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1.
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2.
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   

3.
The nitroblue tetrazolium reduction test (NBT) is an assay based on the activation percentage of neutrophils in peripheral blood, that has been proposed for the follow up of canine leishmaniosis owing to the narrow relationship between the molecules involved in the oxidative burst and the leishmanicidal activity of phagocytes. Domperidone is a drug used for the treatment of canine leishmaniosis having been claimed to stimulate the dogs' cell-mediated immune response. The aim of this study was to evaluate the degree and the lasting of phagocytic activation induced by a 30-day course treatment with Domperidone (0.5 mg/kg/day) in healthy dogs, by using the NBT. A statistically significant increase in the percentages of activated phagocytes was observed in the treated group during treatment, thereafter remaining high for up to one month after the end of treatment. In contrast, untreated dogs maintained the baseline percentage of activated neutrophils all along the study. It is concluded that the NBT is a useful tool for the follow up of the stimulating effects of Domperidone on the neutrophilic response of healthy dogs and that these effects persist for up to one month after treatment with this molecule.  相似文献   

4.
Functional capabilities of morphologically mature (segmented) and immature granulocytes (neutrophils and eosinophils) from bone marrow from cows were studied and compared with similar activities of segmented granulocytes from blood. Phagocytosis of Escherichia coli and postphagocytic oxidative metabolic stimulation, measured by nitroblue tetrazolium (NBT) reduction, were evaluated simultaneously. Phagocytosis was observed readily in segmented neutrophils, neutrophilic bands, and metamyelocytes and rarely in myelocytes. Phagocytosis was not seen in promyelocytes and myeloblasts. Neutrophilic bands and metamyelocytes were phagocytically less active than were segmented neutrophils. Washed segmented bone marrow neutrophils possessed phagocytic activity similar to that of blood neutrophils, whereas the activity of unwashed segmented bone marrow neutrophils was markedly less than that of blood neutrophils. Reduction of NBT was observed only in blood segmented neutrophils and bone marrow segmented neutrophils; the magnitude of NBT reduction was significantly (P = less than 0.005) less in bone marrow neutrophils than in blood neutrophils. Eosinophils were phagocytically less competent than were neutrophils. The NBT reduction was observed only in eosinophils from blood, but not in eosinophils from bone marrow.  相似文献   

5.
The effect of vitamin A deficiency on the activity of peritoneal macrophages (PM) was investigated in noninfected and Newcastle disease virus (NDV)-infected chickens. Day-old chickens with limited vitamin A reserves were fed diets containing either marginal (120 retinol equivalents (RE)/kg) or adequate (1200 RE/kg) levels of vitamin A. At 4 weeks of age, half of the chickens in each group were infected with the La Sota strain of NDV and PM were isolated 11 or 12 days later. These were used for counting the uptake of fluorescein isothiocyanate-labeled yeast cells as an indicator of phagocytic activity and for measuring the reduction of nitroblue tetrazolium (NBT), which provides an estimate of oxygen-dependent killing of microorganisms. Vitamin A deficiency impaired NBT reduction and, to a lesser extent, phagocytosis in both infected and noninfected chickens. NDV infection increased phagocytosis and NBT reduction in normal and, to a lesser extent, in vitamin A-deficient chickens.  相似文献   

6.
Background: The use of soybean oil-based lipid emulsion (SO-based LE) in parenteral nutrition has been reported to impair neutrophil functions in humans and rodents. As yet, little is understood about the effects of SO-based LE on canine immune responses.
Hypothesis: A short-term infusion with SO-based LE affects the phagocytic responses of canine peripheral blood polymorphonuclear neutrophilic leukocytes (PMNs).
Animals: Twenty-four healthy Beagle dogs.
Methods: Experimental study. Dogs were randomly assigned into groups of six and administered a 2-hour IV infusion with 0.9% NaCl solution or sufficient SO-based LE (INTRALIPOS 20%) to supply 40, 100, and 200% of the basal energy requirement (BER). PMN functions were determined after collecting blood samples before, immediately after, and 24 hours after the infusion.
Results: None of the treatments significantly affected the phagocytic capacity of PMNs or circulating leukocyte numbers. The infusion providing 200% of BERs significantly reduced PMN oxidative burst activity, filamentous actin polymerization, and Cdc42 Rho guanosine triphosphatase activity immediately after its delivery. However, these functions were restored to pre-infusion values 24 hours after the infusion. The lower calorie infusions did not have these effects.
Conclusions and Clinical Importance: These results suggest that short-term infusions with a supraphysiological dose of SO-based LE may decrease the immune functions of canine PMNs. However, more long-term studies will be needed to extrapolate the effect of SO-based LE with clinically relevant doses in a practical situation.  相似文献   

7.
A microassay for the luminol-dependent chemiluminescence (CL) response in canine whole blood was developed to measure indirectly the oxidative metabolism of peripheral blood leukocytes. Fifty microliters of blood were mixed with 705 microliters of Hank's balanced salt solution containing 25 mM Hepes and 1.3 x 10(-4) M luminol. This mixture was allowed to equilibrate for 5 min after which 60 microliters of latex beads (0.801 microns diameter) were added as a stimulant, and the CL response was monitored continuously for 5 min at 37 degrees C using a luminometer. The whole blood CL response was significantly correlated (r = 0.784, P less than 0.01, n = 14) with the number of neutrophils in the peripheral blood. Further, the whole blood CL response was abolished by the depletion of neutrophils after passing the blood through an adherence column and by the addition of sodium azide. The relative chemiluminescent light unit (RCLU) was a reliable marker for comparing each peak value in different samples. The coefficient of variation (CV) of repetitive samples was 9.87%, and the CV of 14 normal dogs was 15.7%. This method is useful and applicable for screening the CL response in canine whole blood.  相似文献   

8.
Neutrophils were isolated from the blood and mammary gland of 3 multiparous lactating cows and 3 nulliparous heifers. Neutrophil function was evaluated by phagocytosis and luminol-dependent chemiluminescence. Peroxidase activity was detected by use of transmission electron microscopy. Compared with that for blood neutrophils, percentage of phagocytosis was 9.6% lower for neutrophils isolated from the mammary gland of lactating cows, but this difference was not observed between neutrophils isolated from the mammary gland and from the blood heifers. Similarly, after subtraction of chemiluminescence values in the absence of zymosan, phagocytosing neutrophils from the mammary gland of lactating cows had lower chemiluminescence than did those from the blood of such cows. For heifers, however, chemiluminescent activity by phagocytosing neutrophils obtained from the mammary gland was similar to that of blood neutrophils. Chemiluminescent activity of resting neutrophils from the mammary gland of lactating cows pretreated with cytochalasin B was not inhibited, compared with that of nontreated resting neutrophils (controls). This was attributed to xanthine oxidase activity. Transmission electron microscopy of mammary gland neutrophils from lactating cows revealed peroxidase-positive material associated with milk-fat globule membranes and with phagosomes containing zymosan. Results indicated that ingestion of fat and casein by neutrophils isolated from milk caused a decrease in phagocytic and chemiluminescent activity. Also, luminol-dependent chemiluminescence was not a reliable measure of milk neutrophil function, because of interference by xanthine oxidase.  相似文献   

9.
育成猪对猪链球菌的免疫应答   总被引：1，自引：0，他引：1  
取 7周龄长大杂交猪 12头 ,随机均分为试验组和对照组。试验组按每公斤体重 4.5亿个菌腹部皮下接种猪链球菌 C551 2 6复制猪链球菌病 ,接种后 0、7、14、2 1、2 8d,自前腔静脉采血 ,计数红、白细胞总数和白细胞分类 ,用硝基蓝四氮唑 (NBT)还原试验定量检测嗜中性粒细胞吞噬功能 ,噻唑蓝 (MTT)比色法检测外周血中淋巴细胞转化 ,单向免疫扩散法测定血清 Ig G浓度 ,5 0 %溶血试验测定血清中补体总活性 ,同时 ,用植物血凝素 (PHA)皮肤试验检测迟发型超敏(DTH)反应。结果表明 ,接种后 7d开始 ,试验组白细胞总数升高 ,接种后 14d嗜中性粒细胞百分含量低于对照组 ,淋巴细胞百分含量则升高 ;嗜中性粒细胞还原硝基蓝四氮唑 (NBT)能力均极显著高于对照组 (P<0 .0 1) ,PHA诱导的淋巴细胞转化则极显著降低 (P<0 .0 1) ,DTH只在接种后 2 1d显著低于对照组 (P<0 .0 5 ) ,血清中 Ig G总量接种后 7d降低 (P<0 .0 5 ) ,14d却极显著升高 (P<0 .0 1) ,此后恢复正常 ,血清补体总活性只在接种后 7d降低 (P<0 .0 5 )。可见 ,猪对猪链球菌的免疫应答除了常见的抗原抗体反应外 ,还表现在免疫细胞数及其功能的变化 ,以及补体活性的改变。  相似文献   

10.
Normal canine platelets coated with heterologous or isologous antiplatelet antibody were interacted with viable canine neutrophils in vitro. Platelet phagocytosis was assessed by detecting nitroblue tetrazolium (NBT) reduction, measuring uptake of opsonised 51Cr-labelled platelets, and electron microscopy. The NBT reduction and uptake of 51Cr-labelled opsonised platelets were markedly increased. Electron microscopy revealed phagocytosis of antibody-coated platelets and their degradation intracellularly. Exposure of canine platelets to rabbit anti-canine platelet antibody in vitro produced morphological changes in platelets and caused serotonin release. Serotonin was not released in the absence of antiplatelet antibody or in the presence of normal rabbit gamma-globulin. Morphological changes in the platelets included disappearance of alpha and dense granules and exaggeration of the open canalicular system. These observations indicate that circulating platelets may be vulnerable to an antiplatelet antibody and that antibody-mediated phagocytosis of platelets is an important mechanism in the pathogenesis of immune-mediated thrombocytopenia.  相似文献   

11.
Variables which influence the oxygen-dependent chemiluminescence (CL) response of canine polymorphonuclear leukocytes (PMN) to zymosan were examined in a luminol-dependent CL assay system. Maximal CL responses were obtained when 5 × 106 canine PMN, isolated from heparinized blood, were assayed at 37° C in a Luminometer. The response was enhanced by the addition of 0.05 mM Luminol and inhibited by the addition of 0.06 mM sodium azide and 60 ug superoxide dismutase. Repeatability on a given day was very good; however, day to day variations in CL activity prevented direct comparison of phagocytic activity between days. Opsonization of zymosan in equine serum significantly reduced the CL response by canine PMN as compared to opsonization of zymosan in autologous or homologous canine serum and bovine serum. The present results show that luminol-dependent CL analysis can be used to measure phagocytosis by canine granulocytes in a luminometer and has potential use in clinical situations.  相似文献   

12.
Neutrophil activity in rainbow trout (Oncorhynchus mykiss) is increased upon antigenic stimulation with the Yersinia ruckeri O-antigen bacterin. The characteristics of neutrophil attachment to glass and nitroblue tetrazolium (NBT) staining were used to determine the effectiveness of immunization programs with fingerling rainbow trout. Fish immunized by intraperitoneal injection with doses of 100, 10, or 1 μg of the bacterin showed the highest responses in that order in numbers of glass adherent, NBT-positive neutrophils. Studies on the kinetics of the occurrence of numbers of glass-adherent, NBT-positive staining cells from the fish injected with the 10 μg dose showed the numbers of positive cells were largest on Day 2 after injection. The specific immune response was confirmed by demonstrating the presence of plaque-forming cells by the passive hemolytic plaque assay and the rise in humoral antibody titers by passive hemagglutination 12 days after injection. The effects of immunization in trout could be detected earlier by using the neutrophil glass adherence and NBT reduction assays than by using assays based on observations of the specific immune response.  相似文献   

13.
To compare the efficacy, tolerability and safety of a generic formulation of ciclosporin for human beings with prednisone in the treatment of canine atopic dermatitis), human generic ciclosporin A (hgCsA) (5 mg/kg daily) and prednisone (1 mg/kg daily for seven days, followed by 1 mg/kg every second day) were administered to 13 and seven dogs with atopic dermatitis, respectively, for 42 days. Skin changes were assessed using a modified canine atopic dermatitis extent and severity index (mCADESI-01) and a pruritus intensity scale system. The in vitro functional capacity of phagocytic cells was assessed using the tetrazolium reductase activity and zymosan-stimulated tetrazolium reductase activity tests, as well as measurements of the percentage phagocytic activity and the ingestion capacity of phagocytic cells. Haematological and biochemical parameters were also monitored. There was a greater than or equal to 50 per cent reduction from the baseline in mCADESI-01 scores in 84.6 and 100 per cent of dogs, and a greater than or equal to 50 per cent reduction from the baseline in pruritus scores in 76.9 and 85.7 per cent of dogs, treated with hgCsA and prednisone, respectively. No important adverse physical, haematological or biochemical effects occurred with either drug and no statistically significant changes were detected in any of the four tests assessing the functional activity of phagocytes. The generic formulation of ciclosporin was effective in reducing the severity of physical signs of canine atopic dermatitis and was well tolerated.  相似文献   

14.
The dog is widely used as a translational experimental model studying the host response and new treatments for human endotoxemia. The present study evaluated the applicability of a novel patient-near neutrophil chemiluminescence assay for the measurement of endotoxin activity in human blood when applied to canine blood samples. The assay was observed to be analytically sensitive and specific to endotoxin when tested in vitro, spiked with purified Escherichia coli lipopolysaccharide and live E. coli. The diagnostic sensitivity was sustained during Gram-positive contamination. Finally, it also demonstrated diagnostic potential when able to discriminate dogs with spontaneously occurring endotoxemia from both healthy dogs and diseased dogs without endotoxemia. The rapid patient-near assessment of endotoxin activity in canine blood should facilitate future studies on endotoxemia in both spontaneous disease and in experimental settings.  相似文献   

15.
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16.
Ketamine has been reported to decrease the immune functions of phagocytes. Previously, we observed that the phagocytic capacity and oxidative burst activity (OBA) of canine peripheral blood polymorphonuclear cells (PMNs) were inhibited by the supernatant from canine peripheral blood mononuclear cells (PBMCs) cultures treated with ketamine. In the present study, we examined whether in vitro treatment with ketamine modulates prostaglandin E2 (PGE2) production in PBMCs. Treatment with ketamine or with ketamine-treated PBMCs culture supernatant simultaneously decreased the phagocytic capacity and OBA of PMNs. Ketamine increased PGE2 production by PBMCs. Recombinant PGE2 decreased the phagocytic capacity and OBA of PMNs. AH-6809, an E-prostanoid 2 (EP2) antagonist, restored the phagocytic capacity and OBA of PMNs, decreased by either the ketamine-treated PBMCs culture supernatant or recombinant PGE2. These results suggest that ketamine inhibits the phagocytic responses of canine PMNs, and that this results from the increase in PGE2 produced by canine PBMCs.  相似文献   

17.
The adherence of viable and heat-treated Mycoplasma bovis to bovine peripheral blood neutrophils was studied by specific immunofluorescence staining and flow cytometry. Viable and heat-treated M. bovis cells, adhered to bovine neutrophils in dose-dependent fashion within a 30 min incubation. Fluorescence quenching using crystal violet indicated that unopsonized M. bovis cells remained on the surface of bovine neutrophils without experiencing significant ingestion. The effect of M. bovis adherence on neutrophil microbicidal function was examined by measuring luminol enhanced chemiluminescence (CL). Adherent M. bovis cells did not elicit a bovine neutrophil CL response over a 75 min incubation period. M. bovis inhibited the capacity of bovine neutrophils to mount a CL response. Inhibition occurred whether viable or heat-treated M. bovis cells were used and it occurred when neutrophils were stimulated with opsonized zymosan (OZ) or phorbol myristate acetate (PMA). Inhibition of the PMA stimulated neutrophil CL response required cytadherence by M. bovis cells. These findings suggest that activation of the bovine neutrophil respiratory burst was inhibited at or distal in the pathway to the activation of protein kinase C (PKC), the site of PMA stimulation, and that it was mediated by a direct interaction between the adhering M. bovis cells and the bovine neutrophil membrane.  相似文献   

18.
OBJECTIVE: To examine blood neutrophil counts and luminol-enhanced chemiluminescence (CL) responses in dogs undergoing ovariohysterectomy (OH), premedicated with 2 different drugs. STUDY DESIGN: Randomized clinical study. ANIMALS: Forty-two healthy client-owned bitches. METHODS: Dogs had OH under isoflurane anesthesia with either acepromazine or medetomidine, both in combination with butorphanol, administered as preanesthetic medication. Blood samples were collected when the dog was admitted, at the end of surgery, and the next day (approximately 20 hours after surgery). Blood neutrophils were counted automatically, and neutrophil oxidative activity was assessed by measuring blood CL responses (induced by opsonized zymosan and enhanced by luminol) at 37 degrees C for 40 minutes. RESULTS: Number of circulating neutrophils was significantly increased the day after surgery reflected by enhanced blood CL responses. Neutrophil CL, however, was not significantly altered. No significant differences were detected for perioperative Polymorphonuclear neutrophil (PMN) characteristics between the 2 preanesthetic regimens. CONCLUSIONS: In conclusion, despite clearly increasing the number of circulating neutrophils, OH did not significantly affect neutrophil respiratory burst, as measured by whole-blood CL responses. CLINICAL RELEVANCE: Surgical operation of moderate intensity (e.g., OH) did not significantly alter one of the important immune functions, neutrophil oxidative activity. Further studies are warranted to confirm the significance of this finding, and to assess the value of following this variable in different animal patient populations.  相似文献   

19.
Trans-10, cis-12 conjugated linoleic acid (t10c12-CLA) has been reported to enhance phagocyte function. Clostridium difficile toxin B (TcdB) has been known to inhibit Ras-homologous (Rho) guanosine triphosphatases (GTPases) which play essential roles in neutrophil immune functions. Here, we examined whether in vitro treatment with t10c12-CLA modulates the filamentous actin (F-actin) polymerization, phagocytic capacity, and oxidative burst activity (OBA) of canine peripheral blood polymorphonuclear neutrophilic leukocytes (PMNs) exposed to TcdB. Treatment with t10c12-CLA, but not linoleic acid, enhanced PMN F-actin polymerization, phagocytic capacity, and OBA, while TcdB suppressed these functions. t10c12-CLA reversed the suppressive effects of TcdB on these PMN functions. t10c12-CLA stimulated F-actin polymerization regardless of whether phagocytosis was stimulated by microspheres but only elevated OBA when microspheres were added. We asked whether the effects of t10c12-CLA were associated with changes in the activation of the Rho GTPase Cdc42. Treatment with t10c12-CLA augmented Cdc42 activity in both TcdB-treated and TcdB-naive PMNs during phagocytosis. Thus, t10c12-CLA up-regulates PMN phagocytic responses attenuated by TcdB. This effect is associated with an increase in actin polymerization and may involve the activation of Cdc42.  相似文献   

20.
Proliferation assays performed on peripheral blood mononuclear cells (PBMC) are commonly used in experimental and clinical immunology. A prerequisite for an in vitro assay is the ability to obtain relatively pure populations of mononuclear cells from whole blood, as contaminating polymorphonuclear cells may affect the proliferation of lymphocytes. Purification of canine leucocytes from whole blood is associated with difficulties in obtaining pure lymphocytes in high yields. The aim of this study was to optimize the lymphocyte purification from canine whole blood in terms of total cell recovery and purity, while not influencing the proliferation capacity of the isolated cells. To acquire optimal isolation of canine lymphocytes several density gradient media of different densities and osmolalities were examined. For optimal phagocyte removal, pre-treatment of whole blood with carbonyl iron/arabic gum and/or adherence to fibrinogen pre-coated polystyrene tissue flasks were examined. Lectin-induced proliferation was used as measurement of cell activity of the obtained cell fractions after the different separation procedures. Canine blood pre-treated with carbonyl iron/arabic gum followed by density gradient centrifugation with medium 'G' (density: 1.079 g/cm(3), osmolality: 256 mOsm) and adherence to pre-coated polystyrene tissue flask obtained the best PBMC cultures with a median lymphocyte purity of 88% and a median yield of recovered lymphocytes of 54%. This culture also resulted in the highest proliferation and subsequently the highest stimulation index upon lectin stimulation.  相似文献   

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