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1.
Allogeneic PM/86 melanoma cells of Munich Troll miniature swine have been used for the demonstration of porcine peripheral blood NK cell activity. Compared with the specific lysis of xenogeneic K562-, U937- and Vero-target cells, NK cell-mediated cytotoxicity (NK-CMC) against PM/86 melanoma tumor cells was significantly lower in a 16 h chromium release assay. The target cell susceptibility to peripheral blood NK-CMC of both adult Troll miniature swine and German Landrace sows was very similar. Cold target inhibition assays revealed the allogeneic PM/86 melanoma cells to be the most powerful inhibitors of NK-CMC. Nylon wool non-adherent lymphocytes produced interferon (IFN)-alpha in different quantities upon contact with NK susceptible target cells. The NK effector cells could be stimulated to a higher lytic activity against all susceptible targets by a moderate dose of natural human interleukin-2 (nhuIL-2). The role of NK-CMC in melanoma tumor rejection and/or prevention of metastases is yet unknown in swine although porcine melanoma serves as a good model for the disease in man.  相似文献   

2.
The present study examined the properties of NK activity in Yorkshire swine. The results support other porcine studies which indicate the swine NK system has both similarities and differences to this system in other species. Profiles of NK activity indicated swine NK cells are highly reactive against the YAC-1 lymphoma, the K-562 myeloid leukemia, the P-815 mastocytoma, and the TU-5 virally transformed fibroblast. In contrast, the MOLT-4 and SB leukemias are NK resistant. Kinetic studies indicated that in Yorkshire swine, NK lysis begins 6 h after mixing effectors and targets. The kinetics of the lytic reaction differ both from other breeds of swine and from other species, where cytotoxicity is readily measured in 4-h assays. The delayed lysis was not due to delayed target cell recognition, because Yorkshire swine NK cells are rapidly bound to tumor targets. The delayed lysis seems to be due to a refractoriness in the NK lytic mechanism. This delay may relate to the morphologic finding that the target-binding cell in Yorkshire swine appeared quite different from the large granular lymphocyte (LGL) reported as the NK effector in humans and rodents. Indeed, in light microscopic studies the typical tumor-binding cell in Yorkshire swine is a small, apparently nongranular, lymphocyte. Analysis of NK activity at the single cell level was performed with single effector-tumor conjugates immobilized in agarose. Generally, lysis by target binders paralleled sensitivity to lysis in 51Cr release tests, indicating lysis in agarose may be used as an NK index in swine. Like other species, swine NK cells were found to be nonadherent lymphocytes with a characteristic tissue distribution. Peripheral blood and spleen had the highest levels of NK activity. Lymph node cells displayed a small amount of NK activity which was limited to the YAC-1 target, while thymocytes showed no appreciable NK activity against any of the cell lines tested.  相似文献   

3.
4.
We have analysed natural killer (NK) cytotoxic activity in peripheral blood and ascitic fluids of patients with advanced stage of ovarian epithelial carcinoma. All patients displayed low NK activity in peripheral blood and virtually no cytotoxicity in ascitic fluids. NK activity in ascitic fluids could be substantially augmented after regional administration of virus-modified tumor cell extracts (VMTE), and that in peripheral blood after culture of effector cells with interleukin-2 (IL-2) in vitro. Activated NK cells displayed cytotoxic activity against NK-sensitive and NK-resistant tumor cell lines as well as against fresh ovarian tumors. Parallelism was found between regional NK augmentation and regression of malignant ascites. The latter observation suggests possible NK cell role in defense against ovarian tumors.  相似文献   

5.
Three canine tumour cells were studied for their susceptibility to cytotoxicity by allogeneic canine natural killer (NK) cells: a lymphoma line, 3132 of B cell origin, and two adherent cell lines emanating from the same non-lymphoid tumour isolate, one (A72F) with a fibroblast morphology and one (A72E) with an epithelioid appearance. Both 3132 and A72E have preliminary evidence of retrovirus infection. Unstimulated canine peripheral blood mononuclear cells, used as the source of NK cells, were able to mediate significant lysis of 3132 and A72F cells at effector cell:target cell ratios of under 50:1, although an 18 h incubation was necessary for maximum cytotoxicity. NK activity against the 3132 tumour cells proved to be variable both within a group of dogs as well as on different occasions utilising the same individual donor. The epithelioid form of the A72 tumour cell line, A72E, had gained a marked resistance to NK lysis, although like the 3132 cells, there is preliminary evidence of persistent retrovirus infection in this cell line. Interestingly the A72F cells were as successful as homologous 3132 cells in the cold target inhibition of labelled 3132 cytotoxicity, while A72E did not. This latter result could indicate that not only do A72F and 3132 share NK determinants recognised by the same NK receptor, but the A72E line has lost this important recognition determinant.  相似文献   

6.
Natural killer (NK) cells have been considered to be a group of lymphocytes lacking clonally distributed receptors for antigens typical of T cells and B cells. In some mammalian species, including humans, a subpopulation of CD8+ peripheral blood lymphocytes (PBLs) exhibits NK activity. This NK subpopulation has not been well characterized in mammals and its characterization is particularly poor in the dog. In this study, we demonstrated that a subset of canine CD8+ cells derived from PBLs and lymphokine (IL-2)-activated killers (LAKs) of PBLs that was CD3+, CD4?, CD21?, CD5lo, α/βTCR+, and γ/δTCR? contained substantially higher levels of mRNAs for NK cell-related receptors (NKp30, NKp44, NKG2D, 2B4, and CD16 for PBL, and NKG2D and CD56 for LAK) than the corresponding CD8? cells. This subset of CD8+ lymphocytes derived from LAKs also displayed significantly higher NK cytotoxic activity than the corresponding CD8? cells. In contrast, CD8+ cells derived from nonstimulated PBLs showed very low levels of NK cytotoxic activity. Our results indicate that, in IL-2-stimulated PBLs, canine CD8+ cells are an important subset associated with NK cytotoxic activity.  相似文献   

7.
Natural killer (NK) cells are one of the main cellular components of the innate immune system. They play an important role in the immune response against infections as well as tumour cells and therefore have two major properties: production of immune regulatory cytokines and chemokines as well as cytolytic destruction of particular target cells. The existence of NK cells in swine is well known as well as the phenotype of resting NK cells, but their response following activation by cytokines is still poorly understood. Therefore, we tested the influence of the immune regulatory cytokines IL-2, IL-12 and IL-18 on cytolytic activity, phenotype, IFN-gamma production and the accumulation of perforin in cytoplasm of peripheral blood mononuclear cells (PBMC) as well as purified NK cells. NK cells were enriched from PBMC using a magnetic cell separation (MACS) strategy with monoclonal antibodies against CD3, CD21 and SWC3, thereby removing T-, B- and myeloid cells. Respective fractions were used in flow cytometry (FCM) based cytolytic assays with the human tumour cell line K562 as target. After stimulation with the cytokines described above, the NK cell enriched CD3(-)CD21(-)SWC3(-) fraction showed an evident increase in the cytolytic activity compared to PBMC. This enhanced cytolytic activity was accompanied by a strong enrichment of IFN-gamma producing cells when a combination of all three cytokines (IL-2/IL-12/IL-18) was used; as determined in ELISPOT assays and intracellular staining of IFN-gamma in FCM. Also, the combination of these three cytokines led to an accumulation of perforin in the cytoplasm and an up-regulation of CD25 compared to control cultures incubated in medium without cytokines. The experiments performed clearly indicate a stimulatory role and strong synergistic effects of the investigated cytokines in the activation of porcine NK cells in vitro, inducing IFN-gamma, perforin production and cytotoxicity against target cells.  相似文献   

8.
Cells with the histological and ultrastructural characteristics of large granular lymphocytes (LGL) have been obtained in culture from both cattle and red deer (Cervus elaphus) reacting with 'sheep-associated' malignant catarrhal fever (MCF). Such cells have been derived from thymus, lymph node and spleen suspensions as well as from cerebrospinal fluid cells and cultured cornea. On most occasions their presence was observed only transitorily but by providing the cells with feeder monolayers and, or, interleukin-2, several lines were maintained indefinitely, and some became independent of these factors after prolonged culture. A similar cell line was also derived from a Père David's deer affected with MCF at Whipsnade zoological park. Functionally, cultured LGL were cytotoxic to both primary cell cultures and cell lines and their cytotoxicity was not restricted to histocompatible target cells. These findings suggest that the cultured cells have natural killer cell-like activity and that they are important targets for the agent of MCF in cattle and deer. One cell line derived from a red deer transmitted the disease but none of the cells generated from cattle did.  相似文献   

9.
Natural killer (NK) cells play a role in innate antiviral immunity by directly lysing virus-infected cells and producing antiviral cytokines such as interferon gamma (IFN-γ). We developed a system for characterizing the bovine NK response to foot-and-mouth disease virus (FMDV), which causes a disease of cloven-hoofed animals and remains a threat to livestock industries throughout the world. IL-2 stimulation of PBMC resulted in poor killing of human K562 cells, which are often used as NK target cells, while lysis of the bovine BL3.1 cell line was readily detected. Depletion of NKp46-expressing cells revealed that 80% of the killing induced by IL-2 could be attributed to NKp46+ cells. In order to characterize the response of NK cells against FMDV in vivo, we infected groups of cattle with three different strains of the virus (A24 Cruzeiro, O1 Manisa, O Hong Kong) and evaluated the cytolytic ability of NK cells through the course of infection. We consistently observed a transient increase in cytolysis, although there was variation in magnitude and kinetics. This increase in cytolysis remained when CD3+ cells were removed from the preparation of lymphocytes, indicating that cytolysis was independent of MHC-T cell receptor interaction or γδ T cell activation. In contrast, animals monitored following vaccination against FMDV did not exhibit any increase in NK killing. These data suggest that NK cells play a role in the host immune response of cattle against FMDV, and contrast with the suppression of NK activity previously observed in swine infected with FMDV.  相似文献   

10.
The infectivity and potential immunosuppressive effects of Pseudorabies virus (PRV) was evaluated in swine peripheral blood mononuclear cells (PBMC). Virus progeny titers and viral DNA synthesis at various intervals post-inoculation revealed the replication of PRV in both peripheral blood monocytes and lymphocytes; however, replication in lymphocytes was restricted compared with monocytes. PRV infection resulted in the damage and death of monocytes. Although PRV did not appear to affect the viability of the lymphocytes, PRV infection suppressed lymphocyte functions such as proliferation and interleukin-2 (IL-2) synthesis in response to Concanavalin A. This immunosuppression was dependent upon the multiplicity of infection (MOI) of infectious PRV. UV-inactivated PRV was not immunosuppressive. There was no effect of PRV on natural killer (NK) cell activity. The reduction of lymphocyte proliferation by PRV was not reversible by the addition of supernatant containing porcine IL-2 and non-infected monocytes to the infected cultures. The results from these in vitro studies demonstrate that PRV can infect and cause immunosuppressive effects on swine PBMC. These effects may explain the potential role of PRV in predisposing infected pigs to secondary infection and support the hypothesis that PRV can spread systemically by infected PBMC in blood and lymph.  相似文献   

11.
After the successful use of 3-[4,5-(dimethylthiazol-2-yl)]-2,5-diphenyltetrazolium bromide (MTT) in cell proliferation assays, its use has been established by different workers in cytotoxicity assays and research on leukaemia. In the present study, a colorimetric assay using MTT was adopted to evaluate the cytotoxic activity of chicken intestinal intraepithelial lymphocytes (iIELs), which constitute an important cellular component of the gut-associated lymphoid tissue (GALT). These iIELs are found to exhibit natural killer (NK) cell-like cytotoxic activity, which is spontaneous, non-MHC-restricted, and does not need to be primed. Hitherto, conventional chromium-release assays have been used to evaluate the cytotoxic activity of iIELs, but these assays have disadvantages such as radiation hazards and loss of the cells in washing steps. The mean percentage cytotoxic activity of chicken iIELs evaluated by the colorimetric assay was 90.37±2.53 in a group of 5-week-old chickens and 80.2±3.45 in a group of 8-week-old chickens. These findings established the successful use of a colorimetric assay using MTT for evaluating the cytotoxic activity of chickens iIELs.Abbreviations DMEM Dulbecco's modified Eagle's medium - DMSO dimethyl sulphoxide - E effector cells - GALT gut-associated lymphoid tissue - GM growth medium - iIELs intestinal intraepithelial lymphocytes - MTT 3-[4,5-(dimethylthiazol-2-yl)]-2,5-diphenyltetrazolium bromide - NK cell natural killer cell - OD optical density - RPMI Rosewell Park Memorial Institute medium - T target cells  相似文献   

12.
The morphologic and biological properties of porcine cells mediating natural killer (NK) activity were determined. In a previous study, we demonstrated that lymphocytes from the peripheral blood of pigs greater than 1 week of age possessed NK activity to K562 tumor cells and that lymphocytes from the blood and spleen of pigs greater than 1 day of age were able to mediate natural cytotoxicity against parainfluenza-3 (PI3) virus-infected Vero cells (Yang and Schultz, 1986a). Discontinuous density gradients were used to enrich NK cells. NK cytotoxicity was mainly present in high-density Percoll fractions (50 to 55% and 55 to 60%); little or no NK activity was present in lower density fractions. The NK cell enriched lymphocytes responded to the mitogens PHA, ConA and PWM. NK cells were sensitive to the suppressive effect of corticosteroid, but Protein A did not affect NK activity. The amount of cytotoxicity directly corresponded to the degree of binding that occurred between the NK enriched lymphocyte population and the target cells. Cytochemical and morphological studies demonstrated that these bond cells which are believed to be responsible for the NK activities, were mainly small to medium lymphocytes lacking azurophilic cytoplasmic granules. These findings were confirmed by ultrastructural studies of effector and "target-binding" cells. The results of the present study suggested that the cells mediating NK activity in pigs have the morphological and density characteristics of small and medium sized lymphocytes; findings that differ from those described for NK cells in human and other animal species.  相似文献   

13.
This study was conducted to evaluate immunological changes in peripheral blood leukocytes in pigs that were genetically selected for their improved resistance to mycoplasmal pneumonia of swine (MPS), using MPS vaccine as an antigen. Twelve castrated MPS‐selected Landrace pigs were compared with the same number of pigs from a nonselected line by using a time‐course analysis at the hematological level. After the second sensitization with MPS vaccine, the percentages of B cells, CD4+ T cells, and natural killer (NK) cells in total leukocytes were lower in the selected line than in the nonselected line, whereas the percentage of granulocytes in total leukocytes increased in the MPS‐selected line. We also assessed the proliferative ability of peripheral blood mononuclear cells (PBMCs) stimulated with Mycoplasma hyopneumoniae, lipopolysaccharide or concanavalin A, and found that although the proliferative ability of the PBMC was not different between the two lines at a steady state, the nonselected line showed a significantly higher proliferative ability after sensitization with MPS vaccine than the selected line regardless of antigens used. These results thus indicate that the selection of pigs on the basis of MPS resistance changes their immunophenotype, and would give us beneficial information for the prevention of MPS infection.  相似文献   

14.
A 4-MDa component, recovered from uterine luminal secretions of gilts on d 15 of pregnancy, was assessed for suppression of the lytic responses from natural killer (NK) and lymphokine-activated killer (LAK) effector cells. Each cell type originated from preparations of peripheral blood lymphocytes (PBL), and the LAK cells were generated from the incubation of PBL with interleukin-2. The PBL and LAK cells were cultured for 5 d with and without the 4-MDa component. Following culture, the cells were incubated (22 h) with NK-sensitive K-562 target cells at varying effector:target cell ratios (25:1 to 200:1). Lytic activity was assessed with the chromium-51 release assay. Additional experiments were conducted in order to determine whether suppressor activity of the 4-MDa component was time-dependent and associated with transforming growth factor-beta2 (TGF-beta2). For effector:target cell ratios combined, the 4-MDa component suppressed the lytic activity of PBL but failed to affect the LAK cells. Suppression of NK-mediated lysis occurred by d 3 of the 5-d culture period. In addition, suppressor activity of the 4-MDa component was reversed by a neutralization antibody to TGF-beta2. In conclusion, the 4-MDa component with TGF-beta2 activity suppressed the lytic responses of porcine NK cells.  相似文献   

15.
Cytotoxicity of bovine lymphocytes after treatment with lymphokines   总被引:2,自引:0,他引:2  
Cytotoxic lymphocytes were generated from bovine peripheral blood mononuclear leukocytes after in vitro stimulation with lymphokines that contained interleukin-2. Lymphokine-stimulated cultures were cytotoxic to K562 cells (human natural killer [NK] targets) and YAC-1 cells (mouse NK targets), but not to HSB-2 cells (human NK targets) in a 4-hour, 51Cr-release assay. Cells generated after lymphokine activation also mediated antibody-dependent cellular cytotoxicity to HSB-2 cells. Appearance of effector cells as a function of time in culture, method of stimulation, and cold target competition experiments strongly indicated that direct cytotoxicity and antibody-dependent cellular cytotoxicity may have been mediated by the same cell. Cells generated by similar conditions were able to mediate cytotoxicity against infectious bovine rhinotracheitis virus-infected target cells, especially in an 18-hour assay.  相似文献   

16.
An in vitro 51Cr-release assay was developed to detect the cytotoxicity of natural killer cells (NK) of canine peripheral blood mononuclear leukocytes to canine distemper virus (CDV) target cell membrane-bound antigens. Leukocytes from 23 young (greater than or equal to 1 week of age), CDV-naive gnotobiotic dogs could discriminate between noninfected control and CDV-infected Vero target cells. However, the amount of preinfection NK activity did not positively correlate with the ultimate outcome of the disease process when these same dogs were given virulent R252-CDV. Evaluation of preinfection and postinfection CDV-specific NK activity indicated that infection-associated increases in cytolysis of CDV-infected or noninfected Vero targets did not occur. In vitro infection of peripheral blood leukocytes with CDV did not change the kinetics or magnitude of NK-mediated cytolysis of homologous virus-infected or other NK-susceptible target cells.  相似文献   

17.
We have developed two monoclonal antibodies which detect cell surface antigens present on chicken lymphocytes mediating natural killer (NK) cell activity against the avian tumor cell target. The monoclonal antibodies, K-14 and K-108, stained 17 and 6% of splenic lymphocytes, and 11 and 14% of peripheral blood lymphocytes (PBL), respectively, and fewer than 5% of thymic and bursal lymphocytes. Neither of these monoclonal antibodies stained adherent macrophages or the MC29-virus transformed monocytic cell line. Both monoclonal antibodies significantly inhibited NK cell activity in a standard 4 h 51Cr-release cytotoxicity assay using the LSCC-RP9 tumor cell line as target cells at an effector to target ratio of 50:1. Pretreatment of splenocytes with either monoclonal antibody in the presence of rabbit complement (C) resulted in a significant reduction in NK cell activity. However, the monoclonal antibody K-1 which detects normal chicken macrophages did not interfere with NK cell activity. The monoclonal antibody K-108 significantly blocked Fc receptor-mediated rosette formation of sheep red blood cells coated with IgG antibodies (EA) by 56% while the monoclonal antibody K-14 did not show a significant blocking. These results indicate that the monoclonal antibodies K-108 and K-14 identify different epitopes present on the surface of chicken splenic lymphocytes which mediate spontaneous NK cytotoxicity.  相似文献   

18.
Natural killer (NK) cells have received much attention due to their cytotoxic abilities, often with a focus on their implications for cancer and transplantation. But despite their name, NK cells are also potent producers of cytokines like interferon-gamma. Recent discoveries of their interplay with dendritic cells and T-cells have shown that NK cells participate significantly in the onset and shaping of adaptive cellular immune responses, and increasingly these cells have become associated with protection from viral, bacterial and parasitic infections. Furthermore, they are substantially present in the placenta, apparently participating in the establishment of normal pregnancy. Consequently, NK cells have entered arenas of particular relevance in veterinary immunology. Limited data still exist on these cells in domestic animal species, much due to the lack of specific markers. However, bovine NK cells can be identified as NKp46 (CD335) expressing, CD3(−) lymphocytes. Recent studies have indicated a role for NK cells in important infectious diseases of cattle, and identified important bovine NK receptor families, including multiple KIRs and a single Ly49. In this review we will briefly summarize the current understanding of general NK cell biology, and then present the knowledge obtained thus far in the bovine species.  相似文献   

19.
To understand the influence of crossbreeding on Mycoplasma pneumonia of swine (MPS) resistance and immune characteristics, two crossbred lines were characterized. One crossbred line, LaWa, was generated by crossing the MPS pulmonary lesion selected Landrace line (La) and the highly immune‐selected Large White line (Wa). The second crossbred line, LaWb, was generated by crossing the La line and the nonselected Large White line (Wb). The crossbred LbWb line (nonselected Landrace line × nonselected Large White line) and the La line were used as controls. The LaWa and LaWb lines had an intermediate level of MPS lung lesions between La and LbWb lines, although the difference was not statistically significant. After stimulation with sheep red blood cells (SRBCs), the LaWb and LaWa lines showed immune characteristics similar to that of the La line; the number of monocytes in peripheral blood increased, while B cells, T cells, secretion of SRBC‐specific immunoglobulin G, and interleukin (IL)‐13 decreased. Additionally, the number of natural killer (NK) cells and the expression of IL‐4 and IL‐17 were significantly higher in the LaWb and LaWa lines, respectively. These data suggested that crossbreeding of La and Wa lines resulted in the inheritance of some of the selected immune responses.  相似文献   

20.
Outbred domestic swine or SLA inbred miniature swine were exposed to Ascaris suum either naturally on contaminated lots or by inoculation with UV-irradiated attenuated eggs. Both inbred and outbred swine developed virtually complete protection to a challenge of 10 000 eggs after natural exposure, but inbred swine were less resistant than outbred swine after UV-egg exposure. Flow cytometric analysis of peripheral blood mononuclear cells from these animals, performed to determine changes in cell subsets including helper T-cells, cytotoxic/suppressor T-cells, macrophages, and cells expressing class II major histocompatibility antigens, showed that both outbred and inbred swine had similar responses after parasite exposure. The levels of helper T-cells and cytotoxic/suppressor T-cells did not change after parasite exposure, while there was an appreciable but transient increase in macrophages only in those swine naturally exposed to A. suum. Swine exposed to A. suum, both naturally and by inoculation with UV-eggs, showed an increase in the amount of class II antigens detectable per cell. In a second set of experiments, outbred swine were exposed to A. suum naturally or by repeated experimental inoculation with different doses of normal eggs, and protective immunity and changes in blood cell subsets were determined. The greatest change in blood cell subsets was found at 3 and 5 weeks after initial parasite exposure, when macrophages were elevated moderately in a group of pigs inoculated every other day with 1000 eggs and markedly in a group that was naturally exposed; class II antigen expression was also increased during this period. These increases preceded peak serum antibody responses, which were lower in the naturally-exposed group relative to the experimentally-inoculated group. Both groups had high levels of protective immunity. This suggests than natural exposure to A. suum may activate cells and enhance specific immune responses to give high levels of protection.  相似文献   

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