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1.
Various markers of the inflammatory response were measured in peripheral blood mononuclear cells (PBMCs) and polymorphonuclear neutrophils (PMNs) from 31 dogs with atopic dermatitis (AD). The variables assayed included chemiluminescence, acid hydrolase enzyme concentrations, leukotriene B(4) (LTB(4)) production and complement C3 conversion. The results were compared to those derived from a population of clinically healthy dogs. Dogs with AD exhibited a significant increase in median LTB(4) production in PMNs compared to controls (0.94 versus 0.00 ng/10(6) cells; P<0.01). Significant increases in the median concentrations of intracellular beta-galactosidase (PBMC fraction - 0.42 versus 0.25 mU/10(6) cells; P<0.05) (PMN fraction - 0.47 versus 0.12 mU/10(6) cells; P<0.01) and beta-glucuronidase (PBMC fraction - 0.52 versus 0.27 mU/10(6) cells; P<0.05) were also evident in the AD group. Although median maximum chemiluminescence values for both leucocyte sub-populations were higher in controls, the differences recorded were not significant (P>0.05). However, the median time taken to reach maximum chemiluminescence was significantly shorter in the PMN fraction of dogs with AD (7.00 versus 10.00 min; P<0.01). Atopic dogs had a significant increase in the median percentage conversion of complement C3 to C3b (66.0 versus 57.3%; P<0.01). The results of this study indicate a priming of the inflammatory response in dogs with AD. The role of LTB(4) in the pathogenesis of canine AD and the potential efficacy of leukotriene antagonists in the treatment of this disorder warrant further investigation.  相似文献   

2.
Objective-To evaluate effects of extracellular lactate on viability, shape change, lactate metabolism, and reactive oxygen species (ROS) production in equine polymorphonuclear leukocytes (PMNs). Sample-PMNs isolated from equine venous blood samples. Procedures-PMNs were incubated with 0 to 300mM lactate for 30 minutes before each experiment. Viability was assessed via trypan blue exclusion. Shape change was assessed via flow cytometry and light microscopy. Relative quantification of monocarboxylic acid transporter and lactate dehydrogenase lactate dehydrogenase (LDH) isotype mRNAs was performed with a real-time PCR assay. Effects of lactate at a pH of 7.4 to 6.0 on ROS production in response to phorbol 12-myristate 13-acetate, opsonized zymosan, or N-formyl-methionyl-leucyl-phenylalanine was assessed by luminol-dependent chemiluminescence. Results-Lactate had no effect on viability of PMNs but did alter their size and density. Monocarboxylic acid transporter 1 and lactate dehydrogenase B mRNA values were not altered. Monocarboxylic acid transporter 4 and lactate dehydrogenase A mRNA values were significantly decreased. Lactate incubation of cells significantly decreased PMN-derived luminol-dependent chemiluminescence and induced different sensitivities to stimulants (phorbol 12-myristate 13-acetate, opsonized zymosan, and N-formyl-methionyl-leucyl-phenylalanine). The response ratio to N-formyl-methionyl-leucyl-phenylalanine revealed that PMNs were primed by incubation with up to 50mM lactate, significantly increasing the production of ROS. Incubation with lactate and acidic pH caused a synergistic effect on ROS production. Conclusions and Clinical Relevance-Extracellular lactate potentially has a direct effect on the capacity to produce ROS by equine PMNs, which may be associated with alterations in innate immune functions within a short period after high-intensity exercise.  相似文献   

3.
Decreased apoptotic polymorphonuclear leukocyte rate in dogs with pyometra   总被引：1，自引：0，他引：1  
Polymorphonuclear neutrophil (PMN) apoptosis was examined in three dogs with pyometra by TUNEL assay in a 24-hr incubation period and compared with that in healthy control dogs (n=5). The incidence of apoptotic PMNs in dogs with pyometra was 26.4 +/- 5% and that in healthy dogs was 54.3 +/- 7%. The results indicated that apoptotic PMN rates in dogs with pyometra were significantly lower than those in control dogs (p<0.05), suggesting the prolongation of PMN survival.  相似文献   

4.
In the present study, to investigate the apoptosis of the polymorphonuclear neutrophil (PMN) from healthy dogs, we carried out TUNEL assay and DNA analysis by electrophoresis on dog PMNs. The TUNEL assay indicated that apoptotic PMNs in dogs were 0.15+/-5% before incubation, 0.3+/-5% at 4h incubation, 1+/-6% at 8h, 9+/-4% at 12h and 28+/-5% at 24h, respectively. The ladder formation was much more clearly observed in DNA from PMNs after 24h incubation at 37 degrees C than that before incubation. The results in this study indicated that healthy dog PMNs undergo apoptosis spontaneously within hours to days, and that the apoptosis of PMNs might be related to the high turnover of these circulating cells in dogs.  相似文献   

5.
A luminol-dependent chemiluminescence (LDCL) assay was used to assess the response of polymorphonuclear leukocyte (PMN) preparations from 4 species of ruminants (ie, cattle, sheep, goats, and antelopes) and 6 species of nonruminants (ie, swine, dogs, cats, rabbits, horses, and persons) to both opsonized and nonopsonized preparations of living and heat-killed Pasteurella haemolytica and Staphylococcus aureus and to opsonized and nonopsonized heat-killed strains of each bacterium in the presence of sterile culture supernatant (leukotoxin) from P haemolytica. The LDCL responses of PMN preparations from each of the species studied were greater for living than for heat-killed S aureus. The most efficient LDCL emission was observed with reaction mixtures containing opsonized living S aureus. Regardless whether they contained killed or living bacteria, the opsonized S aureus preparations elicited LDCL emissions more efficiently than did the corresponding nonopsonized preparations. Living P haemolytica cells and their sterile culture supernatant inhibited the LDCL emissions of phagocytically stimulated PMN preparations from ruminants, but not those from nonruminants. The LDCL response of ruminant PMN to nonopsonized living P haemolytica was characterized by the development of a peak response at 10 minutes of incubation followed by a precipitous decrease and a subsequent complete cessation of chemiluminescence. The peak LDCL response was higher for opsonized living P haemolytica than for nonopsonized living bacteria, and the increased response lasted longer. However, opsonization of living P haemolytica with the serum samples tested only temporarily spared the ruminant PMN preparations from the detrimental effects of leukotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

6.
Activation of polymorphonuclear cells (PMNs) leads to the formation of superoxide, which is in turn dismutated to H2O2 by superoxide dismutase (SOD) and is partly responsible for oxygen-dependent microbicidal activity. However, no comparative information is available on the effect of SOD inhibition before PMN activation to allow simulation of the SOD defects that are known to occur in some ruminants. This paper attempts to examine the degranulative and phagocytic responses in buffalo, cattle and goat PMNs exposed to diethyldithiocarbamate, a known SOD inhibitor. The activity of glutathione peroxidase and reductase was increased in the presence of SOD inhibitor. On activation, H2O2 production increased significantly (p<0.01), while SOD inhibition before the activation of PMNs caused a significant decline in the production of H2O2 (p<0.05) in all the species studied. There was a significant increase (p<0.05) in the phagocytosis of Candida albicans spores by buffalo PMNs activated with opsonized zymosan. Activation of bovine PMNs after exposure to the SOD inhibitor resulted in a significant decline (p<0.05) in phagocytic activity; in the other species, the two values only approached significance. Among the activators, opsonized zymosan caused a significant increase in phagocytic activity as compared to lipopolysaccharide, particularly in the PMNs of buffaloes (p<0.05). Increased fungicidal activity (p<0.05) occurred with opsonized zymosan-activated PMNs of all the species studied. The fungicidal activity was found to decline in PMNs exposed to SOD inhibitor before activation (p<0.05). Interestingly, the phagocytic activity of caprine PMNs was found to be lower than that of PMNs from cattle (p<0.05).  相似文献   

7.
In cows, interferon-tau (IFNT) regulates maternal recognition around days 15-19 after artificial insemination (AI). The present study hypothesized that if key target genes of IFNT are clearly upregulated in earlier stages of pregnancy, these genes could be use as indices of future pregnancy in cows. Therefore, we determined the expression of these genes in peripheral blood mononuclear leukocytes (PBMCs) and polymorphonuclear granulocytes (PMNs) during the maternal recognition period (MRP). Twenty multiparous Holstein cows were subjected to AI on day 0 and categorized into the following groups: pregnancy (Preg, n = 9), embryonic death (ED, n = 5) and non-pregnancy (NP, n = 6). Progesterone levels in the Preg group were higher than those in the NP group on days 12-21. ISG15 and OAS-1 (IFN-stimulated genes: ISGs) mRNA in PBMCs on day 8 was higher in the Preg group than in the NP group, and these mRNAs in PMNs was higher in the Preg group on day 5 than in the NP and ED groups. Interleukin-10 (IL-10, Th2 cytokine) mRNA expression increased on day 8 in the PBMCs of pregnant cows. Tumor necrosis factor α (TNFα, Th1 cytokine) mRNA expression was stable in all groups. In an in vitro cell culture experiment, IFNT stimulated mRNA expression of ISGs in both PBMCs and PMNs. IFNT stimulated IL-10 mRNA expression in PBMCs, whereas IFNT increased TNFα mRNA levels in PBMCs in vitro. The results suggest that ISGs and IL-10 could be responsive to IFNT before the MRP in peripheral blood immune cells and may be useful target genes for reliable indices of pregnancy before the MRP.  相似文献   

8.
Apoptosis is essential in eliminating neutrophils (polymorphonuclear leukocytes: PMNs) in animals. The suppression of PMN apoptosis is believed to be beneficial in eradicating pathogens and is implicated in the pathogenesis of human inflammatory diseases. In the present study, canine PMNs were stimulated with recombinant human granulocyte colony-stimulating factor (rhG-CSF) to investigate the in vitro effect on the apoptosis of canine PMNs. Apoptotic cell rates were assessed by flow cytometry in relation to the ability of PMNs to produce reactive oxygen species (ROS). Canine PMN apoptosis was markedly suppressed by rhG-CSF treatment, in association with the retention of the PMN ability to produce ROS. The addition of cycloheximide abolished this suppression by rhG-CSF. Moreover, canine PMNs, which were stimulated by rhG-CSF, expressed high levels of anti-apoptotic mcl-1 gene mRNA, as quantified by real-time polymerase chain reaction method. The results suggest that PMNs, stimulated by G-CSF, could work effectively over a longer period to eliminate pathogens, and that the prolongation of the PMN life-span might occasionally aggravate tissue injuries in dogs. In addition, the suppression of PMN apoptosis seems to be mediated by the induction of anti-apoptotic mcl-1 gene expression.  相似文献   

9.
Ferret polymorphonuclear cells (PMNs) and peripheral blood mononuclear cells (PBMCs) were separated from whole blood by density gradient centrifugation. Using a 50% Percoll solution (density=1.066), PMNs and PBMCs were successfully isolated after centrifugation; the purities of the PMNs and PBMCs were 94.2% and 95.6%, respectively. To evaluate the function of isolated ferret PMNs, we measured the superoxide generation with a MCLA-dependent chemiluminescence assay. The isolated ferret PMNs responded to phorbol 12-myristate 13-acetate (PMA) with kinetics similar to that of human PMNs. The ferret PMNs did not respond to N-formyl-Met-Leu-Phe (fMLF), unlike human PMNs, which rapidly responded. Thus, authors established a method for the rapid separation of highly purified populations of functional PMNs from the whole blood of ferrets.  相似文献   

10.
Zymosan-induced and luminol-aided chemiluminescence (CL) of whole blood from beagle dogs was estimated for the function of polymorphonuclear leucocytes (PMNs). Whole blood (0.1 ml) was examined directly and results were obtained within 20 min. A phagocytic function of PMNs can be estimated from the peak CL counts and the number of PMNs in a specimen, and the opsonic activity can also be estimated by the peak time showing peak CL after the addition of non-opsonized zymosan. The optimal temperatures to keep diluted whole blood for the CL measurement was around 13 degrees C. Thus, this method offers information concerning the functions of phagocytic cells in whole blood.  相似文献   

11.
The purpose of this study was to optimize conditions for high throughput measurement of radical oxygen species (ROS) production and expression of tissue factor, also termed procoagulant activity, by canine leukocytes. Granulocytes and mononuclear cells were separated by density gradient centrifugation from peripheral blood collected on several occasions from three healthy large breed dogs. To determine optimal conditions for ROS production, granulocytes were incubated for 1 or 3h in PBG (PBS containing 0.5% BSA and 5mM glucose) or RPMI containing 10% fetal bovine serum (FBS); lipopolysaccharide (LPS), zymosan, peptidoglycan (PGN) and phorbol myristate acetate (PMA) were used as stimuli. ROS was assessed by conversion of the nonfluorescent dye dihydrorhodamine 123 to fluorescent rhodamine 123 by radical species released into the media. To identify optimal conditions for expression of tissue factor, mononuclear cells were incubated for 5h in RPMI containing different concentrations of heat-inactivated FBS (HI-FBS), and LPS, zymosan, PGN or PMA as stimuli. Expression of tissue factor was determined using a one-stage recalcification assay performed in an automated nephelometric coagulation analyzer. Neither LPS nor zymosan increased ROS production by granulocytes incubated in PBG media. In contrast, granulocytes incubated in RPMI had dose-dependent increases in ROS production in response to zymosan and PGN. ROS production was significantly increased by incubation with concentrations of LPS of 0.01microg/ml or greater, and by zymosan concentrations of 0.1microg/ml or greater. ROS production in response to incubation with PMA was significantly increased starting at 10(-7)M, and was significantly greater for cells incubated in RPMI than cells incubated in PBG. LPS-, zymosan- and PGN-stimulated procoagulant activity increased in a dose-dependent manner, whereas PMA-stimulated procoagulant activity peaked at 10(-7)M. Increasing concentrations of HI-FBS significantly increased LPS-, zymosan- and PGN-induced procoagulant activity of mononuclear cells. Results obtained in this study indicate production of ROS by canine granulocytes is optimal when these cells are incubated for 3h in RPMI with LPS (0.1microg/ml), zymosan (10 microg/ml), PGN (10 microg/ml), and PMA (10(-7)M). Furthermore, canine mononuclear cells express procoagulant activity in response to LPS, zymosan, PGN, and PMA, and responses to LPS, zymosan and PGN are enhanced by the addition of HI-FBS. These findings suggest that HI-FBS retains important serum proteins that facilitate interactions between each of these bacterial or yeast derived products and the mononuclear cells. Consequently, future studies regarding the regulation of procoagulant activity by canine mononuclear cells should be performed in the presence of HI-FBS. Both assays utilized in this study allow high throughput of samples, and therefore are appropriate choices for rapid screening of conditions and/or therapeutic interventions affecting the canine inflammatory system.  相似文献   

12.
Effects of polymorphonuclear neutrophil leukocytes (PMN) on mammary tissue of lactating cows were studied in vitro. The PMN were isolated from mammary glands of nulliparous heifers given an injection of 5 micrograms of Escherichia coli endotoxin. Mammary tissue was obtained from noninfected quarters of 5 lactating Holstein cows and was cultured in supplemented medium 199. Mammary explants were treated by addition of intact or lysed PMN (10(5), 10(6), 10(7)/ml) or PMN (10(5), 10(7)/ml) which were allowed to phagocytose opsonized zymosan. Controls included cultures of mammary tissue alone, PMN alone, and mammary tissue plus zymosan. Cultures were incubated at 37 C for 3, 8, or 24 hours. Tissue from 1 randomly selected culture/treatment was weighed and processed for microscopy. Tissue from remaining cultures was incubated with [3H]amino acids or [14C]acetate to determine rates of protein and fatty acid synthesis. Media from all cultures were assayed for activity of the lysosomal enzyme, N-acetyl-beta-D-glucosaminidase. An increase (P less than 0.02) in the activity of this enzyme was detected in the medium of explant cultures treated with 10(7) phagocytosing PMN/ml at 3 and 8 hours and with 10(7) intact or lysed PMN/ml at 8 hours. Treatment did not inhibit (P greater than 0.05) rates of protein or fatty acid synthesis. Microscopic examination indicated that epithelial cell damage resulted from treatment with 10(6) and 10(7) intact, lysed, or phagocytosing PMN/ml. Greatest morphologic damage resulted from treatment with phagocytosing PMN.  相似文献   

13.
The objective of this in vitro study was to evaluate the immunomodulatory effects of recombinant human granulocyte‐macrophage colony‐stimulating factor (rhGM‐CSF) on polymorphonuclear cell (PMN) function in dogs with cancer. PMNs were harvested from dogs with naturally developing cancer as a pre‐clinical model to evaluate the immunomodulatory effects of rhGM‐CSF on PMN phagocytic and cytotoxic functions, cytokine production and receptor expression. Some aspects of cancer‐related PMN dysfunction in dogs with cancer were restored following incubation with rhGM‐CSF including PMN phagocytosis, respiratory burst and LPS‐induced TNF‐α production. In addition, rhGM‐CSF increased surface HLA‐DR expression on the PMNs of dogs with cancer. These data suggests that dysfunction of innate immune response in dogs with cancer may be improved by rhGM‐CSF. The results of this study provided a pathophysiologic rationale for the initiation of clinical trials to continue evaluating rhGM‐CSF as an immunomodulatory therapy in dogs with cancer.  相似文献   

14.
Haemophilus somnus-induced interference with bovine neutrophil functions   总被引：6，自引：0，他引：6  
The effect of Haemophilus somnus on bovine polymorphonuclear leukocyte (PMN) function was examined in vitro with whole cells and fractions extracted from the surface of this bacterium. The ability of PMNs to iodinate protein and ingest Staphylococcus aureus was significantly inhibited in the presence of live cells, heat-killed whole cells or supernatant fluid from heat-killed cells, but not in the presence of washed, heat-killed cells. None of the fractions inhibited nitroblue tetrazolium (NBT) reduction by PMNs. The PMN inhibitory factors were further characterized. The material that inhibited S. aureus ingestion was found to be a heat-stable cell surface material of greater than 300 000 MW. The fraction inhibiting iodination of protein was found to be less than 10 000 MW.  相似文献   

15.
OBJECTIVE: To determine the effects of porcine mammary secretions on polymorphonuclear (PMN) leukocyte function and to relate concentrations of estradiol-17beta and cortisol in mammary secretions to PMN cell function. SAMPLE POPULATION: Mammary secretions from 10 healthy sows and blood PMN leukocytes from 27 healthy sows. PROCEDURE: Mammary secretions were collected within 24 hours after parturition (colostrum) and 12 to 13 days later (milk). Chemoattractant properties were assessed by use of a cell migration assay. Phagocytic capacity of PMN cells in colostrum and milk was assessed by recording chemiluminescence following phagocytosis of Escherichia coli or zymosan. Estradiol-17beta and cortisol concentrations were determined by use of radioimmunoassays. RESULTS: Chemoattractant properties of colostrum and milk were significantly greater than that of zymosan-activated serum. However, chemoattractant properties did not differ significantly between the 2 types of secretions. The capacity of PMN cells in colostrum to phagocytose either zymosan or E. coli was less, compared with cells in milk, and the ability of cells in either type of mammary secretion to phagocytose E. coli was greater than the ability to phagocytose zymosan. Concentrations of estradiol-17beta and cortisol were greater in colostrum, compared with milk. No clear relation was evident between PMN cell activity and hormone concentrations in mammary secretions. CONCLUSIONS AND CLINICAL RELEVANCE: Although chemoattractant properties of colostrum and milk did not differ, the phagocytic capacity of PMN cells in colostrum was significantly less than that of cells in milk. This may predispose sows to coliform mastitis during the early postparturient period.  相似文献   

16.
During bacterial-mediated diseases, neutrophils (PMNs) play a critical role in defending the host against invading pathogens. PMN production of reactive oxygen species (ROS) contributes to the bactericidal capabilities of these cells. ROS are produced intracellularly and can be released extracellularly. The aberrant extracellular release of ROS, however, has been reported to induce injury to host tissues during mastitis and other inflammatory-mediated diseases of cattle. The acute phase response, which occurs shortly after infection or tissue injury, is characterized by the induction of a large number of plasma proteins referred to as acute phase proteins (APP). alpha1-Acid glycoprotein (AGP) is an APP that increases in response to infection or injury in cattle and humans. The precise function of AGP is unknown, but it has been reported to possess anti-inflammatory properties. The objective of this study was to evaluate the effects of bovine AGP on PMN pro-inflammatory responses, including respiratory burst activity and cytokine production. Bovine AGP dose-dependently inhibited zymosan-induced PMN extracellular release of superoxide anion and hydrogen peroxide without affecting the capacity of PMN to engulf and kill Staphylococcus aureus. Moreover, AGP exerted its effect on ROS production regardless of whether PMNs were exposed to AGP prior to or after activation. In contrast to respiratory burst activity, AGP enhanced PMN production of IL-8. The precise mechanism by which AGP regulates PMN functions remains unknown, but data presented in this study suggest that AGP may have a complex role by differentially regulating PMN pro-inflammatory activities.  相似文献   

17.
It was investigated whether beta-adrenoceptor antagonists could disturb the interaction between cytotoxin preparations isolated from Pasteurella haemolytica and bovine polymorphonuclear leukocytes (PMNs). The toxicity of the cytotoxin preparation was evaluated by measuring the chemiluminescence response and the viability of the cells after incubation with the cytotoxin. No effect on cell viability was detected when PMNs were incubated with 63 micrograms cytotoxin per ml while the chemiluminescence response was diminished by approximately 30%. The beta-adrenoceptor antagonists alprenolol (10(-5) M) and propranolol (5 X 10(-7) - 5 X 10(-6) M) were able to attenuate this effect of cytotoxin on the chemiluminescence response of PMNs. It seemed unlikely that propranolol and alprenolol diminished the effect of cytotoxin on the chemiluminescence response of PMNs by their beta-adrenoceptor blocking potency because other beta-adrenoceptor antagonists used were without effect. Also, the membrane stabilizing characteristics of the beta-adrenoceptor antagonists used were probably not responsible for the diminished interaction between PMNs and the cytotoxin. Whether beta-adrenoceptor antagonists could be used in vivo to prevent or treat P. haemolytica infections in bovines remains to be examined.  相似文献   

18.
Neutrophil (PMN) contribution to the acute inflammatory processes may lead to an excessive generation of reactive oxygen metabolites species (ROS) and secretion of granule enzymes. We compared the effects of either phorbol myristate acetate (PMA) or N-formyl-methionyl-leucyl-phenylalanine (fMLP) in combination with a pre-treatment by cytochalasin B (CB) on the production of ROS and the release of total and active myeloperoxidase (MPO) by isolated equine PMNs. The ROS production was assessed by lucigenin dependent chemiluminescence (CL) and ethylene release by α-keto-γ-methylthiobutyric acid (KMB) oxidation. In the supernatant of activated PMNs, total equine MPO was measured by ELISA and active MPO by the SIEFED (Specific Immunologic Extraction Followed by Enzymatic Detection) technique that allows for the study of the interaction of a compound directly with the enzyme. The stimulation of PMNs with CB-fMLP only modestly increased the release of MPO, but more than 70% of released MPO was active. PMA stimulation markedly increased the production of ROS and release of MPO, but more than 95% of released MPO was inactive. When PMNs were pre-incubated with superoxide dismutase (SOD) prior to PMA activation, the lucigenin enhanced CL, which is linked to the superoxide anion (O2−) production, was much more decreased than KMB oxidation, linked to the hydroxyl-like radical production. The addition of SOD prior to the activation of PMNs by PMA also limited the loss of the activity of released MPO. These results confirm the key role of O2− generation in the ROS cascade in PMN and reveal its critical role on MPO inactivation.  相似文献   

19.
During mastitis and other bacterial-mediated diseases of cattle, neutrophils play a critical role in the host innate immune response to infection. Neutrophils are among the earliest leukocytes recruited to the site of infection and contribute to host innate immune defenses through their ability to phagocytose and kill bacteria. The bactericidal activity of neutrophils is mediated, in part, through the generation of reactive oxygen species (ROS). Extracellular release of ROS can induce injury to host tissue as well, and aberrant release of ROS has been implicated in the pathogenesis of certain inflammatory-mediated diseases. Due to their essential role in bacterial clearance and implicated involvement in the pathogenesis of other diseases, there is much interest in the study of neutrophil-generated ROS. Several assays have been developed to measure ROS production, however, many of these have not been evaluated with bovine neutrophils. The objectives of the current study were to evaluate different assays capable of measuring bovine neutrophil ROS, and to compare the results of assays never previously tested with bovine neutrophils to those obtained from more well-established assays frequently used with these cells. Eight different assays were evaluated, including: luminol, isoluminol, and methyl cypridina luciferin analog (MCLA) chemiluminescence assays; Amplex Red, dihydroethidium (DHE), dichlorodihydrofluorescein diacetate (CM-H(2)DCFDA), and dihydrorhodamine 123 fluorescence assays; and the cytochrome c absorbance assay. The assays were evaluated in the context of their abilities to detect ROS produced in response to two agonists commonly used to induce neutrophil activation, phorbol 12-myristate, 13-acetate (PMA) and opsonized zymosan. Diphenyleneiodonium chloride, a NADPH oxidase inhibitor, was used to assess the specificity of the assays to detect ROS. The ability of these assays to discriminate between intra- and extracellular ROS and to specifically detect distinct ROS was evaluated using superoxide dismutase and catalase, which scavenge extracellular superoxide and hydrogen peroxide, respectively. With the exception of the DHE assay, all assays detected bovine neutrophil ROS generation elicited by PMA and zymosan. PMA, but not zymosan, was able to stimulate neutrophil generation of ROS at levels that were detectable with DHE. The MCLA chemiluminescence assay was the only assay that detected ROS produced in response to each of the lowest concentrations of PMA and zymosan tested. To our knowledge, this is the first study to evaluate DHE-, MCLA-, Amplex Red-, and isoluminol-based assays for the measurement of bovine neutrophil ROS, and the most comprehensive comparative study of ROS assays under similar experimental conditions.  相似文献   

20.
Peanut agglutinin (PNA) and surface immunoglobulin (SIg) were investigated as markers for T and B lymphocytes in blood and lymphoid tissues of dogs of various ages. In the blood study, 4 age groups (n = 8 dogs/group) were used. The mean (+/- SD) percentages of PNA-positive (PNA+) cells were 68.4 +/- 8.6% (group 1, less than 1 year old), 70.3 +/- 9.2% (group 2, 1 to 2 years old), 72.0 +/- 3.7% (group 3, 5 to 6 years old), and 63.8 +/- 10.1% (group 4, 10 to 11 years old). The mean percentages of SIg-positive (SIg+) cells in blood were 32.1 +/- 10.6% (group 1), 43.2 +/- 7.0% (group 2), 34.3 +/- 4.8% (group 3), and 35.0 +/- 6.8% (group 4). The mean total percentages of PNA+ and SIg+ cells were 100 +/- 6.0% (group 1), 113.5 +/- 4.9% (group 2), 106.3 +/- 5.3% (group 3), and 98.9 +/- 9.2% (group 4). The proportions of PNA+ and SIg+ cells in dogs of group 2 were significantly (P less than 0.05) different from those in dogs of the other groups. Serial changes in PNA+ and SIg+ cells were investigated in blood of 6- to 29-week-old pups (n = 8). A significant (P less than 0.05) transient decrease in PNA+ cells and a corresponding increase in SIg+ cells was observed in pups between 14 and 17 weeks old. Lymphoid tissue specimens and blood samples were obtained from 2- to 6-month-old dogs (n = 11) and from 6- to 12-month-old dogs (n = 10).(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

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