.001), and in stage IV gastric cancer vs. stage I patients (p = 0.037). The activation index was higher in all gastric cancer cases compared to control (p = 0.001).Conclusion. All patients with gastric adenocarcinoma, irrespective of the stage, revealed changes in the monocyte chemiluminescence activity, i.e. a longer time to maximum in spontaneous chemiluminescence and larger area under the curve of spontaneous and induced chemiluminescence, the activation index. Maximal monocyte spontaneous chemiluminescence intensity diminished in stage IV gastric cancer compared to the control cohort. Immune activity reflected in monocyte chemiluminescence correlates with the stage of gastric adenocarcinoma.

Aspirin Resistance as a Result of Impaired Interaction of Platelets and Neutrophils in Patients with Coronary Heart Disease

Aim. To study the relationship between the levels of synthesis of reactive oxygen species (ROS) by platelets and neutrophils in patients with coronary heart disease (CHD) before and after coronary artery bypass grafting (CABG), depending on sensitivity to acetylsalicylic acid (ASA).Material and methods. The study included 95 patients with coronary artery disease who are indicated for CABG surgery. The control group consisted of 30 healthy donors. The antiplatelet therapy was stopped for at least 5 days before CABG. In the postoperative period, from the first day, all patients were received 100 mg of an enteric form of acetylsalicylic acid (ASA). Resistance to ASA was determined at the level of platelet aggregation with arachidonic acid ≥20% by optical agregometry at least at one observation point: before CABG, on 1-3 day and on 8-10 day after surgery. We evaluated the spontaneous and induced lucigenin-dependent chemiluminescence (CL) of platelets (ADP induction) and neutrophils (zymosan induction) by the exit time to maximum intensity (Tmax), maximum intensity (Imax) and area (S) under the CL curve.Results. 70.5% sensitive (sASA) and 29.5% resistant (rASA) to ASA patients were revealed. Prior to CABG, in sASA patients, the Imax of spontaneous and zymosan-induced neutrophil CL and CL platelet activity was increased relative to control values. Tmax of spontaneous platelet CL, Imax and S under the ADP-induced platelet CL curve were lower in sASA patients, if to compare with rASA patients. On the 1st and 8-10th day after CABG in sASA patients, the CL indicators of neutrophil and platelet activity also remained elevated compared to control values. On the 1st day after CABG decreased levels of S under the spontaneous CL curve of neutrophils in rASA patients was established compared with sASA patients, and increased levels of Imax and S under the curve of induced neutrophil CL were detected in comparison with the control range. In rASA patients, the values of Tmax of spontaneous platelet CL decreased in relation to the values detected in the control group and sASA patients. On the 8–10th day after CABG, most indicators of spontaneous and zymosan-induced CL neutrophils in rASA patients were also increased compared to control values. In rASA patients a positive correlation of Imax-induced CL was found (r=0.83) on the 1st day after CABG and negative correlations of Tmax of spontaneous CL (r=- 0.75) and S under the curve induced CL (r=-0.70) on the 8-10th day were detected between platelets and neutrophils.Conclusion. In sASA patients with coronary heart disease before and after CABG, a high level of synthesis of superoxide radical by neutrophils and platelets was detected. The relationship between the levels of the synthesis of superoxide radical by neutrophils and platelets was found only after CABG in rASA patients. Increased synthesis of superoxide radical due to metabolic and regulatory relationships in neutrophils and platelets stimulates pro-inflammatory processes in coronary artery disease and determines the sensitivity of platelets to ASA.

Chemiluminescence activity of neutrophil granulocytes under the influence of magnetic nanoparticles of ferrihydrite (<i>in vitro</i>)

The article presents the results of studying and evaluating the impact of magnetic nanoparticles of ferrihydrite on neutrophil granulocytes in human blood ( in vitro ) in order to determine their bio compatibility and eco toxicity. The subject soft here search were blood neutrophil granulocytes of 29 conditionally-healthy donors of blood, as well as magnetic nanoparticles of ferrihydrite (NP), the preparation dose in minimum concentration reached 25 mg, in maximum concentration it reached 50 mg per 10 6 cells/ml. We implemented the sol of magnetic NP, obtained by biogenic synthesis in International Scientific Centre for Studying Extreme States of an Organism. Functional activity of blood neutrophil granulocytes has been determined by luminol-dependent chemiluminescence. Magnetic NP were introduced into pilot samples straight before chemiluminescent analysis, and also after the incubation with in 30 minutes under 37 °С entigrade. As a result of thee stimation of the early response of neutrophil granulocytes to the influence of minimum concentration of magnetic NP in vitro we found statistically true decrease of the intensity (1.6 times), the area under the curve (2.1 times) in zymosan-induced chemiluminescent response, the activation index (2.3 times). When evaluating the late response of neutrophil granulocytes to the influence of maximum concentration of magnetic NP in vitro we have found statistically true lowering of the time of reaching the peak (10 times) of spontaneous chemiluminescence. More over we marked consider able lowering of maximum intensity 6 times and the reduction of the area under the curve of zymosan-induced chemiluminescence 5.6 times under the influence of magnetic NP under the lowering of activation index 3.7 times. The authors determined that magnetic NP were intensively decreasing the functional activity of neutrophil granulocytes. The intensity of the impact is higher under the preliminary incubation of the cells with magnetic NP. At the same time, short effect of magnetic NP to neutrophil granulocytes can be a modulating one and depends on the initial level of cell reactivity. We revealed that magnetic NP influence concerns only activated cells.

In Vivo Effects of Human Bone Marrow Mesenchymal Stromal Cells on the Development of Experimental B16 Melanoma in Mice.

Experiments on F1(CBA×C57BL/6) mice with experimental metastatic melanoma B16 F10 showed that single intravenous injection of xenogeneic bone marrow mesenchymal stromal cells (BM-MSC) in a dose of 106 cells/mouse significantly increased 100-day survival rate of tumor-bearing animals. In contrast, administration of BM-MSC in a dose of 2×106 cells/ mouse reduced survival rates in comparison with the biocontrol (injection of B16 cells alone, 5×105 cells/mouse). This phenomenon can be related to in vivo participation of BM-MSC in reprogramming of resident tissue macrophages, including tumor microenvironment, towards pro- (M1) or anti-inflammatory (M2) phenotype. This is indirectly confirmed by the data on switching from activation to inhibition of ROS-producing activity of blood mononuclears and peritoneal macrophages in tumor-bearing mice in the test of luminol-dependent zymosaninduced chemiluminescence.

Activation of the body’s natural defenses reserve of mice treated with various physico-chemical agents

The purpose of this work is to reveal the activation of natural defenses reserve in mice after treatment with different physico-chemical agents in vivo using previously developed technology of adaptive response induction. Physical agents were represented by X-rays, carbon ions, infrared light, He-Ne laser light, famine and chemical agents – by immunomodulator CaCl2 and anti-inflammatory drug ibuprofen. The following tasks were set: assessment of cytogenetic damage using a micronucleus test, the weight index of lymphoid organs (thymus and spleen) and the level of ROS production in whole blood through the method of luminol-dependent zymosan-induced chemiluminescence. SHK mice were irradiated according to the scheme of adaptive response. Analysis of data on the number of cytogenetic damage in bone marrow showed that pretreatment of the animals with all investigated agents and subsequent exposure to X-rays or carbon ions at a dose of 1.5 Gy has led to a decrease in radiosensitivity compared to the nontreated animals. Similar results were observed when analyzing weight index of lymphoid organs. Determination of level of ROS production has shown that the activation index calculated according to the relation of induced to spontaneous light area, was significantly higher in all groups of mice, indicating activation of the natural defenses reserve as compared to the group exposed only at a dose of 1.5 Gy. The obtained results confirm the assumption of revealing activation of the natural defense of the organism with the help of the adaptive response induction technology.

Modifying Effect of Autotransfusion of Mesenchymal Stromal Cells on the Production of Reactive Oxygen Species and Cytokines by Mononuclear Cells in Patients with Chronic Heart Failure.

We studied in vivo modifying effect of autotransfusion of human bone marrow mesenchymal stromal cells on ROS generation and production of cytokines (TNFα,TNFβ, IL-1α, IL-10, IFNγ, and GM-CSF) and PGE2 by mononuclear cells of patients (N=21) with chronic heart failure. These parameters were evaluated prior to (control) and after (immediately and on day 14) intravenous administration of stromal cells in doses of 100-200×106. Immediately after autotransfusion, significant increase of in vitro zymosan-induced chemiluminescence of blood mononuclear cells from 10 patients was observed. At later terms after autotransfusion (day 14), inhibition of chemiluminescent activity of blood mononuclear cells was revealed in 50% patients. We discuss possible mechanisms of involvement of transplanted autologous bone marrow mesenchymal stromal cells in reprogramming of blood mononuclear phagocytes from the pro- to anti-inflammatory phenotype under conditions of their in vivo interaction manifesting in transition from activation to inhibition of ROS-producing activity of macrophages and significant suppression of in vitro LPS-induced production of TNFα and GM-CSF by blood mononuclears against the background of significantly elevated TNFβ, IL-10, and IL-1α concentrations.

Syngeneic and Xenogeneic Transplantations of Mesenchymal Stromal Cells Modify the Production of Reactive Oxygen Species by Blood Mononuclears of Mice.

In vivo modifying effects of bone marrow mesenchymal stromal cells of humans and laboratory mice on ROS production by mouse blood mononuclears are studied by luminol-dependent zymosan-induced chemiluminescence after syngeneic and xenogeneic transplantation into systemic blood flow. The chemiluminescent activity of mouse blood mononuclears has increased early (1 day) after syngeneic (mouse mesenchymal stromal cells) and xenogeneic (human mesenchymal stromal cells) transplantation. Later, 7-21 days after syngeneic and xenogeneic transplantation, the chemiluminescent activity of mouse mononuclears is suppressed. The probable mechanisms of involvement of the transplanted mesenchymal stromal cells in reprogramming of the blood mononuclear phagocytes from proinflammatory (M1) to anti-inflammatory (M2) phenotype under conditions of their in vivo interactions are discussed; a frequent manifestation of this reprogramming is transition of the phase of activation into inhibition of ROS-producing activity of macrophages.

The respiratory explosion activity of neutrophils in blood from patients with Graves' disease manifestation


 Objective. To study respiratory burst activity in neutrophilic granulocytes in the onset of Graves’ disease (GD).
 Material and methods. Twenty-six females aged 18—55 years (mean age, 40.7±13.2 years), with the diagnosis of Graves’ disease verified for the first time, were enrolled in this study. Spontaneous and zymosan-induced chemiluminescence (CL) was assessed using a CL3606 36-channel analyzer for 90 min. The following parameters of the chemiluminescence curve were determined: time to attain the maximum (Tmax), the maximum intensity (Imax), and the area under the curve (S).
 Results. Patients with GD and lucigenin-dependent CL of neutrophils exhibited decreased zymosan-induced CL and the increased Imax of luminol-dependent spontaneous and zymosan-induced CL of neutrophils. Association between the anti-TPO level in blood serum with Tmax (r=–0,70; р=0.036) and Imax of luminol-dependent induced CL (r=–0.72; р=0.030) was found. The maximum level of synthesis of secondary reactive oxygen species (ROS) was found to be elevated in patients with GP both at relative rest and upon antigen-induced respiratory burst.
 Conclusion. In patients with GD, respiratory burst activity in neutrophils increases due to synthesis of secondary reactive oxygen species. Upon the onset of GD, already at the stage of subclinical thyrotoxicosis, the association between the indices of respiratory burst in neutrophils and thyroid hormones is lost but there emerges a relationship between the CL response and anti-TPO concentration.

Combined Effect of Low-Intensity Helium-Neon Laser and X-Ray Radiation on in Vivo Cellular Response of the Whole Blood and Lymphoid Organs in Mice.

We studied the effect of exposure to helium-neon laser (dose range 0.16-50 mJ/cm2) on activation of natural protection reserve in mice using the adaptive response test. DNA comets method revealed a protective response manifested in DNA damage level in whole blood leukocytes of mice and in lymphoid organs by the thymus and spleen weight index; preexposure to laser did not induce the adaptive response. ROS level in the whole blood was assessed by the level of zymosan-induced luminol chemiluminescence. In mice subjected to adaptive laser irradiation in doses of 0.16-5 mJ/cm2 followed by X-ray irradiation in a dose of 1.5 Gy, the activation index calculated as the ratio of induced to spontaneous area of luminescence was by 1.4 times lower than that in non-irradiated animals, which attested to reduced ROSgeneration reserve capacity of neutrophils.

Хемилюминесцентная и энзиматическая активность нейтрофильных гранулоцитов у больных распространенным гнойным перитонитом в зависимости от исхода заболевания

Aim was to study the chemiluminescent activity and the activity of NAD- and NADP-dependent dehydrogenases of neutrophils depending on the outcome of the widespread purulent peritonitis (WPP). 51 patients with a mean age of RSE 54.2 ± 19.2 years were observed. As a control 75 healthy people of similar age range were examined. Lucigenin- and luminol-dependent chemiluminescence activity and activity of NAD(P)-dependent dehydrogenases peripheral blood neutrophils were studied. In patients with a favorable outcome of WPP the maximum intensity increased and the magnitude of the activation index of lucigenine-dependent spontaneous chemiluminescence reduced. Regardless to the outcome of WPP in patients with increased activation index and maxima of the luminol-dependent spontaneous and zymosan-induced chemiluminescence. At the unfavorable outcome of the disease in neutrophils NAD- dependent isositrate dehydrogenase and anaerobic lactate dehydrogenase reaction activity increased. Regardless to the outcome WPP in patients neutrophils aerobic reaction of lactate dehydrogenase, glucose-6-phosphate dehydrogenase and NADP-dependent glutamate dehydrogenase activity reduced but levels of NADH-dependent reaction of malate dehydrogenase, NADH-dependent glutamate dehydrogenase and NADP-dependent isocitrate dehydrogenase activity increased. With the reduction of the intensity ofplastic processes and imbalances enzymatic activity in nitrogen metabolism in patients with WPP at unfavorable outcome of the disease increases the activity of enzymes that characterize the level of anaerobic and aerobic respiration. In the absence of marked changes in the activity of enzymes that characterize the level of energy processes in cells of patients with favorable outcome of the WPP, increases the intensity of spontaneous lucigenin-dependent chemiluminescence and reduced neutrophil activation index.

Effects of 5-chloro-2-methyl-4-isothiazolin-3-one and other candidate biodiesel biocides on rat alveolar macrophages and NR8383 cells

Biocides are added to biodiesels to inhibit and remove microbial growth. The effects of 5-chloro-2-methyl-4-isothiazolin-3-one (CMIT), a candidate biodiesel biocide, were studied using freshly isolated rat alveolar macrophages (AM) and NR8383 cell line. CMIT markedly inhibited phagocytic oxidative burst as measured by zymosan-induced chemiluminescence, and cellular cytokine secretion as measured by zymosan-induced TNF-α secretion. The 50% inhibition concentration (LC(50)) for CMIT was 0.002-0.004mM for both cellular functions. AM exposed to CMIT for as little as 2min showed markedly inhibited functions that persisted for at least 5h. Sodium metabisulfite was able to partially neutralize the inhibitory activity of CMIT. Cysteine and glutathione, when present at a molar ratio of 2-1 or higher against CMIT, were effective neutralizers, while serine, histidine, alanine, and albumin were without effect. When the AM testing system was used to compare the toxicity of CMIT against three other candidate biodiesel biocides, methylene dithiocyanate (MDC) was found to be of comparable toxicity to CMIT, 2-methyl-4-isothiazolin-3-one (MIT) was much less toxic, and dimethyl acetylenedicarboxylate (DMAD) was non-toxic. Because AM is among the first cell-type exposed to inhaled biodiesel aerosols, the result suggested that CMIT present in biodiesel may produce respiratory effects, and further investigations including animal studies are warranted.

Measurement of whole blood phagocyte chemiluminescence in a microtitreplate format.

Zymosan-induced luminol-enhanced chemiluminescence of phagocytes was measured in whole blood using a microtitreplate format. A consistent reaction response curve could be obtained using a normal blood sample, with low intra-assay variations in peak light index, cumulated response as well as time taken to reach the peak. Variations were common when tests were done on different days for the same individual, or when different persons were compared simultaneously. Reactions were hastened and enhanced if continuous shaking at 37 degrees C was applied. The interval between time zero and peak was the most consistent variable, with a coefficient of variation of 12% in inter-assay analysis. Because of its simplicity and ease of operation, the method is potentially useful in studying phagocyte functions in pathological states.

Effect of Fluconazole on Phagocytic Response of Polymorphonuclear Leukocytes in a Rat Model of Acute Sepsis

Recently, fluconazole (FLZ) has been shown to improve survival and reduce multiorgan failure in experimental and clinical septic shock. The mechanism by which FLZ affords protection against sepsis remains obscure. This study examines the effect of FLZ on phagocytic activity of polymorphonuclear leukocytes (PMNs) in a rat model of septic shock by inducing fecal peritonitis in male Wistar rats using intraperitoneal instillation (1 mL/kg) of fecal suspension in saline (1:1 w/v). Sham control rats received sterile fecal suspension and vehicle treatment. FLZ was administered in the doses of 0, 3, 10, and 30 mg/kg by gavage 30 minutes before fecal instillation. The samples of peritoneal fluid were collected 8 hours following fecal inoculation for the evaluation of phagocytic response of PMNs using zymosan-induced luminol-dependent chemiluminescence (CL). Fecal peritonitis caused massive infiltration of PMNs in the peritoneal cavity (ANOVA F4.45 = 6.322, P < .001). Although FLZ reduced the infiltration of PMNs, this effect was neither significant nor dose dependent. The actual CL response was significantly higher in the peritoneal fluid of rats subjected to peritonitis, which was significantly and dose-dependently attenuated by FLZ treatment (ANOVA F4.45 = 11.048, P < .001). Normalization of CL response for 1000 PMNs revealed that FLZ dose-dependently albeit insignificantly reduced the activity of PMNs. The high dose of FLZ caused 2.29-fold decrement in the area under curve (AUC) pertaining to cumulative CL response. The findings of this study suggest that FLZ protects rats against septic shock by inhibiting PMN-mediated inflammatory cascade without compromising their phagocytic activity.

Differences in the changes of allergen-specific IgE serum levels and the chemiluminescence of peripheral blood phagocytes in patients with allergic rhinoconjunctivitis during the ragweed season

The objective of this study was to compare the changes in the values of allergen-specific serum IgE levels and zymosan-induced whole blood chemiluminescence (CL) in 41 patients who had exclusively only ragweed allergy in the season of acute symptoms of disease in July, August and September. All patients had allergic rhinitis or rhinoconjunctivitis. Each patient was investigated as a self-control. The ragweed-specific IgE levels were measured by enzyme immunoassay (EIA). The luminol amplified zymosan-induced CL of whole human blood was detected. The allergen-specific serum IgE levels showed slight, but not significant, gradually increasing elevations during the whole season. On the other hand, significant increases were found in the values of the basal but especially in the zymosan-stimulated CL of peripheral blood phagocytes during the acute phase of allergy. Both the basal and the zymosan-induced CL reflected significantly the activated state of the immune system. These observations clearly show that there are well detectable signs of the systemic activation of the immune system in allergic rhinoconjunctivitis beside the local alterations. In addition, the measurements of the basal and zymosan-induced CL of peripheral phagocytes could clearly reflect the clinical state of disease in vitro.

Effect of long-term exposure to ozone on functional activity of human phagocytes.

Functional activity (biocidal properties) of whole blood phagocytes from humans exposed to ozone was studied by measuring spontaneous and zymosan-induced luminol-dependent chemiluminescence. Generation of reactive oxygen species and biocidal properties of phagocytes depended on the time of ozone exposure and development of bronchopulmonary diseases. The data suggest that generation of reactive oxygen species must be assayed for elaboration of individual patient management programs.

Effects of native and oxidized low-density lipoproteins on macrophage chemiluminescence

Effects of low-density lipoproteins (LDL) obtained from healthy donors and patients with hypercholesterolemia on spontaneous luminol-dependent and zymosan-induced chemiluminescence of rat macrophages were studied. Unlike LDL from healthy donors, native LDL from patients with hypercholesterolemia inhibited spontaneous chemiluminescence of macrophages. Simultaneous incubation with endotheliocytes from the umbilical vein led to the appearance of inhibitory effect of LDL from healthy donors (incubation for 24 h) and potentiated this effect of LDL from patients with hypercholesterolemia (incubation for 6 and 24 h). The inhibitory effect was more pronounced in LDL incubated with umbilical endotheliocytes under ischemic conditions then after aerobic incubations. This corresponded to higher oxidation of LDL confirmed by accumulation of thiobarbituric acid-reactive substances, increased fluorescence, and high electrophoretic mobility in agarose gel. These data suggest that the model system of spontaneous and zymosan-induced chemiluminescence of macrophages can be used for evaluating the degree of oxidation and potential atherogenicity of LDL.