Natural killer (NK) activity of porcine blood lymphocytes against allogeneic melanoma target cells

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.     

Enhancement by interferon of chicken splenocyte natural killer cell activity against Marek's disease tumor cells

A non-immune natural killer-type cell population (NK) from 6-to 12-week old chickens was able to kill MSB-1 Marek's disease (MD) tumor cells in vitro; as measured by the 51Cr-release cytotoxicity assay. Removal of T cells, B cells, adherent cells, or any combination of the three populations of cells did not result in diminished levels of cytotoxicity of the remaining spleen cells against MSB-1 cells. The cytotoxicity of chicken NK cells could be rapidly augmented by polyinosinic-polycytidylic acid (poly I:C) and by the Cal 11914 strain of Newcastle disease virus (NDV), but not by the TCND strain of NDV which is not an interferon (IFN) inducer, indicating that IFN play a role in augmentation of the NK activity in chickens.     

Natural killer cell activity in untreated and treated dogs with lymphoma

Natural killer (NK) cell activity and function were determined for 11 untreated and treated dogs with lymphoma. Concurrent chromium release and single cell binding assays, methods used to measure overall cytotoxic activity and that from individual cells, respectively, were performed at effector-to-target cell ratios of 50:1 and 100:1, with incubation periods of 12 and 16 hours. Significant reduction was achieved in overall activity for untreated dogs, using a 16-hour incubation period and an effector-to-target ratio of 100:1 (P less than 0.05). Decreased activity (P less than 0.025) was also achieved for those dogs that were administered combination chemotherapy, consisting of such drugs as cyclophosphamide, vincristine, prednisone, and doxorubicin. There was no significant difference in binding or cytotoxic activity by individual cells in the untreated or treated dogs, compared with the healthy controls. Short- or long-term treatment with glucocorticoids did not influence overall NK cll activity or individual cell cytotoxicity. The overall cytotoxic activity in untreated dogs was reduced, but these dogs had relatively normal numbers of NK cells compared with paracontrols. This suggests that a defect in recycling or the ability to kill targets repetitively, may be involved. A similar defect was found in NK cells of dogs treated aggressively with combination chemotherapy.     

Development and functional characterization of monoclonal antibodies recognizing chicken lymphocytes with natural killer cell activity.

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.     

Antimicrobial activity of chicken NK-lysin against Eimeria sporozoites

NK-lysin is an antimicrobial and antitumor polypeptide that is considered to play an important role in innate immunity. Chicken NK-lysin is a member of the saposin-like protein family and exhibits potent antitumor cell activity. To evaluate the antimicrobial properties of chicken NK-lysin, we examined its ability to reduce the viability of various bacterial strains and two species of Eimeria parasites. Culture supernatants from COS7 cells transfected with a chicken NK-lysin cDNA and His-tagged purified NK-lysin from the transfected cells both showed high cytotoxic activity against Eimeria acervulina and Eimeria maxima sporozoites. In contrast, no bactericidal activity was observed. Further studies using synthetic peptides derived from NK-lysin may be useful for pharmaceutical and agricultural uses in the food animal industry.     

Induction of lymphokine-activated killer cells of equine origin: specificity for equine target cells.

The in vitro stimulation of peripheral blood mononuclear cells (PBMC) with interleukin 2 (IL-2) results in the development of potent cytotoxic effector cells, referred to as lymphokine-activated killer (LAK) cells. LAK cells are capable of lysing a wide variety of autologous, allogeneic and xenogeneic tumor cells. The exact mechanism of target cell recognition by LAK cells remains unknown. LAK cell activity has been reported for a variety of domesticated species except the horse. We report here that IL-2-stimulated equine PBMC, which fail to lyse either human or murine tumor cell lines, exhibit potent cytolytic activity against an equine tumor cell line, EqT8888. Cytolytic activity against the EqT8888 cells required 3 days of incubation with IL-2, was mediated primarily by T-cells, and was not restricted by major histocompatibility complex antigens. Though LAK activity could only be demonstrated using equine-derived target cells, xenogeneic targets could be lysed in a lectin-mediated cytotoxicity assay. The xenogeneic targets also failed to block LAK cell-killing of the EqT8888 cells in a cold-target competition assay. These results indicate that LAK cells in the horse appear to utilize a species-specific recognition mechanism during target cell lysis.     

Induction of specific cytotoxic T-cell activity against xenogeneic target cells in carp (Cyprinus carpio)

OBJECTIVE: To investigate the induction of cytotoxic T cells in carp (Cyprinus carpio) after inoculation of fish with 2 xenogeneic line cells and to examine specificity of the cytotoxic activity. ANIMALS: 22 carp. PROCEDURE: Fish were inoculated with mouse myeloma line cells P3.NS-1/1Ag4.1 (NS-1) or chicken Marek's disease tumor-derived lymphoma line cells (MDCC MSB-1). Cytotoxic activity of immune lymphocytes was evaluated by incubating effector cells with homologous and heterologous target cells. Populations of effector cells were identified by blocking T-lymphocytes from effector cells, using anti-carp T-cell monoclonal antibody and complement. RESULTS: Lymphocytes in blood, spleen, and head kidney of carp inoculated with NS-1 cells or MDCC MSB-1 cells had dose-dependent cytotoxic effects against homologous target cells but not against heterologous target cells. Lymphocytes from noninoculated carp did not have cytotoxic effects. Depletion of T-lymphocytes in spleen cells from NS-1-inoculated carp resulted in a decrease of cytotoxic activity against NS-1 cells. Cytotoxic activity of spleen lymphocytes from NS-1-inoculated or noninoculated carp was not evident when cytotoxic tests were performed after addition of anti-NS-1 carp serum. CONCLUSIONS AND CLINICAL RELEVANCE: Inoculation with xenogeneic target cells induces a specific cytotoxic T-cell response in carp. Thus, cell-mediated immunity plays a role in defense against infection of parasitic organisms such as protozoa and helminths.     

Natural killer cell activity in chickens exposed to Marek's disease virus: inhibition of activity in susceptible chickens and enhancement of activity in resistant and vaccinated chickens

Chickens of 2 genetic lines (lines P and N) were inoculated with a pathogenic strain of Marek's disease (MD) virus (MDV) and chronologically examined for disease response and natural killer (NK) cell expression. The NK cell reactivity was assayed in an in vitro cytotoxicity assay in which effector cells from the spleen of test chickens were reacted with 51Cr-labeled LSCC-RP9 target cells. Chickens of line P developed progressive debilitating disease and a high incidence of gross tumors and death. The NK cell reactivity of line-P chickens infected with MDV was significantly lower than that of uninfected control hatchmates. In contrast, NK cell levels were significantly elevated in MDV-inoculated line-N chickens that were resistant to MD and in chickens of lines P or N that had been inoculated with herpesvirus of turkeys (HVT). NK cell levels were also elevated in line P if chickens were vaccinated with HVT before infection with MDV. Inhibition of NK reactivity in susceptible chickens and elevation of reactivity in naturally resistant or vaccinated chickens may indicate a role for the NK cell system in regulating resistance to MD.     

豫北地区临床分离鸡大肠杆菌的体外抑菌作用测定

为了有效地治疗鸡大肠杆菌病,采用微量倍比稀释方法测定了17种抗菌药物对临床分离的豫北地区15株鸡大肠杆菌的体外最小抑菌浓度（MIC）,并根据其MIC及MIC范围（MICRange）使用SPSS 13.0中Probit过程计算出17种抗菌药物的MIC50和MIC90。结果表明：多粘菌素B的抑菌作用最强,MIC50、MIC90分别为0.11、0.87μg/mL;加替沙星的抑菌作用次之,MIC50、MIC90分别为2.53、3.88μg/mL,其它3种药物恩诺沙星、左氧氟沙星、环丙沙星的抑菌作用相当,但不及加替沙星,MIC50、MIC90分别为10.11-11.79μg/mL、15.16-21.13μg/mL;多西环素和阿莫西林等12种抗菌药物的抑菌作用较小,MIC50、MIC90分别为18.53-388.50μg/mL和30.59-713.42μg/mL。     

Effects of canine distemper virus on natural killer cell activity in dogs

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.     

重组鸡IL-2的抗球虫作用

鸡白细胞介素2(Ch IL-2)是一种重要的细胞因子,对Ch IL-2的抗球虫作用做了初步研究,分别在14日龄和21日龄给实验雏鸡胸部肌肉注射不同剂量的重组Ch IL-2蛋白,28日龄口服接种1×105个活球虫卵囊,结果表明,重组Ch IL-2蛋白能降低相对卵囊产量、减少盲肠病变、提高相对增重。其中以注射250μg重组Ch IL-2蛋白的试验组抗球虫效果最好,相对卵囊产量为23.26%,盲肠病变记分分别为0.4,相对增重率为97.80%,综合抗球虫指数(AC I)达到192.8。抗球虫效果良好,明显的优于其他试验组。     

Host environment as a modulating factor of swine natural killer cell activity

The large granular lymphocyte (LGL) population includes such heterologous effector cells as the natural killer (NK), lymphokine activated killer (LAK), antibody dependent cellular cytotoxic (ADCC) and non-MHC restricted T cells. These LGL subpopulations have all been associated with NK activity. In some species, enhanced NK activity is correlated with exposure to viral, bacterial and parasitic agents. Consequently, the host environment could serve as a modulatory factor of NK activity in laboratory animals. During our investigation of tumor regression in melanoma swine, we observed marked differences in the NK activity of peripheral blood lymphocytes collected from two separate groups of Sinclair melanoma miniature swine maintained under different conditions. Group A pigs were vaccinated and extensively treated for endo- and ectoparasites while group B swine were not. In addition, chronic exposure to infectious and parasitic diseases have been documented in the group B swine. Peripheral blood NK activity was assessed by standard in vitro 4-h chromium release assays. The NK activity of group B swine was markedly exaggerated when compared to group A swine. Thus, the significance of NK activity may be distorted as a result of the modulating effect of pathogen exposure.     

Natural killer cells in normal horses and specific-pathogen-free foals infected with equine herpesvirus.

Peripheral blood mononuclear cells (PBMC) from an adult horse and from foals demonstrated natural killer (NK)-type cytotoxicity against a range of xenogeneic and allogeneic cell targets. The human tumour cell line, Chang liver was consistently the most susceptible. Chang liver, rabbit kidney (RK-13), equine sarcoid (ES) and embryonic equine kidney (EEK) cells were more susceptible when presented to horse PBMC than monolayer cultures. Embryonic equine lung (EEL) and murine YAC-1 cells conversely, were more susceptible in a trypsinized state. Horse PBMC demonstrated higher levels of NK-type activity against EEK, EEL and RK-13 cells infected with equine herpesvirus 1 (EHV-1) compared with uninfected cells. Similarly, EEK and EEL cells infected with Semliki forest virus (SFV) were more susceptible. Cytotoxicity against EHV-1-infected EEK cells developed faster, between 4 and 8 h of incubation and reaching a maximum at 24 h. By contrast, cytotoxicity against uninfected fibroblasts was not significant until approximately 16 h of incubation with maximum cytotoxicity observed between 32 h and 48 h. Specific pathogen-free (SPF) foals were inoculated with live EHV-1. PBMC isolated from these foals at different days after inoculation did not display appreciably reduced or elevated NK cytotoxicities against Chang liver cells and EHV-1-infected EEK targets, when compared with that of a PBMC reference from a healthy adult horse.     

A monoclonal antibody against chicken thrombocytes reacts with the cells of thrombocyte lineage

A new mouse monoclonal antibody (mAb), HUKT was raised against chicken peripheral blood thrombocytes. The mAb HUKT appeared to detect a specific marker on the surface of chicken thrombocytes. Flow cytometry (FCM) analysis revealed that it did not react with cells from the normal thymus, bursa of Fabricius, six kinds of chicken cell lines, chicken erythrocytes or human platelets. In addition, HUKT(+) cells in peripheral blood leukocytes (PBL) were CD45(low), Bu-1a(-) and CD3(-) cells. Immunoblotting analysis showed that the molecule recognized by HUKT is a monomer with an apparent molecular weight of 150 kDa under non-reducing and reducing conditions. Tissue distribution studies revealed that only cells of thrombocyte lineage in bone marrow and embryonic blood cells were stained by HUKT. The HUKT mAb presented here may be useful for both ontogenetic studies of thrombocyte lineage and immunological studies in the chicken.     

Concanavalin A-induced suppressor cell activity in intestinal mucosal leukocytes obtained from healthy cows

Leukocytes isolated from the intraepithelium, lamina propria, and aggregated lymphoid follicles (ALF) of the bovine small intestinal mucosa were activated by concanavalin A (conA) to generate suppressor activity against the proliferative response of autologous and allogeneic leukocytes to conA, phytohemagglutinin, and pokeweed mitogen. Freshly obtained intraepithelium, lamina propria, and ALF leukocytes, preincubated with 25 to 50 micrograms/ml of conA for 24 to 48 hours, were able to inhibit the mitogenic responses of autologous and allogeneic lymphocytes when cocultured at a ratio greater than or equal to 1:1 (suppressor cells to responder cells). Depletion of adherent cells (monocytes/macrophages) by sequential plastic and gel adherence completely abrogated the conA-induced suppressor activity of all 3 leukocytes populations. However, reconstitution with autologous or allogeneic monocytes/macrophages during the induction phase restored the inducible suppressor activity. Addition of recombinant human interleukin-2 at a concentration as low as 5 U/ml during the responder phase enabled the responder population to recover the response apparently impaired by the conA-treated ALF leukocytes. At a concentration of 10 U/ml, human interleukin-2 was not only able to restore the responder population's response to phytohemagglutinin stimulation, but highly enhanced its proliferative ability. Although quantitative variations were observed between different cell populations and cells obtained from individual cows, the extent and general pattern of the inducible suppressor activity were similar in cells from cows studied.