首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献，搜索用时 46 毫秒
1.
Two lymphoblastoid cell lines were isolated from different pigs and were maintained in culture for over 100 passages or 20 months. These cell lines were characterized by their cell surface antigens, ability to stimulate a mixed lymphocyte reaction and production of immunoglobulin. When tested against a panel of monoclonal anti-cell surface antigen antibodies, only those monoclonal antibodies which detect porcine class I or II molecules reacted against the lymphoblastoid cell lines in a microcytotoxicity assay. The two pig cell lines could stimulate peripheral blood mononuclear cells in a mixed lymphocyte reaction. P-SC(1) and P-16(2) also demonstrated a dependency upon the presence of 2-mercaptoethanol for cell division. The secretion of pig immunoglobulin by P-SC(1) or P-16(2) was first demonstrated by ELISA using a polyclonal anti-swine IgG (heavy and light chain) serum. By the use of monoclonal anti-IgA, IgG or IgM antibodies in an enzyme-linked assay on Western blots of P-SC(1) or P-16(2) lysate/supernatant, the two cell lines were demonstrated to be producing a whole monomeric IgA molecule and a mu chain.  相似文献   

2.
This study was carried out to develop and characterize mouse monoclonal antibodies (mAbs) against chicken CD25 (chCD25), the alpha chain of the interleukin-2 (IL-2) receptor. A recombinant chimeric chCD25/IgG4 fusion protein was expressed in Chinese hamster ovary (CHO) cells and isolated from spent cell culture medium by protein G affinity chromatography. Purified chCD25 protein was used to immunize mice, from which 54 stable hybridomas secreting chCD25 mAbs were produced. Two mAbs, chCD25-32 and chCD25-54, with high binding affinity for chCD25-expressing CHO cells were selected for further characterization. By flow cytometry, both mAbs detected cells in the spleen, bursa of Fabricius, intestinal duodenum, and immunostained established chicken T cell, B cell, and macrophage cell lines. Both mAbs reacted with a 55 kDa protein on Western blots of lysates from concanavalin A (Con A)-stimulated spleen mononuclear cells. Intraperitoneal injection of chickens with bacterial lipopolysaccharide increased the percentage of chCD25(+) spleen cells by approximately 4-fold compared with untreated animals. In vitro stimulation of spleen cells with Con A increased the percentage of chCD25(+) cells by up to 50-fold compared with cells treated with medium alone. Finally, the chCD25-32 mAb suppressed IL-2-driven spleen cell proliferation and reduced IL-2-induced nitric oxide production. These mAbs may be useful for future investigation of chicken regulatory T cells.  相似文献   

3.
Porcine IL-12Rbeta2 gene was cloned from mRNA preparation of mitogen-activated peripheral blood mononuclear cells (PBMCs), and its complete nucleotide sequence was determined. To confirm the biological function, the entire open reading frame (ORF) was re-cloned into a mammalian expression vector, pcDNA3.1/Zeo(+), at the downstream of CMV promoter, and introduced to a Th1-like human lymphoma cell line, Jurkat E6-1. Antibiotic-resistant cells retaining the expression construct were selected then, isolated by the limiting dilution method. An established clone (10B10) constitutively expressed chimeric IL-12Rs composed of intrinsic (human) beta1 and extrinsic (porcine) beta2 subunits, and produced interferon (IFN)-gamma in response to IL-12 of both species with optimal PHA/PMA stimulation. The production of IFN-gamma was observed as early as 42 h after culture and appeared to be dose-dependent within the range between 20 and 2000 pg/ml. Thus, this clone not only reacts with IL-12 of both species but also provides a useful tool for quick and sensitive detection of IL-12 bioactivity.  相似文献   

4.
A bioassay for bovine interleukin-1 (IL1) activity is described. The assay is based on the IL1-stimulated proliferation of a mouse T-lymphocyte cell line, D10(N4)M. Bovine mononuclear cells stimulated with lipopolysaccharide produce an interleukin-1-like activity which stimulated the growth of the D10(N4)M cell line in a dose-dependent manner. The stimulatory activity was neutralised by a combination of both anti-human IL1 alpha and anti-human IL1 beta sera. The quantity of IL1-like activity released from the mononuclear cells increased asymptotically with increasing lipopolysaccharide dose.  相似文献   

5.
Feline separated mononuclear cells (SMC) were obtained from peripheral blood by ficoll-diatrizoate gradient separation. SMC were further fractionated on nylon wool columns into nylon wool adherent cells (NWAC) and nylon wool effluent cells (NWEC). The three cell populations, SMC, NWAC and NWEC, were characterised using direct immunofluorescent staining for surface immunoglobulin (sIg) as a B cell marker and neuramidase treated guinea pig erythrocyte-rosette formation (E-rosettes) and mitogen-induced lymphocyte blastogenesis (LB) as possible T-cell markers. Feline SMC consisted of 30.1 +/- 4.0% sIg+ cells 36.6 + 5.4% E-rosette forming cells and 33.3% null cells i.e. cells which were sIg- and non E-rosette forming. Fractionation of SMC on nylon wool columns yielded NWEC which were significantly enriched for T cells in that they contained 68.6 +/- 2.9% E-rosette forming. Fractionation of SMC on nylon wool columns yielded NWEC which were significantly enriched for T cells in that they contained 68.6 +/- 2.9% E-rosette forming cells. NWAC were 51.0% +/- 10.8% sIg+, approximately 20% of cells were lost. The LB responsiveness of NWEC to concanavalin A (Con A) and phytonaemagglutinin-P (PHA-P) was enhanced compared to SMC. NWAC were non-responsive to Con A and PHA-P at all concentrations tested. It was concluded that nylon wool column fractionation of feline SMC was an efficient procedure for T cell enrichment and that the enriched cells retained the properties of E-rosette formation and blastogenesis by mitogens.  相似文献   

6.
Marek's disease virus (MDV)-transformed lymphoblastoid cells (MDCC-MSB1, -PA9 and -RP1) added to chicken splenic lymphocytes after treatment with mitomycin, suppress the lymphoproliferative response to T-cell mitogens (concanavalin A or phytohemagglutinin) by 40-70%. This suppressive activity was observed in syngeneic as well as in allogeneic combinations of cell lines and responder lymphocytes. The suppressive effect disappeared when the addition of MD-transformed cell lines to the responder cultures was delayed for 24 h. Treatment with glutaraldehyde, instead of mitomycin, greatly weakened the suppressive activity of the MD lymphoblastoid cells. A reduction of interleukin 2 (IL-2)-like activity produced by responder lymphocytes was observed after mixing with mitomycin-treated lymphoblastoid cells, but also, although slightly less, with the same glutaraldehyde-treated cells. Nevertheless no membrane fluorescence was observed, using INN-CH16 monoclonal antibody on MDV-induced lymphoblastoid cell lines to check up on the presence of IL-2 receptor-like structure. All the three lines exhibited a CD4+, CD8- phenotype.  相似文献   

7.
B-congenic chickens differ in macrophage inflammatory responses   总被引：1，自引：0，他引：1  
The influence of the chicken major histocompatibility (B) complex (MHC) on monocyte and macrophage recruitment and activation was examined using fully developed 15I5-B congenic White Leghorn lines (ten backcross generations). The phagocytic activity of Sephadex-elicited peritoneal macrophages for sheep red blood cells (SRBCs) was highest in lines 15.7-B2 and 15.P-B13 and lowest in 15.15I-B5 and 15.N-B21. The same pattern of phagocytic activity was obtained when LPS (E. coli) was used as the in vivo elicitor-activator of peritoneal macrophages. Lines with B2 and B13 haplotypes had elevated percentages of phagocytic macrophages and a higher internalization activity per cell than did B5 and B21 congenic chickens. Differential peritoneal macrophage function between congenic lines was further supported by quantitation of superoxide anion release. B2 and B13 haplotypes were associated with high activity in contrast with B5, which was low, and 15I5 (B15) and B21 which were intermediate for superoxide anion release by macrophages. In vitro activation of blood monocytes with LPS resulted in similar line differences for SRBC phagocytic activity as were observed with in vivo Sephadex and LPS activation. In contrast, chemotaxis of blood mononuclear leukocytes to f-met-leu-phe produced a reciprocal response pattern among the haplotypes. Cells from lines with haplotypes B5 and B21 were superior to those of B2, B13, and B15 congenic lines in their directed migration towards this chemoattractant. All functional differences occurred despite similarities among lines in the cellular profiles of both elicited peritoneal exudate cells and isolated blood mononuclear cells.  相似文献   

8.
We established 9 cell lines from 63 tumor cases of enzootic bovine leukosis and studied their properties. Cells of all lines formed small clumps and floated in culture medium, indicating growth. Four of the 9 cell lines were surface immunoglobulin (SIg)-positive, but the remaining 5 line cells were negative for SIg or, if SIg was detected, the percentage of SIg-positive cells was very low. Tests for the properties of the cells with monoclonal antibodies to lymphocytes revealed that the established line cells are B-lymphocytes. Morphological observation also revealed that they had the morphology of B-lymphoblastic cell. The results of E and EAC rosette assay were negative, but 6 of 8 cell lines were positive for EA rosetting. All the 9 cell lines reacted with MoAb C-143, which recognizes the tumor-associated antigen (TAA) of the EBL tumor cell. All 9 cell lines produced bovine leukosis virus (BLV). These results suggest that the 9 cell lines are tumor cells derived from B-lymphocytes of EBL.  相似文献   

9.
A simple but sensitive method for measuring the in vitro antibody response of cells from pig peripheral blood to a specific antigen is described. Peripheral blood mononuclear cells from lysozyme immunized pigs are cultured in the presence of lysozyme for five days, then washed and transferred to lysozyme coated wells of a microelisa plate and incubated for 18 hr. After washing the cells off the microelisa wells, the amount of pig anti-lysozyme immunoglobulin produced was measured by an enzyme-linked immunosorbent assay (ELISA). This assay allowed for the monitoring of the appearance of antibody-producing cells in the peripheral blood. In addition, the in vitro response of individual pigs to lysozyme correlated with serum antibody titers to lysozyme but not with the in vitro proliferative response of T cells to lysozyme. The assay provides an in vitro system for studying immunodeficiency in piglets and the general mechanisms behind a low immune response to a specific antigen.  相似文献   

10.
The monoclonal antibody (MAb), C5B6, recognizes the CD11c/CD18 molecule on the surface of bovine peripheral blood monocytes. C5B6 was reactive with 69-83% monocytes, all granulocytes, and less than 5% of lymphocytes from cattle. Of the lymphocyte series, the antibody had specificity for large lymphocytes and two lines expressing T cell markers, but was not reactive with small lymphocytes, thymocytes, a tumor cell line of B-cell lineage, an interleukin 2 (IL2)-dependent T cell line, fibroblasts, or human, sheep, goat or pig peripheral blood mononuclear cells. No dual fluorescence was seen using C5B6 and antibodies to bovine IgM, CD2, CD4 or CD8. Immunoprecipitation of 125I labeled peripheral blood mononuclear cells with C5B6 antibody defined two bands: 150,000 and 95,000 Da. Antibody to the beta chain (CD18) of the leukocyte adhesion receptor family precipitates the 95 kDa beta subunit and the three associated alpha subunits (180, 165 and 150). The bands obtained using MAb C5B6 correlated with the p150/95 bands observed using an antibody that precipitated the alpha and beta chains of the leukocyte adhesion receptor family. Functionally, the primary but not the secondary proliferative response to alloantigens was inhibited by C5B6 MAb. No effect was seen using C5B6 MAb in cytotoxicity assays or in the secondary proliferative response to Brucella abortus or bovine herpes virus type 1.  相似文献   

11.
Interferon-gamma (IFN-gamma) is a major effector cytokine of the immune system with an expression pattern strictly restricted to cells of the lymphoid lineage. Several years ago, we reported that, during early pregnancy, the trophectoderm of the pig blastocyst, which represents a monolayer of polarized epithelial cells secretes high amount of IFN-gamma in a transient and developmentally regulated manner. In an effort to study the molecular basis of this atypical IFN-gamma gene expression, a pig trophectoderm cell line, TBA B4-3, was established in our laboratory. These cells developed a polarized phenotype with high transepithelial electrical resistance (TER) when grown on a microporous membrane. We found that treatment of polarized TBA B4-3 cells with the strong PKC agonist PMA induced, 3-4 days later, a transient IFN-gamma mRNA expression and vectorial IFN-gamma protein secretion. In order to better understand IFN-gamma gene regulation in TBA B4-3 cells, we examined in this system the effect of several drugs and factors known to affect the inducibility of this cytokine in T lymphocytes, the main source of IFN-gamma in the immunocompetent animal. We found that cyclosporine A (CsA) treatment of TBA B4-3 cells induces a partial inhibition of IFN-gamma secretion, thus indicating a minor role for the calcineurin signaling pathway in IFN-gamma expression. In addition, we found that although PMA alone can induce IFN-gamma secretion, the calcium ionophore A23187 synergizes with PMA for induction. We also analyzed by Southern blot the methylation status of a CpG dinucleotide in the 5' flanking region of IFN-gamma promoter and found that it was unmethylated in TBA B4-3 cells and in several pig epithelial cell lines that do not express IFN-gamma thus indicating the absence of correlation between demethylation and the ability to express IFN-gamma. Taken together, these results indicate that the mechanisms involved in IFN-gamma induction in TBA B4-3 cells are atypical compared to those presently known to operate in the T cell lineage.  相似文献   

12.
In order to study the biological characteristics of somatic cells of Mashen pig, the ear marginal fibroblast cell lines of Mashen pig were established from 3 days old Mashen pig by direct explant method,and the biological characteristics of cell lines were detected. The results showed that the cell lines obtained from Mashen pig were in conformity with basic properties of typical fibroblasts, the shape of adherent cells were typical spindle, star and polygon, the time of grew into monolayer cells was 10 to 13 d, the growth curve was typical S shaped. The frequency of metaphase chromosome number (2n=38) was about 84% of the total number of cells, and it had met the establishment standard of fibroblast cell line. The transfection efficiency of green fluorescent protein(pEGFP-C1)transfected by lipofectamine-mediated method was 25%. The successful establishment of the fibroblast cell lines of Mashen pig had opened up a new method for the conservation of genetic resources.  相似文献   

13.
Two monoclonal antibodies were prepared which react specifically with pig serum immunoglobulin and with the population of B lymphocyte-bearing surface immunoglobulin. Comparison of our monoclonal antibodies with reagents specific for gamma, mu and alpha immunoglobulin chains in double immunodiffusion and immunoelectrophoresis revealed that the monoclonal antibodies recognise IgM in pig serum and mu chain or mu chain-like molecules on B lymphocytes. The monoclonal antibodies, designated LIG 2 and LIG 4, reacted positively with adult pig sera but not with fetal or precolostral sera or with sera from other animal species. LIG 2 and LIG 4 reacted with 15 per cent of cells from the peripheral blood lymphocyte population, 20.2 per cent of spleen cells and 20 per cent of lymph node cells, but did not react with pig erythrocytes, granulocytes or cells isolated from thymus, or with the lymphocytes of other species. Positive reactions were also found on lymphatic and intestinal tissue sections. No genetic polymorphism was found in the pig population revealed by the monoclonal antibodies. The monoclonal antibodies LIG 2 and LIG 4 may be useful for studying the pig immune system, especially as a standard reagent for measuring pig serum IgM and for the identification of positive B lymphocytes.  相似文献   

14.
This study was carried out to develop and characterize mouse monoclonal antibodies (mAbs) against chicken CD83 (chCD83), a membrane-bound glycoprotein belonging to the immunoglobulin superfamily that is primarily expressed on mature dendritic cells (DCs). A recombinant chCD83/IgG4 fusion protein containing the extracellular region of chCD83 was expressed in Chinese Hamster Ovary (CHO) cells and isolated from the spent cell culture medium by protein G affinity chromatography. The extracellular region of the chCD83 protein was purified and used to immunize mice. A cell fusion was performed, from which 342 hybridomas were screened for mAbs to chCD83. Two mAbs, chCD83-159 and chCD83-227, stained the greatest percentage of chCD83-transfected CHO cells and were selected for further characterization. By flow cytometry, both mAbs reacted with a chicken macrophage cell line, HD11. Both mAbs also recognized a single 53 kDa protein on Western blots of lysates from lipopolysaccharide-stimulated spleen mononuclear cells or unstimulated HD11 cells. Immunostaining of chicken secondary lymphoid organs identified chCD83(+) cells with morphologic and subtissue localization properties comparable to mammalian DCs. In vitro stimulation of spleen mononuclear cells with concanavalin A (Con A) decreased the percentage of chCD83(+) cells compared with cells treated with medium alone. Interestingly, spleen cells treated with Con A in the presence of chCD83-227 mAb exhibited decreased percentage of MHCII(+) cells compared with cells treated with an isotype-matched negative control mAb. These chCD83 mAbs may be useful for future investigations of chicken immune cell maturation and mechanisms of action.  相似文献   

15.
为研究马身猪体细胞的生物学特性,试验以3日龄马身猪耳缘组织为材料,采用组织块贴壁法建立马身猪耳缘组织成纤维细胞系,并对体外获得的细胞系进行生物学特性检测。结果表明,获得的马身猪耳缘组织成纤维细胞系符合成纤维细胞的基本特征,细胞贴壁生长,呈典型的梭形、星形和多边形,细胞汇合成单层的时间为10~13 d,生长曲线呈S型,中期染色体二倍体（2n=38）占主体,约占细胞总数的84%,符合细胞建系的要求;脂质体（LipofectamineTM 2000）转染绿色荧光蛋白质粒（pEGFP-C1）的转染效率为25%。马身猪耳缘组织成纤维细胞系的成功建立为地方猪种遗传资源的保护开辟了新的方法。  相似文献   

16.
  相似文献   

17.
Bovine peripheral blood mononuclear cells (PBM's) were depleted of monocytes by three techniques: plastic adherence, passage through Sephadex G-10, and carbonyl iron treatment followed by buoyant density separation over Ficoll-Hypaque (FH). Although the resulting cell populations differed in their T and B cell ratios and percentages of residual monocytes, these preparations were generally more responsive to phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM) than control cells containing monocytes. Passage of PBM's over a column of Sephadex G-10 and subsequent negative selection on plastic dishes previously coated with F(ab′)2 anti-immunoglobulin or peanut agglutinin (PNA) resulted in highly enriched populations of T cells bearing receptors for PNA (99% PNAR+) and B cells (84% surface-immunoglobulin+, 10% PNAR+, 6% null), respectively. The percentage of monocytes remaining in either cell preparation was less than 0.1%. Reactions of these isolated lymphocyte subpopulations demonstrated that bovine T cells can be strongly activated by PHA, Con A and PWM without apparent need for auxiliary B cells or monocytes. Stimulation of T and B cell populations with PWM produced a pattern of reactivity which was interpreted to indicate that PWM may also activate B cells slightly, perhaps requiring T cell help. The use of this simple panning technique for lymphocyte separation, with its large capacity and specificity, has general application to the further study of cellular interactions.  相似文献   

18.
Two mouse monoclonal IgM antibodies, B.1 and B.2, have been produced using the mouse myeloma cell line Sp2/0-Ag 14 and spleen cells from mice immunized with chicken bursa cells. The binding of the monoclonal antibodies to cells in suspension or tissue sections was demonstrated by means of the unlabeled peroxidase-antiperoxidase method. B.1 recognizes 61% of the bursa cells, 10-14% of the cells of spleen and of the peripheral mononuclear blood leukocytes and 1% of the thymus cells. The B.1+ cells are regarded as B cells. Their location in tissue sections corresponds with the known B-dependent areas of lymphoid organs. Competitive binding and double marker experiments proved that the B.1 antigen is distinct from surface immunoglobulin (Ig). In the bursa all B.1+ cells are also Ig+, whereas in the thymus, spleen and blood only about 90% of the B.1+ cells show this conformity. B.2 mainly recognizes so called reticular epithelial and reticular cells of the bursa (36%), thymus (20%) and spleen (13%). The B.2+ cells represent the second major cell population of the bursa.  相似文献   

19.
Long-term growth of T cell cultures requires addition of Interleukin 2 (IL-2). In order to maintain bovine cultures, optimal conditions for bovine IL-2 production were defined using peripheral blood mononuclear cells (PBM). Irradiation and preculture enhanced IL-2 production possibly by reducing suppressor activity. IL-2 activity was also detected in Bovine Herpesvirus Type 1-stimulated cultures. Unlike mitogen-stimulated cultures, a wide variation in IL-2 activity was seen between supernatants produced by virus-stimulated cells from different animals indicating the clonal nature of antigen specific cells from individuals. Bovine IL-2-dependent cells used to quantitate IL-2 activity were characterized as: PNA, esterase negative, H4+ (anti Ia-like), B29+ (anti-pan T cell), and C5- (anti-monocyte). The observations that bovine IL-2 can maintain activated murine cells, CTLL-20 and HT-2, could lead to the replacement of rat IL-2 with bovine IL-2 in long-term murine cultures. Conditions described here result in large volumes of active medium.  相似文献   

20.
The ability of small RNA interference (RNAi) to reduce specific gene expression was tested using interleukin-10 (IL-10) and interferon-gamma (IFN-gamma) production by cultured swine blood mononuclear cells stimulated by Escherichia coli lipopolysaccharide or concanavalin A. Antisense (AS) phosphorothioate oligodeoxynucleotides (ODNs) corresponding to a sequence in the region of the AUG initiation codon of swine IL-10 or IFN-gamma mRNA inhibited production of IL-10 (>or=93.5%) and IFN-gamma (>or=99%) mRNAs. Interleukin-10 and IFN-gamma protein production was inhibited more than 95% by the AS ODNs. Scrambled and sense ODNs RNAi used as negative controls did not alter mRNA expression for either cytokine but slightly reduced IL-10 protein production. Cytokine-specific and control RNAi did not inhibit beta(2)-microglobulin mRNA expression in mitogen-stimulated blood mononuclear cells. Thus AS ODNs RNAi specifically inhibit expression of pig IL-10 and IFN-gamma mRNAs by cultured, mitogen-stimulated blood mononuclear cells and may be an attractive alternative method for studying cytokine function.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号