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1.
The objective of this study was to determine whether porcine peripheral blood leukocytes and intestinal intraepithelial leukocytes can mediate antibody-dependent cell-mediated cytotoxicity and spontaneous cell-mediated cytotoxicity against target cells infected with transmissible gastroenteritis virus. Peripheral blood leukocytes collected from six young adult pigs and intraepithelial leukocytes from a further five pigs were used as effector cells in chromium release assays against PK-15 cells persistently infected with transmissible gastroenteritis virus. Both peripheral blood leukocytes and intraepithelial leukocytes were capable of mediating antibody-dependent cell-mediated cytotoxicity and spontaneous cell-mediated cytotoxicity against PK-15 transmissible gastroenteritis cells. While the peripheral blood leukocytes mediated lower levels of specific 51Cr release in spontaneous cell-mediated cytotoxicity than in antibody-dependent cell-mediated cytotoxicity, the intraepithelial leukocytes were more effective in spontaneous cell-mediated cytotoxicity than antibody-dependent cell-mediated cytotoxicity.  相似文献   

2.
A sub-population of circulating porcine gammadelta T cells express cell surface antigens associated with antigen presenting cells (APCs), and are able to take up soluble antigen very effectively. Functional antigen presentation by gammadelta T cells to memory helper T cells was studied by inbred pig lymphocytes immunised with ovalbumin (OVA). After removing all conventional APCs from the peripheral blood of immunised pigs, the remaining lymphocytes still proliferated when stimulated with OVA. When gammadelta T cells were further depleted, OVA specific proliferation was abolished, but reconstitution with gammadelta T cells restored proliferation. The proliferation was blocked by monoclonal antibodies (mAb) against MHC class II or CD4, and by pre-treatment of gammadelta T cells with chloroquine. These results indicate that a sub-population of circulating porcine gammadelta T cells act as APCs and present antigen via MHC class II.  相似文献   

3.
Fifteen pigs were inoculated with African swine fever virus in a study of the pathogenesis of the disease. All pigs surviving the first two weeks developed high circulating antibody titers against African swine fever virus and persistent viremia. Hemolytic complement levels declined to 50 to 70 hemolytic complement 50 (CH50) units/ml from mean preinoculation levels of 120 CH50 units/ml. Immune deposits consisting of African swine fever antigen, host immunoglobulin G, and native C3 were found in the glomeruli of surviving pigs. All pigs surviving two weeks after inoculation developed leukocyte-bound antibody functionally characteristic of immunoglobulin E (IgE). Antigen-specific degranulation of antibody-coated leukocytes produced secondary platelet aggregation and vasoactive amine release. The results suggest that the IgE-basophil-platelet loop acting via amplification by leukocyte-derived platelet-activating factor participates in the immune complex deposition process in African swine fever.  相似文献   

4.
T cell‐mediated cellular immunity and humoral immunity are equally important for the prevention of diseases. To assess activation of human and mouse cellular immunity, early activation markers of lymphocytes are often used in flow cytometry targeting expression of CD69 molecules. Response of humoral immunity against infection or vaccination has been well investigated in pigs, but that of cellular immunity has been largely neglected due to lack of direct evaluation tools. Thus, in pig research a proper assay of antibody reacted with porcine CD69 is still unavailable. In the present study, two anti‐porcine CD69 mAb‐producing mouse hybridomas, 01‐14‐22‐51 (IgG2b–κ) and 01‐22‐44‐102 (IgG2a–κ), both showing fine reactivity with phorbol 12‐myristate 13‐acetate (PMA) and ionomycin‐stimulated porcine peripheral blood lymphocytes in flow cytometry, were established. When porcine peripheral blood lymphocytes were activated with PMA and ionomycin and analyzed by flow cytometry, it was found that both mAbs generated in this study stained about 70% of lymphocytes. In contrast, after an identical procedure, only 5% and 13.5% of lymphocytes were stained with anti‐interferon‐γ mAb and anti‐tumor necrosis factor‐α mAb, respectively. These results indicate that evaluation of cellular immunity activation turns more sensitive after using our newly generated mAbs.  相似文献   

5.
Acute inflammatory diseases, such as colic, septicemia and endotoxemia are common in equines and have been shown to be correlated to vascular injury and thrombosis. In humans with similar thrombotic conditions, P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1)-mediated platelet-leukocyte adhesion contributes to the pathogenesis of these disorders through the generation of inflammatory mediators and tissue factor. As such, we hypothesized that a P-selectin-PSGL-1 (platelet-leukocyte) interaction, similar to that in humans, may also exist in the horse. The objective of this study was to investigate phenotypic and morphological properties of equine platelet activation with a focus on CD62P (P-selectin) expression and CD62P mediated platelet-leukocyte interactions. To study high levels of platelet activation, we used 1 U/ml thrombin to induce secondary, irreversible aggregation in both human and equine platelets. Addition of glycyl-L-prolyl-L-arginyl-L-proline amide (GPRP) prior to thrombin activation blocked fibrin polymerization, allowing the use of flow cytometry to study alpha-granule expression as a measure of platelet activation. Thrombin activation resulted in high levels of activation, measured as P-selectin expression, in both humans and equines. Interestingly, our research illustrates that in healthy horses, P-selectin is also constitutively expressed on 20-25% of resting platelets. This finding is in direct contrast to humans, in which P-selectin expression is negligible (<5%) in the absence of agonist activation. The high baseline level of P-selectin expression among equine platelets may suggest that they are primed for leukocyte adhesion, possibly resulting in prothrombotic conditions. This phenomenon could be of significant clinical relevance, as it may be related to the rapid clinical decline often seen in equine patients with colic and endotoxemia, where vascular injury and thrombotic complications compromise patient survival. Based on these findings, further investigation into the mechanisms of platelet P-selectin-mediated inflammation and platelet-leukocyte mediated vascular injury in the horse appears warranted.  相似文献   

6.
The influence of platelets and platelet lysate on superoxide anion production and beta-glucuronidase release from neutrophils was investigated. Neither the suspension of unstimulated platelets from healthy calves nor its lysate had any influence on O2- production and enzyme release On the other hand, a great rise in superoxide anion production and beta-glucuronidase release was observed in the case of platelets from bronchopneumonic calves. It is suggested that platelets of bronchopneumonic calves can activate polymorphonuclear leukocytes, causing their degranulation and aggravating superoxide anion production.  相似文献   

7.
A low-density cell population was isolated from skin explants of pigs and characterized as a highly enriched dendritic cell (DC) population based on phenotypical and functional properties. The skin-derived DCs were identified by their characteristic ultrastructural properties as well as by consistent co-expression of the CD1 and SWC3a antigens that clearly differentiate them from other porcine leukocytes. These cells exhibit higher expression of porcine MHC class II (SLAII) and CD80/86 antigens as compared to macrophage/monocyte cells. They consistently expressed the S100 beta antigen at high levels and did not express the lymphoid markers CD3, CD4 or CD8. Within this population of skin-derived DCs there was variable expression of CD11c, CD14 and CD16. Functional characterization of this DC population revealed that they are efficient in uptake and processing of soluble protein antigens and in endocytosis of small (0.02 microm) but not large (2 microm) polystyrene beads. Further, these cells were efficient inducers of primary allogeneic responses and in stimulating antigen-specific and mitogen-induced proliferation and IFN gamma responses in autologous lymphocytes. This study provides important information to further characterize the cutaneous DCs and develop models to analyze the role of these cells in immune responses in vivo.  相似文献   

8.
The objective of the present study was to analyze, by flow cytometry, changes in PBMC subsets in pigs having postweaning multisystemic wasting syndrome (PMWS), a new condition associated to porcine circovirus type 2 (PCV2) infection. Thirteen acutely PMWS affected pigs were selected from a farm seronegative to porcine reproductive and respiratory syndrome virus (PRRSV) and to Aujeszky's disease virus (ADV); 11 clinically healthy pigs were selected from a high health farm with no history of PMWS and free of the major swine pathogens, and used as a control group. All pigs were necropsied, and tissue samples were fixed in formalin; blood with EDTA anticoagulant was used to perform the flow cytometric analysis. PBMC were incubated with mAb against porcine CD3, CD4, CD8, CD25, CD45, IgM, SWC3, and SLA-Class II. Flow cytometric analysis showed substantial changes in leukocyte subsets in the peripheral blood of PMWS-affected pigs, which were characterized by an increase of monocytes, a reduction of T (mainly CD4(+)) and B-lymphocytes, and the presence of low-density immature granulocytes. Altogether, these changes would suggest an inability of acutely PMWS-affected pigs to mount an effective immune response.  相似文献   

9.
Interleukin 4 (IL-4) is an important regulatory cytokine produced by activated T lymphocytes and mast cells, and regulates the growth and differentiation of cells such as B and T lymphocytes. In the present study, recombinant thioredoxin (Trx)-porcine IL-4 (pIL-4) fusion protein was prepared by Escherichia coli (E. coli), and by using this protein as an immunogen, monoclonal antibodies (mAbs) against pIL-4 were produced to establish a basis for a research on immune responses in pigs. Six stable hybridoma cell lines were successfully established and specific binding of each mAb to recombinant pIL-4 produced by E. coli and insect cells infected with recombinant baculovirus was shown by enzyme-linked immunosorbent assay (ELISA) and/or immunoblot analysis. Isotype analyses of these mAbs revealed that the subclass of 5 out of 6 mAbs was IgG1 and the rest was IgG2b. Further, assessment of their epitopes by competition binding assay indicated that the mAbs obtained in this study bound to 4 different epitopes. The recombinant proteins and mAbs produced in this study will be useful tools for the assessment of porcine immune system.  相似文献   

10.
Thromboembolism is a major cause of morbidity and mortality in dogs with immune-mediated hemolytic anemia (IMHA). To the authors' knowledge, the role of platelets in thromboembolic events associated with IMHA has not been extensively investigated. In the study reported here, we evaluated cell membrane expression of P-selectin with flow cytometry to determine whether platelets circulate in an activated state in association with primary IMHA. Median P-selectin expression for 20 dogs with primary IMHA was 8.1-fold greater, compared with values for 20 healthy dogs. Fifteen of 20 dogs (75%) with IMHA had P-selectin median fluorescence intensity (MFI) values that exceeded the reference interval for healthy dogs. Additionally, P-selectin MFI after activation of platelets with phorbol myristate acetate was 2.1-fold greater for dogs with IMHA than for healthy control dogs. Despite treatment of all dogs with immunosuppressive therapy and 18 dogs with subcutaneously administered low-dose unfractionated heparin, 7 dogs developed clinical signs consistent with thromboembolism. These data provide support for the hypothesis that platelets circulate in an activated state in many dogs with IMHA.  相似文献   

11.
The acute phase protein response is a well-described generalized early host response to tissue injury, inflammation and infection, observed as pronounced changes in the concentrations of a number of circulating serum proteins. The biological function of this response and its interplay with other parts of innate host defence reactions remain somewhat elusive. In order to gain new insight into this early host defence response in the context of bacterial infection we studied gene expression changes in peripheral lymphoid tissues as compared to hepatic expression changes, 14–18 h after lung infection in pigs. The lung infection was established with the pig specific respiratory pathogen Actinobacillus pleuropneumoniae. Quantitative real-time PCR based expression analysis were performed on samples from liver, tracheobronchial lymph node, tonsils, spleen and on blood leukocytes, supplemented with measurements of interleukin-6 and selected acute phase proteins in serum. C-reactive protein and serum amyloid A were clearly induced 14–18 h after infection. Extrahepatic expression of acute phase proteins was found to be dramatically altered as a result of the lung infection with an extrahepatic acute phase protein response occurring concomitantly with the hepatic response. This suggests that the acute phase protein response is a more disseminated systemic response than previously thought. The current study provides to our knowledge the first example of porcine extrahepatic expression and regulation of C-reactive protein, haptoglobin, fibrinogen, pig major acute phase protein, and transferrin in peripheral lymphoid tissues.  相似文献   

12.
The objective of this study was to develop a novel competitive ELISA to measure the acute phase protein serum amyloid A (SAA) in pigs using species-specific reagents. Polyclonal antibodies were produced in rabbits immunised with recombinant porcine SAA (rSAA) expressed in Escherichia coli. Both the rSAA and polyclonal antibodies were used to develop a novel competitive assay that was analytically and clinically validated. This assay had within- and between-run coefficients of variation of 8.6% and 25%, respectively, and demonstrated a high level of accuracy as determined by linearity-under-dilution (correlation coefficient, r=0.965). The analytical and functional limits of detection were 3.3 and 105.02mg/L, and the upper and lower quantification limits were 66.9 and 2.8mg/L, respectively. Statistically significant differences (P<0.0001) were found between the concentrations of SAA in healthy and diseased pigs. This novel assay precisely and sensitively measures SAA levels in pigs and will facilitate the more accurate assessment and study of the acute phase response in this species.  相似文献   

13.
OBJECTIVE: To evaluate platelet surface-associated P-selectin, mean platelet component concentration (MPC), mean platelet component distribution width (MPCDW), mean platelet volume (MPV), and platelet distribution width (PDW) for detection of activated platelets in dogs with septic and nonseptic inflammatory disease. ANIMALS: 20 healthy dogs and 20 dogs with septic and nonseptic inflammatory disease. Procedures-Platelet surface-associated P-selectin (expressed as the median fluorescence intensity [MFI] of the platelet population), MPC, MPCDW, MPV, and PDW were determined in 20 healthy adult dogs, and reference ranges were calculated. These parameters were also determined in 11 dogs with nonseptic and 9 dogs with septic inflammatory disease and evaluated to determine which parameters were useful for detection of activated platelets. Results-12 dogs with inflammatory disease had P-selectin greater than the upper limit of the reference range, whereas 16 dogs with inflammatory disease had MPC lower than the lower limit of the reference range. All dogs in which P-selectin was greater than the upper limit of the reference range had MPC lower than the lower limit of the reference range. The correlation coefficient for P-selectin and MPC was 0.62. Differences in the MPCDW, MPV, and PDW in most dogs with inflammatory disease (compared with healthy dogs) were found; however, the correlation coefficients for P-selectin and MPCDW, MPV, and PDW were low. CONCLUSIONS AND CLINICAL RELEVANCE: Platelet surface-associated P-selectin and MPC appeared to be useful to detect activated platelets in most dogs with septic and nonseptic inflammatory disease.  相似文献   

14.
The measurement of serum pepsinogen concentrations by enzymatic method and immunoassay provides diagnostic values and should be helpful in the detection of gastric diseases related to a rise of blood pepsinogen. In the present study, the correlation between a conventional enzymatic method and a recently developed radioimmunoassay (RIA) for serum pepsinogen A was investigated. A total of 123 sera samples of porcine foetuses (n = 28), adult healthy pigs (n = 56), pigs with parakeratosis (n = 25) and pigs with ulceration of the pars oesophagea (n = 14) were tested. Overall, there was a slight correlation between the two methods (r = 0.60). In relation to individual animal groups, the correlations (r) were 0.39 (P>0.05), 0.74 (P<0.001), 0.19 (P>0.05) and 0.34 (P>0.05) in foetuses, healthy pigs, pigs with parakeratosis and pigs with ulcers, respectively. In both methods, pepsinogen concentrations (means+/-SE) were significantly higher (P<0.05) in pigs with parakeratosis (1778 +/- 86.00 mUTyr/L; 690 +/- 53.00 ng/mL) and in pigs with ulcers (2026 +/- 153.00 mUTyr/L; 1747 +/- 94.00 ng/mL) when compared to healthy pigs (935 +/- 58.00 mUTyr/L; 275 +/- 35.00 ng/mL). The proteolytic method gave a significant increased activity (P<0.05) in foetuses (1150 +/- 82.00 mUTyr/L) vs. (935 +/- 58.00 mUTyr/L) in healthy adult pigs, indicating an additional proteolytic activity in the sera of foetuses or neonates.  相似文献   

15.
Innate immune recognition of pathogens involves various surface receptors and soluble proteins that precede agglutination, complement activation, phagocytosis, and the adaptive immune response. Mannan-binding lectins (MBLs), ficolins (FCNs) and surfactant protein A (SP-A) are soluble collagenous lectins that bind surface structures of various bacteria, viruses and fungi. Some single nucleotide polymorphisms (SNPs) in collagenous lectin genes of humans and other species, including pigs, have been implicated in variation in susceptibility to infectious and inflammatory diseases. In this study we determined the frequencies of 13 SNP alleles of MBL-A, MBL-C, ficolin-α, ficolin-β, and SP-A in 1324 healthy pigs and 461 pigs diagnosed with common infectious diseases at necropsy. For comparison, we also analyzed 12 other SNP alleles in several other innate immune genes, including galectins and TLRs. Several SNPs within genes encoding porcine MBL-A, MBL-C and SP-A were more frequent in pigs diagnosed at necropsy with various diseases or pathogens. These findings suggest that several collagenous lectin SNPs are associated with disease susceptibility and therefore might be genetic markers of impaired innate immune function.  相似文献   

16.
Interactions between Salmonella typhimurium and peripheral blood leucocytes from healthy, Salmonella-free pigs were investigated in vitro. Both granulocytes and monocytes phagocytized FITC-labelled heat-killed Salmonella bacteria as shown by flow cytometry. Phagocytosis in whole blood and isolated leucocytes was measured as acquired fluorescence in the leukocytes and was both time and dose related. Living, serum-opsonized Salmonella bacteria induced a dose-dependent oxidative burst in PMNs and monocytes as measured by luminol-enhanced chemiluminescence (LC). When opsonized in normal serum the Salmonella bacteria, in the range of 2-5 x 10(7) cfu, induced a LC response in monocytes comparable to the level of responses induced by phorbol myristate acetate (PMA) and opsonized zymosan, and the Salmonella-induced response was only marginally reduced by superoxide dismutase (SOD). Intracellular killing of Salmonella by monocytes was assessed from plate colony counts of lysed monocytes and showed that Salmonella typhimurium was able to survive and proliferate in adherent monocytes in vitro despite a reduction in intracellular cfu during the first hour's incubation in cells from some pigs. Experiments with the exhaustion of oxidative burst in non-adherent monocytes were performed by prestimulation with PMA, heat-killed Salmonella or buffer. Prestimulation with PMA led to a strong reduction in oxidative burst induced by living opsonized Salmonella bacteria, whereas prestimulation with heat-killed bacteria gave rise to an enhanced response. In these experiments intracellular killing of the added living Salmonella gave variable results, in that monocytes from two out of three pigs showed no essential change in intracellular bactericidal activity, but with cells from one pig a less pronounced bactericidal activity was found after prestimulation with PMA.  相似文献   

17.
Shan T  Wang Y  Wu T  Liu C  Guo J  Zhang Y  Liu J  Xu Z 《Journal of animal science》2009,87(3):895-904
Sirtuin1 (Sirt1) is a NAD-dependent deacetylase that plays important roles in a variety of biological processes. In the current study, we examined tissue-specific and different expression pattern of porcine Sirt1 and the effect of resveratrol (RES) on expression of Sirt1 in porcine adipocytes. The full-length complementary DNA sequence of porcine Sirt1 was 4,024 bp (GenBank accession no: EU030283), with a 2,226-bp open reading frame encoding a 742-AA protein (a predicted molecular mass of 80.9 kDa; GenBank accession no. ABS29571). Comparison of the deduced AA sequence with the corresponding sequences of human, dog, cattle, and mouse Sirt1 showed 82 to 92% similarity. Furthermore, the porcine Sirt1 was highly expressed in porcine brain, to a lesser degree in spleen and white adipose tissue, and had low but detectable expression in liver. In subcutaneous adipose tissue and omental adipose tissue, expression of the porcine Sirt1 mRNA was greater in adult pigs than in young pigs (P < 0.01). In vitro, exposure of cultured adipocytes to 40 and 80 micro M RES for 24 h increased mRNA levels of porcine Sirt1 by 47.86% (P < 0.01) and 91.04% (P < 0.01), respectively. Accordingly, lipid accumulation and NEFA release were decreased (P < 0.05), respectively. After cultures were treated with RES for 48 h, the mRNA level of porcine Sirt1 was increased by 103.84% (P < 0.01) and 148.79% (P < 0.01), respectively. Lipid accumulation was decreased and NEFA release was increased (P < 0.05), respectively. These results provide information needed for manipulating Sirt1 expression in regulating fat deposition in pigs.  相似文献   

18.
Five acute phase proteins (APPs) [C-reactive protein (CRP), serum amyloid A (SAA), haptoglobin (Hp), pig-MAP and albumin] were measured in pigs with naturally occurring infections by porcine reproductive and respiratory syndrome virus (PRRSV), Aujeszky's disease virus (ADV), porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae, and in animals with tail and ear bites, arthritis and other acute inflammatory processes. Healthy specific pathogen-free (SPF) pigs were used as controls. In PRRSV-infected pigs, all APPs with the exception of pig-MAP exhibited significant changes compared with controls. In animals affected with ADV only Hp presented changes of statistical significance, whereas pigs with PCV2 showed marked modifications in all APPs tested. Animals affected with Mycoplasmosis showed concentrations of all positive APPs significantly above levels obtained in SPF pigs, though albumin concentrations did not differ from controls. Finally, all APPs studied showed substantial changes in pigs with acute inflammation. The results indicated that an acute phase response was developed in the different diseases studied, this response being higher in animals with clinical signs and concurrent bacterial processes. Haptoglobin would be the APP that better reflects pathological states; however, to get more complete and valuable information it might be advisable to perform APPs profiles including another APP, such as CRP or SAA.  相似文献   

19.
This study evaluated the effects of adding soluble fibre to the diet of healthy weaner pigs and weaner pigs experimentally infected with enterotoxigenic Escherichia coli (ETEC) in a model of post-weaning colibacillosis. Bodyweight gain, intestinal changes and proliferation of ETEC were measured 7 days following weaning. The basal diet consisted of pregelatinised rice fortified with animal protein. Addition of guar gum to this diet elevated the soluble fibre content from 1 to 6 per cent, and was associated with reduced bodyweight gains, increased large intestinal weights and fermentation, and increased proliferation of ETEC in the small intestine. The optimal levels and type of dietary fibre used for weaner pig diets require further evaluation.  相似文献   

20.
A high molecular-mass proteolytic enzyme of Actinobacillus pleuropneumoniae serotype 1, was purified from culture supernatants (CSN) by using DEAE-cellulose and sepharose-4B-gelatin chromatography. In 10% SDS-polyacrylamide gels copolymerized with porcine gelatin, the protease showed a single band of activity of > 200 kDa. However, minor molecular-mass proteolytic bands were observed when the protease was electrophoresed in the presence of either 5% beta-mercaptoethanol, 50 mM dithiothreitol, or 0.25 M urea. Furthermore, when the > 200-kDa purified protein was passed through a sucrose gradient, several bands with proteolytic activity were found: 62, 90, 190, and 540 kDa. The proteolytic activity was increased in the presence of calcium or zinc and was not affected after being heated at 90 degrees C for 5 min. Proteolytic activities were also observed in CSN from all A. pleuropneumoniae serotypes and biotypes. The purified protease hydrolyzed porcine IgA and IgG in vitro. In addition, by immunoblot the protease was recognized by serum of naturally infected pigs with serotypes 1 and 5, and by serum of pigs experimentally infected with serotypes 1, 2, 8, or 9. Serum of a pig vaccinated with CSN of a serotype 3 strain also recognized the protease, but not sera of pigs vaccinated with a bacterin (serotype 1). Proteins from CSN of all the serotypes, which were precipitated with 70% (NH4)2SO4, were recognized by a polyclonal antibody raised against the purified protease. Taken together these results indicate that an antigenic protease is produced in vivo by all the serotypes of A. pleuropneumoniae. The results indicate that proteases could have a role in the disease and in the immune response of pigs infected with A. pleuropneumoniae.  相似文献   

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