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1.
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   

2.
The aim of this study was to determine whether cholesterol, added to the cell growth medium or to cell suspension buffer, could protect Acholeplasma laidlawii cells against the toxic effects of hydrogen peroxide (H2O2). Variable concentrations of cholesterol (0.05–1.0 mg/ml) were added to the A. laidlawii suspension buffer and to the growth medium. Cells were then washed carefully and incubated with 0.001% (v/v) H2O2 at 37°C for 30 min and the viability was determined. The results indicated that cells were more viable in the presence of cholesterol than were cells grown in the absence of cholesterol. In addition, the oxygen uptake rate resulting from the oxidation of 5.5 mmol/L glucose was 2-fold and 4-fold higher for cells grown in medium supplemented with 0.05 and 0.50 mg/ml cholesterol, respectively, compared to cells grown in a medium with no added cholesterol. These findings indicate that cholesterol might play a role in protecting Mollicutes against the oxidative damage caused by H2O2. Deceased October 25, 2001  相似文献   

3.
The effects of acellular milk on the activity of the microbicidal cationic enzymes of the polymorphonuclear cells of goats were studied in an attempt to explain the phenomenon by which PMN functions fail in mastitis. Assays were undertaken on the myeloperoxidase, lysozyme and elastase activities in a polymorphonuclear cell (PMN) lysate, both in the presence and absence of acellular milk from homologous species. There was a significant decrease (p<0.05) in the activity of lysozyme, myeloperoxidase and elastase in the presence of acellular milk. Superoxide and H2O2 production following activation of caprine PMNs by lipopolysaccharide (LPS) was significantly reduced (p<0.05) in the presence of acellular milk. Thus, the microbicidal function of PMNs is significantly impaired in the presence of acellular milk and this may contribute to the development of mastitis in dairy animals.  相似文献   

4.
Oxidative stress can induce apoptosis of granulosa cells and lead to follicular atresia, thereby reducing the number of pigs giving birth. The aim of this study was to investigate the protective effect of Periplaneta americana peptide (PAP) on the apoptosis of the granulosa cells of pig ovaries (PGCs) induced by hydrogen peroxide (H2O2) via FoxO1. PGCs were treated with H2O2 to establish a cell apoptosis model. Cell viability was measured using the cell counting kit-8 (CCK-8) assay, and cell apoptosis was detected using flow cytometry. The malondialdehyde (MDA) level and nitric oxide (NO) content were detected to reflect the oxidative stress. Western blotting, qRT-PCR and overexpression were undertaken to determine the expression of FoxO1 and caspase-3, and immunofluorescence was used to detect FoxO1 in the nucleus and cytoplasm. PGCs were treated with 100 μM H2O2 for 6 hr, which resulted in oxidative damage and apoptosis and an apoptosis rate for PGCs of 32.95%. Next, PGCs were treated with 400 μg/ml PAP for 24 hr to repair the apoptosis induced by H2O2. PAP improved cell viability in H2O2-stimulated PGCs, the increased MDA level and NO content caused by H2O2 stimulation were reversed and the apoptotic rate of PGCs was reduced. The qRT-PCR and Western blotting results indicated that PAP decreased the H2O2-induced apoptosis and the expression of FoxO1 and caspase-3 in PGCs. The effect of PAP was the same following FoxO1 overexpression. FoxO1 was expressed in the nucleus when stimulated by H2O2 or overexpression; however, it migrated to the cytoplasm following PAP treatment. PAP decreased the apoptosis of PGCs induced by H2O2 by regulating FoxO1 expression and nuclear translocation.  相似文献   

5.
In this study, the effect of reproductive hormones and substances with hormonal activity on the oxidative burst activity of blood polymorphonuclear leucocytes (PMN) high yielding dairy cows was evaluated. Different concentrations of: progesterone, oestradiol 17β, FSH, LH, GnRH, cortisol and PGF2α were incubated in vitro for 4 h with PMN of seven high milk yielding cows, during the period of anoestrous postpartum. Controls were run in parallel in which each hormone was replaced by its solvent. After incubation with hormones the competence of PMN to generate H2O2 was monitored by flow cytometry. A down‐regulation on the oxidative burst activity of PMA‐stimulated PMN was observed when cells were incubated with progesterone. Significant (p ≤ 0.001) differences between control and progesterone incubated cells were observed from 6.56 μg/ml. The same predisposition was observed when PMNs were incubated with cortisol. Besides for all concentrations employed, a decrease in the burst activity was observed, only beyond 0.19 mg/ml, statistical differences between the results obtained by the control and the cortisol incubated cells were obtained. Concerning oestradiol 17β, an increase on H2O2‐production was observed when PMN were incubated with 15 pg/ml and 45 pg/ml of this steroid (p ≤ 0.05), followed by a depression of the cell’s activity when unphysiological concentrations were employed. Significant (p ≤ 0.05) differences between the obtained with the control and oestradiol 17β incubated cells were observed only in the highest concentration of oestradiol. No statistical differences were observed in the metabolic burst activity of PMN incubated with FSH, GnRH and LH when compared with the results obtained by the control.  相似文献   

6.
Polymorphonuclear neutrophils (PMN), which comprise over 70% of the somatic cells in goat milk, are a major cellular component of innate immunity in the goat mammary gland. However, the function of milk PMNs is modified after diapedesis compared to PMNs in blood. As many aspects of PMN activity depend directly on intracellular Ca2+ concentration ((Ca2+)i), the present study aimed to determine the changes in Ca2+ homeostasis of milk PMNs from lactating goats compared to autologous blood PMNs, and to examine the significance of these variations to the immuno-competency of milk PMNs. The intracellular Ca2+ store of freshly prepared milk cells was estimated from the elevation of (Ca2+)i after ionomycin treatment, which was found to be significantly less than blood PMNs. Replenishment of the intracellular Ca2+ store in milk cells after intracellular Ca2+ depletion by Bapta-AM followed by spiking with 2.5 mM Ca2+ for 20 min was also compared to that of blood PMNs, showing that after depletion/spiking the intracellular Ca2+ store in milk cells was much less than blood PMNs. The production of superoxide anion (O2?) in vitro in response to (Ca2+)i-dependent or (Ca2+)i-independent modulators was used to evaluate the relevance of altered Ca2+ homeostasis on the immuno-competency of milk cells compared to blood PMNs. The results indicated that milk cells produced similarly low levels of O2? as blood PMNs when treated with ionomycin. However, the amount of O2? produced by milk cells in response to phorbol 12-myristate 13-acetate (PMA) stimulation, although greater than ionomycin treatment, was significantly less than that of blood PMNs. The capacity for O2? production by both cell types in response to PMA reverted to the resting state with use of the protein kinase C (PKC) inhibitor, staurosporine. In conclusion, the current study demonstrated an irreversible shortage of intracellular Ca2+ in the milk PMNs of lactating goats compared to blood PMNs. It also showed that preliminary O2–production, primed by ionomycin treatment, remained unchanged in milk PMNs, despite the shortage in intracellular Ca2+, but decreased O2? production capacity, mediated via the PKC pathway, in milk PMN. It is suggested that the defects in Ca2+ homeostasis in milk PMNs of lactating goats is partially attributable for the post-diapedesis functionality modifications.  相似文献   

7.
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   

8.
The immuno therapeutic potential of hydro-methanolic extract of Azadirachta indica (A. indica) was studied during bovine clinical mastitis (CM). The somatic cell count (SCC), total bacterial count (TBC), milk differential leukocyte count (DLC), hydrogen peroxide (H2O2), superoxide anion (O2 −) production and interleukin- 2 (IL-2) and gamma interferon (IFN-γ) cytokines expression were studied before and after intramammary infusion of A. indica extract in diseased cows. The results revealed that A. indica treatment significantly (P < 0.05) decreased the SCC, TBC, milk neutrophil percent and significantly (P < 0.05) enhanced milk lymphocyte percent, H2O2 and O2 − production by milk cells. The IL-2 and IFN-γ were expressed in normal healthy cows and diseased cows after A. indica treatment, whereas both the cytokines could not be expressed in cows treated with antibiotic and in untreated diseased cows. The results of the present study indicated anti inflammatory, antibacterial and immunomodulatory potential of the herb, these activities could be due to the presence of bioactive principle in the extract. This is a preliminary trial indicated beneficial effect of the herb against bovine mastitis it can be developed as an alternative therapy where the use of antibiotics is normally restricted.  相似文献   

9.
A pair of primers directed to 16S-23S rDNA interspacer (ITS) was designed directed to Brucella genetic sequences in order to develop a polymerase chain reaction (PCR) putatively capable of amplifying DNA from any Brucella species. Nucleic acid extracts from whole-blood from naive dogs were spiked with decreasing amounts of Brucella canis RM6/66 DNA and the resulting solutions were tested by PCR. In addition, the ability of PCR to amplify Brucella spp. genetic sequences from naturally infected dogs was evaluated using 210 whole-blood samples of dogs from 19 kennels. The whole-blood samples collected were subjected to blood culture and PCR. Serodiagnosis was performed using the rapid slide agglutination test with and without 2-mercaptoethanol. The DNA from whole blood was extracted using proteinase-K, sodium dodecyl sulphate and cetyl trimethyl ammonium bromide followed by phenol–chloroform purification. The PCR was capable of detecting as little as 3.8 fg of Brucella DNA mixed with 450 ng of host DNA. Theoretically, 3.8 fg of Brucella DNA represents the total genomic mass of fewer than two bacterial cells. The PCR diagnostic sensitivity and specificity were 100%. From the results observed in the present study, we conclude that PCR could be used as confirmatory test for diagnosis of B. canis infection.  相似文献   

10.
Tea catechins (TC) are polyphenols that have potent antioxidant activity. The objectives of this study were to determine the effects of TC on antioxidant status of hepatocytes challenged with H2O2. Primary hepatocytes of goat were exposed to 1 mm H2O2 without or with 5, 50 and 500 μg/ml TC. The cells were harvested at 48 h post‐treatment to determine effects of TC on proliferation, apoptotic features and membrane integrity of cells, and expression of genes and activities of antioxidant enzymes. H2O2 exposure caused damage to cells (p < 0.001). A lower concentration of TC (5 μg/ml) displayed a protective effect by inhibiting exorbitant cell proliferation and DNA degradation. Both H2O2 exposure and TC pre‐incubation affected expression of antioxidant enzymes at mRNA and protein levels (p < 0.001). The activities of catalase (CAT) (p = 0.027), CuZn‐superoxide dismutase (CuZn‐SOD) (p < 0.001) and glutathione peroxidase (GPx) (p < 0.001) increased with TC pre‐incubation followed by H2O2 challenge. Changes of CuZn‐SOD activity induced by H2O2 and TC basically paralleled the changes in the corresponding mRNA and protein levels, but the correlation in CAT and GPx expression displayed slightly different patterns at different concentrations of TC. These findings infer that oxidative stress can induce deleterious cellular responses and this unfavourable condition may be alleviated by treatment with TC.  相似文献   

11.
The aim of this work was to quantify NO, O2? and ONOO? production during heparin‐induced capacitation of cryopreserved bovine spermatozoa. A time dependent hyperbolic increase was observed for heparin‐dependent capacitation, O2 uptake, and NO production. Conversely, O2? production was increased during the first 15 min of incubation, showing a decrease from this time until 45 min. At 15 min of heparin incubation, a threefold increase in O2 consumption (5.9 ± 0.6 nmol/min × 107 cells), an enhancement in NO release (1.1 ± 0.2 nmol/min × 107 cells), and a five‐fold increase in O2? production (1.3 ± 0.07 nmol/min × 107 cells), were observed. Peroxynitrite production rate was estimated taking into account NO and O2? generation and the second‐order rate constant of the reaction between these species. To conclude, heparin‐induced capacitation of cryopreserved bovine spermatozoa activates (i) mitochondrial O2 uptake by high ADP levels due to increased energy requirements, (ii) NO production by a constitutive NOS and (iii) O2? production by a membrane‐bound NAD(P)H oxidase. The products of both enzymes are released to the extracellular space and could be involved in the process of sperm capacitation.  相似文献   

12.
Eleven double-muscled calves of the Belgian White and Blue breed and eleven Friesian calves have been investigated at rest, during exercise on a treadmill (11% incline; speed 1.3 m.sec-1) and 10 and 30 minutes after the end of this exercise. Blood gases and acid-base status were determined in mixed venous and arterial blood sampled from the pulmonary and the carotid artery respectively. Expired gases were collected in a balloon. The time of collection, volume of expired gases and fractional O2 and CO2 concentrations in expired gases were measured.In double-muscled calves, inadequate oxygen intake and carbon dioxide elimination were demonstrated by the increase in the carbon dioxide tension (PaCO2) and in the hydrogen ion concentration [H+]a and the decrease in the oxygen tension (PaO2) in arterial blood during exercise. In Friesian calves, an adequate increase in oxygen intake occurred and no acidosis was recorded. A metabolic acidosis explained by only a 1.5-fold increase in the cardiac output and by the small increase in haemoglobin concentration was recorded in double-muscled calves.It was concluded that some aspects of the cardio-pulmonary and metabolic responses to exercise in double-muscled calves can be related to their inability to greatly increase their O2 consumption.  相似文献   

13.
Oxidative stresses induced by reactive oxygen species (ROS) have been shown to be involved in several physiological and pathophysiological processes, such as cell proliferation and differentiation. Steroid hormones can protect cells against apoptosis or induce cell proliferation by several mechanisms. Among androgenic hormones, dihydrotestosterone (DHT) is generated by a 5α-reduction of testosterone. Unlike testosterone, DHT cannot be aromatized to estradiol, therefore DHT is considered a pure androgenic steroid. This study was conducted to examine the effect of DHT (10-7 M) on H2O2 (10-3 M) -induced injuries in mouse embryonic stem (ES) cells. H2O2 induced ROS generation and increased lipid peroxide formation and DNA fragmentation. These effects of H2O2 were inhibited by pretreatment with DHT. H2O2 also increased the phosphorylation of p38 MAPK, SAPK/JNK and nuclear factor kappa B (NF-κB), but DHT blocked these effects. Moreover, H2O2 decreased DNA synthesis and the levels of cell cycle regulatory proteins [cyclin D1, cyclin E, cyclin-dependent kinase (CDK) 2, and CDK 4]. These effects of H2O2 were inhibited by pretreatment with DHT. In conclusion, DHT may partially prevent H2O2-induced cell injury through inhibition of ROS and ROS-induced activation of p38 MAPK, SAPK/JNK and NF-κB in mouse ES cells.  相似文献   

14.
本研究以沙打旺抗病和感病品种为材料,接种埃里砖格孢孢子悬浮液后分别施加外源H2O2及其清除剂AsA,通过统计发病率、病情指数及测定POD、PPO、CAT、SOD、Glu和Cht六种病程相关蛋白酶活性,研究H2O2对沙打旺抗黄矮根腐病的影响。结果表明,接种埃里砖格孢后2种沙打旺H2O2含量高于未接种植株,且感病品种在38 h H2O2含量最高,为929 μmol/g,抗病品种最高值出现在24 h,为986 μmol/g。抗病、感病沙打旺发病率、病情指数在H2O2处理后最低,施加AsA最高。H2O2处理各种酶活性都有不同程度的增加,且对抗病品种酶活性提高大于感病品种,表明外施H2O2可以减缓沙打旺黄矮根腐病的发生和侵染。表明H2O2与沙打旺抗病性紧密相关,并通过调节病程相关酶活性来增强沙打旺对黄矮根腐病的抗性,减少侵染率。  相似文献   

15.
Measurement of hydrogen peroxide (H2O2) concentration and pH in exhaled breath condensate (EBC) is useful for detection and monitoring of asthma in humans. In contrast, limited information on the use of these parameters for the investigation of lower airway inflammation (LAI) is available for horses. Aims of the current study were to investigate the intra- and inter-day variations of EBC H2O2 concentration and pH in horses and establish any relationship(s) with LAI. Both intra- and inter-day variability of EBC H2O2 concentration were large, while those of pH were small. No significant difference in the intra-day or inter-day H2O2 concentrations or pH measurements were found in control or LAI horses, except for inter-day H2O2 concentration in horses with LAI (p = 0.019). There was no significant difference in EBC pH or H2O2 concentration between control and LAI horses, however a trend for a reduced pH in horses with LAI was observed.  相似文献   

16.
Summary

Calves (n = 2) born to dams with experimentally induced brucellosis, and calves (n = 4) born to dams with naturally occurring infection were examined by the delayed‐type hypersensitivity (DTH) test for possible B. abortus infection. The results were compared with the serum agglutination test, complement fixation test, and Coombs test. Five calves were nursed by their dams for 8–10 weeks after birth. One calf was separated from its dam and fed artificial milk.

Three to five months after birth, four calves tested seropositive in the serologic tests. Antibodies were detected in one calf as early as 1 week after birth. The calf fed on artificial milk was seronegative 4–5 weeks after birth. All calves reacted to the DTH test antigen from week 12 until the end of the experiment, even though serologic tests were negative. We conclude that the DTH test is a valuable technique for diagnosing Brucella in calves born to infected dams.  相似文献   

17.
Butylated hydroxyanisole (BHA) is a synthetic phenolic compound consisting of a mixture of two isomeric organic compounds: 2-tert-butyl-4-hydroxyanisole and 3-tert-butyl-4-hydroxyanisole. We examined the effect of BHA against hydrogen peroxide (H2O2)-induced apoptosis in primary cultured mouse hepatocytes. Cell viability was significantly decreased by H2O2 in a dose-dependent manner. Additionally, H2O2 treatment increased Bax, decreased Bcl-2, and promoted PARP-1 cleavage in a dose-dependent manner. Pretreatment with BHA before exposure to H2O2 significantly attenuated the H2O2-induced decrease of cell viability. H2O2 exposure resulted in an increase of intracellular reactive oxygen species (ROS) generation that was significantly inhibited by pretreatment with BHA or N-acetyl-cysteine (NAC, an ROS scavenger). H2O2-induced decrease of cell viability was also attenuated by pretreatment with BHA and NAC. Furthermore, H2O2-induced increase of Bax, decrease of Bcl-2, and PARP-1 cleavage was also inhibited by BHA. Taken together, results of this investigation demonstrated that BHA protects primary cultured mouse hepatocytes against H2O2-induced apoptosis by inhibiting ROS generation.  相似文献   

18.
Respiratory infection of cattle with bovine herpesvirus type 1 (BHV-1) predisposes cattle to secondary pneumonia with Mannheimia haemolytica as part of the bovine respiratory disease complex (BRD). One cell type that has received limited investigation for its role in the inflammation that accompanies BRD is the respiratory epithelial cell. In the present study we investigated mechanisms by which BHV-1 infection of respiratory epithelial cells contributes to the recruitment and activation of bovine polymorphonuclear neutrophils (PMNs) in vitro. Primary cultures of bovine bronchial epithelial (BBE) cells were infected with BHV-1 and assessed for cytokine expression by real-time PCR. We found that BHV-1 infection elicits a rapid IL-1, IL-8 and TNF-α mRNA response by BBE cells. Bovine PMNs exhibited greater adherence to BHV-1 infected BBE cells than uninfected cells. The increased adherence was significantly reduced by the addition of an anti-IL-1β antibody or human soluble TNF-α receptor (sTNF-αR). Pre-incubation of bovine PMNs with conditioned media from BHV-1 infected BBE cells increased PMN migration, which was inhibited by addition of an anti-IL-1β antibody, sTNF-αR, or an IL-8 peptide inhibitor. Conditioned media from BHV-1 infected BBE cells activated bovine PMNs in vitro as demonstrated by PMN shape change, production of reactive oxygen species and degranulation. PMNs also exhibited increased LFA-1 expression and susceptibility to M. haemolytica LKT following incubation with BHV-1 infected BBE cell conditioned media. Our results suggest that BHV-1 infection of BBE cells triggers cytokine expression that contributes to the recruitment and activation of neutrophils, and amplifies the detrimental effects of M. haemolytica LKT.  相似文献   

19.
In dairy cows, hydrogen peroxide (H2O2) produced from a low‐molecular‐weight compound in milk from inflamed quarters was lower than that in milk from un‐inflamed quarters. In milk of delivery grade, characteristics of H2O2 production in milk with high electrical conductivity (EC) were examined in this study. Milk samples were collected from a total of 230 cows at 1‐month intervals, and the EC of skimmed milk was determined. Based on the highest and the lowest EC of a cow's quarter milk, the inter‐quarter difference of ≥0.6 mS/cm (mean + t0.01 SE) was taken as a high EC. Milk with high EC was found in 52 quarters. In cows with milk of high EC, H2O2 production in milk with normal EC was higher than that in milk with high EC in the same animal but was lower than that in the control population. In milk with high EC, the decrease of H2O2 production correlated with the increase in EC. The production of H2O2 decreased in particular when the inter‐quarter difference exceeded 0.8 mS/cm. In milk collected from the same quarter 1 month before, EC changed from normal to high, and H2O2 production decreased. In milk from the other three quarters, EC remained normal and H2O2 production remained unchanged. We concluded that milk with high EC appeared in low H2O2‐producing cows. The results suggest that the degree of decrease in H2O2 production reflects the extent of quarter abnormality.  相似文献   

20.
We investigated the effect of oxidative stress induced by hydrogen peroxide (H2O2) on lipid peroxide (LPO) level and nuclear maturation in porcine oocytes cultured with or without cumulus cells. After 22 h of pre‐culture, oocytes with attached cumulus cells (COC group) or denuded oocytes (DO group) were cultured with H2O2, and intra‐oocyte H2O2 and LPO levels were quantitatively analyzed using immunofluorescence. This is the first report evaluating LPO levels in porcine oocytes. After H2O2 supplementation, the DO group showed severe accumulation of H2O2 and LPO in the oocytes. Similarly, while inhibition of progression of nuclear maturation was observed in both groups, the effect was more severe in the DO group. These results demonstrate that cumulus cells reduce the accumulation of H2O2 stress in oocytes. Furthermore, we attempted to reduce the oxidative stress by H2O2 with L‐carnitine, a H2O2 scavenger. L‐carnitine decreased H2O2 and LPO levels in the oocytes in both groups, and improvement in the progression of impaired nuclear maturation was observed. These effects were different by the presence of cumulus cells. Our results provide that L‐carnitine is useful for alleviating H2O2‐induced oxidative stress by reducing LPO levels and improving the progression of nuclear maturation.  相似文献   

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