共查询到20条相似文献,搜索用时 15 毫秒
1.
2.
Amplification of human immunoglobulin has many potential applications such as analysis of clonality, isolation of immunogenic antigens and antigen-specific immunotherapy. Here we describe a method for amplification of human immunoglobulin heavy and light chains from single B lymphocytes or plasma cells. Cells are isolated by FACS, and Ig is amplified by semi-nested RT–PCR. The method is versatile, sensitive and reliable: it provides appropriately paired heavy and light chains, requiring as little as 2 days to produce amplified Fab DNA from human tissues. 相似文献
3.
A distinct subset of human CD4+ cells with a limited alloreactive T cell receptor repertoire 总被引:5,自引:0,他引:5
Y Morishita H Sao J A Hansen P J Martin 《Journal of immunology (Baltimore, Md. : 1950)》1989,143(9):2783-2789
We describe a subset of CD4+/CD3+ human T lymphocytes that demonstrated a remarkably limited TCR repertoire responding to alloantigen stimulation. These cells have been characterized previously by their granular morphology and expression of CD11b but not CD28. Whereas multiple CD28+/CD4+ alloproliferative cloned cell lines generated by culture at limiting dilution immediately after isolation from peripheral blood each had a unique TCR-beta gene rearrangement, 19 of 21 CD11b+/CD4+ clones showed identical TCR-beta, and gamma gene rearrangements. In conventional MLR, the CD11b+/CD4+ cells responded poorly after stimulation with some HLA-class II Ag, and staining with a TCR Id-specific antibody and DNA blot hybridization suggested that the responding CD11b+/CD4+ cells typically contained predominant clonal populations. Clones of CD11b+/CD4+ cells with different TCR gene rearrangements showed closely similar patterns of responses when stimulated by a panel of allogeneic PBMC, but the response pattern did not correspond to that of any known HLA-class II Ag. These findings indicate that CD11b+/CD4+ cells have a limited alloproliferative repertoire characterized by predominant recognition of a limited number of undefined determinants that appear to be expressed in association with multiple distinct HLA-class II Ag. Our results suggest that CD11b+/CD4+ cells are selected for clonal reactivity by processes distinct from those for CD28+/CD4+ cells. 相似文献
4.
N Hashimoto K Takatsu Y Masuho K Kishida T Hara T Hamaoka 《Journal of immunology (Baltimore, Md. : 1950)》1984,132(1):129-135
A covalent conjugate of avidin with ricin subunit A-chain (avidin-RA) was prepared by using N-succinimidyl 3-(2-pyridyldithio)propionate as a coupling agent. Selective cytotoxic activity after the combined treatment of spleen cells with biotinylated antibody and avidin-RA was demonstrated by the fact that the responsiveness to LPS was selectively abrogated by pretreatment of the cells with biotinylated rabbit anti-mouse immunoglobulin (MIg) antibody, but not with biotinylated anti-Thy-1.2 antibody. Neither the biotinylated antibody alone nor avidin-RA alone was effective in decreasing the responses to mitogens. Moreover, a high anti-DNP PFC response elicited by DNP-KLH-primed BALB/c mouse spleen cells stimulated in vitro with DNP-KLH was mostly abrogated by the pretreatment of the cells with biotinylated anti-MIg antibody and avidin-RA. Again, neither the biotinylated antibody alone nor avidin-RA alone was effective in decreasing the anti-DNP PFC response. This cell-killing method with the use of biotinylated antibody and avidin-RA was applied and evaluated in experimental systems in which the helper action of T cells on B cells was mediated by T cell-replacing factor (TRF) or was performed by the direct interaction of T cells with B cells (cognate interaction). When DNP-KLH-primed splenic B cells, pretreated with biotinylated F(ab')2 fragment of DCF1 male anti-BALB/c-B IgG antibody against acceptor site(s) for TRF followed by treatment with avidin-RA, were stimulated with DNP-OVA in the presence of monoclonal TRF, the anti-DNP PFC response was significantly decreased, whereas the same treated B cells responded well to stimulation with DNP-PPD in the presence of Tbc-primed T cells (cognate interaction). These results indicate that B cells responsible for the cognate interaction and those having TRF acceptor site(s) belong to a distinct subpopulation of B cells, and that the cytocidal action of the noncovalent conjugate of the antibody and RA formed from the biotinylated antibody and avidin-RA via an avidin-biotin complex has immunologic selectivity, eliminating only the latter subset of B cells recognized by the antibody. 相似文献
5.
Effect of T cell-derived lymphokines containing B cell differentiation factor(s) for IgG (BCDF gamma) on gamma-specific mRNA in murine B cells 总被引:5,自引:0,他引:5
S Jones Y W Chen P Isakson J Layton E Pure C Word P H Krammer P Tucker E S Vitetta 《Journal of immunology (Baltimore, Md. : 1950)》1983,131(6):3049-3051
Cytoplasmic RNA was isolated from cells cultured with LPS and LPS plus a T cell-derived supernatant (SN) (PK 7.1) containing B cell differentiation factors. The steady state levels of isotype-specific mRNA were assessed by Northern blot analysis with gamma-specific CH3 probes. It was demonstrated that the SN induces an increase in the level of mRNA for gamma 1 and a concomitant decrease in the levels of mRNA for gamma 2b and gamma 3. 相似文献
6.
C M Balch P A Dougherty L B Vogler E W Ades S Ferrone 《Journal of immunology (Baltimore, Md. : 1950)》1978,121(6):2322-2328
A newly defined human B cell differentiation antigen, designated as BDA, has been defined and partially characterized. BDA is expressed on normal human B cells and lymphocytic leukemia cells at all stages of known differentiation (pre-B cells to plasma cells). It is distinct from DR (Ia-like) determinants and other known B cell surface constituents. 相似文献
7.
A lectin activity that selectively induces different functions of human lymphocytes has been described in a PBS crude extract obtained from the seeds of Artocarpus integrifolia (jackfruit). Both unfractionated peripheral blood mononuclear cells and purified T cells are strongly stimulated to proliferate by this extract, whereas purified B cells are not. However, the lectin induced a potent polyclonal activation of B cells measured by a reverse hemolytic plaque assay using a multivalent anti-human Fab antibody. 相似文献
8.
D L Kastner T M McIntyre C P Mallett A B Hartman A D Steinberg 《Journal of immunology (Baltimore, Md. : 1950)》1989,143(8):2761-2767
This study examines Ig VH utilization in murine lupus with emphasis on the relative contribution of 3' and 5' gene families. We used in situ hybridization with 35S-labeled ssRNA probes to detect VH expression in individual spleen cells. Cells were taken from unmanipulated animals, and were not stimulated in vitro. This approach allows analysis of VH usage among only those B cells which have undergone activation in vivo, while minimizing the potential for skewing in vitro. We compared usage of the 3' 7183 and Q52 families with the more 5' J558 family in adult NZB, MRL-lpr/lpr, and nonautoimmune NIH Swiss mice. VH utilization in the autoimmune strains was proportionate to VH family size, and was not biased toward the 3' families when compared with the Swiss repertoire. Moreover, 3' skewing did not develop in NZB mice with increasing age. Thus, systemic autoimmunity is not associated with impaired normalization of the adult repertoire away from the 3' bias of early ontogeny. Instead, our data support a stochastic model for VH gene usage in the activated B cells and plasma cells of adult lupus mice. 相似文献
9.
Maës J Caspi Y Rougeon F Haimovich J Goodhardt M 《Journal of immunology (Baltimore, Md. : 1950)》2000,165(2):703-709
It has recently become clear that recombination of Ig genes is not restricted to B cell precursors but that secondary rearrangements can also occur under certain conditions in phenotypically immature bone marrow and peripheral B cells. However, the nature of these cells and the regulation of secondary V(D)J recombination in response to B cell receptor (BCR) stimulation remain controversial. In the present study, we have analyzed secondary light chain gene rearrangements and recombination activating gene (RAG) expression in the surface IgM+, IgD- murine B cell line, 38C-13, which has previously been found to undergo kappa light chain replacement. We find that 38C-13 cells undergo spontaneous secondary Vkappa-Jkappa and RS rearrangements in culture, with recombination occurring on both productive and nonproductive alleles. Both 38C-13 cells and the Id-negative variants express the RAG genes, indicating that the presence of RAG does not depend on activation via the 38C-13 BCR. Moreover, BCR cross-linking in 38C-13 cells leads to a rapid and reversible down-regulation of RAG2 mRNA. Therefore, 38C-13 cells resemble peripheral IgM+, IgD- B cells undergoing light chain gene rearrangement and provide a possible in vitro model for studying peripheral V(D)J recombination. 相似文献
10.
The mode of action of T-cell-suppressor factor (TsF) induced by ultraviolet B (UVB) preirradiation in terms of interaction with several cytokines was studied. Suppression of murine contact photosensitivity (CPS) to 3,3',4',5-tetrachlorosalicylanilide (TCSA) by preirradiation of the sensitizing site to low doses of UVB was caused by antigen-specific suppressor T cells (Ts) and was not associated with the generation of efferent limb-acting suppressor cells. TsF released by Ts inhibited the proliferation of immune lymph node (LN) cells in vitro and reduced interleukin (IL)-2 production of these cells in an antigen-specific fashion without affecting the IL-2 receptor (IL-2R) expression. Both rIL-2 and rGM-CSF have the ability to restore CPS responses in the UVB-preirradiated mice when administered after but not before photosensitization. However, rIL-2 but not rGM-CSF counteracted the in vivo inhibitory effect of TsF. rGM-CSF did not affect the density of I-A+ epidermal Langerhans cells (LCs). It was suggested that TsF inhibited IL-2-mediated immune T-cell proliferation, while rGM-CSF reconstituted the CPS by enhancing the function of photodamaged LCs. These results indicate multiple steps of the UVB-induced immunosuppression circuit, each of which seems to be controlled by different immunomodulators. 相似文献
11.
BCGFII activity on activated B cells of a purified murine T cell-replacing factor (TRF) from a T cell hybridoma (B151K12) 总被引:13,自引:0,他引:13
N Harada Y Kikuchi A Tominaga S Takaki K Takatsu 《Journal of immunology (Baltimore, Md. : 1950)》1985,134(6):3944-3951
Experiments were performed to examine a growth-promoting activity on B cells or B leukemic cells of T cell-replacing factor (TRF) produced by a murine T cell hybridoma (B151K12) which constitutively produces TRF. The cellfree supernatant (CFS) from B151K12 cells (B151-CFS) could induce terminal differentiation of pre-activated B cells or in vivo passaged chronic B leukemia cells, BCL1, into immunoglobulin-secreting cells, while it did not exert a nominal lymphokine activity such as BCGFI (now known as BSFpl), IL 2, or gamma-interferon. However, it promoted [3H]thymidine uptake of dextran sulfate (DXS)-stimulated normal B cells and in vivo passaged BCL1 cells, suggesting that it also has BCGFII activity. We tried extensively to purify and to separate the TRF active molecule from the BCGFII active molecule by using many types of purification procedures. The purification scheme consisted of ammonium sulfate precipitation, DEAE-cellulose chromatography, Blue-Sepharose chromatography, hydroxylapatite chromatography, and gel permeation with fast protein liquid chromatography (FPLC). It was revealed that the BCGFII active molecule was hardly separable from the TRF during the entire purification procedure. The TRF as well as BCGFII active materials were glycoprotein with an apparent m.w. of 50 to 60 Kd on gel permeation chromatography and 18 Kd on SDS-PAGE under reducing conditions. The BCGFII active materials were hardly separable from the TRF active one, even after a reverse-phase FPLC, in which both BCGFII and TRF activities were recovered in the fractions eluted at 44 to 48% acetonitrile in 0.1% trifluoroacetic acid (TFA). Furthermore, the absorption of TRF and BCGFII active materials by using BCL1 cells removed not only TRF but also BCGFII activity. Moreover, B cell-specific monoclonal antibody (9T1), which can preferentially block TRF-dependent plaque-forming cell responses, also inhibited the expression of BCGFII activity to BCL1 cells. Taking all of the results together, we conclude that the TRF from B151K12 cells promotes growth of appropriately activated, such as DXS-stimulated normal cells and BCL1 tumor cells. These results suggest that B151-TRF may act on B cells as B cell growth and differentiation factors. 相似文献
12.
Secondary antigen stimulation usually produces IgG antibodies with hypermutated V segments. Studying a strong secondary response to the polynucleotide antigen poly(dC), however, we found a highly selective IgG antibody (mAb dC7) with only one mutation (a conservative Leu to Ileu substitution) throughout the whole VH domain. To investigate the roles of VH and VL domains in selective binding by this mAb, we prepared its VH, VL and single-chain Fv (scFv) fragments. A bacterial expression system produced soluble monomeric V region proteins. CD spectra confirmed that they had the beta-secondary structure expected for Ig domains. Both the scFv and VH fragments bound to single-stranded non-protonated poly(dC) and to ssDNA but not to protonated, more structured poly(dC) or dsDNA. The VL domain alone did not bind to nucleic acids, but VL association modified the VH binding, giving the scFv a 10-fold higher affinity than the VH for poly(dC) and greatly increasing the cytosine-dependent selectivity. Non-ionic interactions were prominent in the Fv reaction with a (dC)( n) sequence. Ionic interactions were revealed in Fv cross-reactions with ssDNA, and were more prominent in binding of either poly(dC) or ssDNA by VH alone, consistent with the lesser base selectivity of the VH. Thus, the Fv and VH alone bind to a single antigen, poly(dC), but mechanistic differences result from additional subsites in the Fv. Generation of a selective IgG with very few CDR mutations in either VH or VL, which was accompanied by IgM antibodies with unmutated V regions, also suggests that nucleic acid binding activity is a property of the B cell repertoire even before immunization. 相似文献
13.
D N Wang H T Chen J Liao P N Akolkar S K Sikder F Gruezo E A Kabat 《Journal of immunology (Baltimore, Md. : 1950)》1990,145(9):3002-3010
Nine groove-type mAb to alpha(1----6)dextran were cloned and sequenced. Together with previous reports from this laboratory, the VH and VL of 34 mAb have been sequenced, in which 10 VH19.1.2 and 11 VH9.14.7 combined with the V kappa-Ox1 gene to form two major families of anti-alpha(1----6)dextrans. The same D minigene (DFL16) was used by all VH19.1.2 and VH9.14.7 mAb; however, the patterns of JH and J kappa usage are quite different. VH19.1.2 mAb used only JH3 and J kappa 2, whereas VH9.14.7 mAb used three JH (JH1, JH2, and JH3) and all four active J kappa (J kappa 1, J kappa 2, J kappa 4, and J kappa 5). Relative uniformity in the lengths of VH CDR3 and the junctional sequences is seen in both families. Some mAb from different mouse strains share common structural features. The differences in idiotypic specificities and in the amino acid sequences suggest that VH19.1.2 and VH9.14.7 may differ in the conformation of CDR1 and CDR2. Combining with V kappa-Ox1 gene to generate groove-type combining sites to the single site-filling epitope of alpha(1----6)dextran, the two VH chains may require certain conformations of CDR3. Whether such conformational requirements influence the choice of J minigenes, the selection of the length of VH CDR3 and the sequences at junctions, are discussed. 相似文献
14.
Poonam Malhotra Manish Adhikari Saurabh Mishra Sumit Singh Piyush Kumar Shravan Kumar Singh 《Free radical research》2016,50(11):1265-1278
Radiation exposure to immune system induces imbalance in cytokines expression involved in Th1/Th2 homeostasis perturbations. In the present study, N-acetyl tryptophan glucoside (NATG), a bacterial secondary metabolite, was evaluated for its possible radioprotective potential to immune system using J774A.1 murine macrophages. In this study, expression of IFN-γ, TNF-α, IL-10, IL-2, IL-12, IL-13 and IL-17A cytokines was analyzed in irradiated and NATG pretreated cells using ELISA assay. Results of the study indicated that irradiated macrophages (NK-1R+?cells) pretreated with NATG showed higher (p?.05) survival at all observed time-intervals (2?h-48?h) as compared to irradiated (20Gy) cells that were not pretreated with NATG. However, NATG pretreatment to irradiated HEK293T cells (that did not express NK-1Receptor) did not provide significant survival, suggesting NK-1R involvement in NATG-mediated radioprotection. Cytokine expression analysis demonstrated that NATG pre-treated plus irradiated J774A.1 murine macrophages exhibited increased IFN-γ levels (~90%) with significant decrease in TNF-α at 24h as compared to irradiated cells. Further, significant decrease (~20%) in IL-10 and IL-2 (~26%) levels was observed in irradiated macrophages pretreated with NATG as compared to only irradiated cells. A sharp improvement in IL-17A (~92%) and IL-12 (~116%) expression was observed in irradiated macrophages pretreated with NATG as compared to only irradiated cells. Hence, NATG pre-treatment to irradiated macrophages induced IFN-γ, IL-17A and IL-12 expression, but suppresses TNF-α, IL-10 and IL-2 expressions. Conclusively, NATG pretreatment overcomes radiation-induced Th2 immune response by improving Th1 responsive cytoprotective cytokines IFN-γ, IL-17A and IL-12 in irradiated macrophages possibly by NK-1R antagonistic mechanism, and thus contributes to radioprotection. 相似文献
15.
16.
Rikke S?e Michael T Overgaard Anni R Thomsen Lisbeth S Laursen Inger M Olsen Lars Sottrup-Jensen Jesper Haaning Linda C Giudice Cheryl A Conover Claus Oxvig 《European journal of biochemistry》2002,269(8):2247-2256
Murine pregnancy-associated plasma protein-A (PAPP-A) cDNA encoding a 1545 amino-acid protein has been cloned. We have also identified and cloned cDNA that encodes a novel variant of PAPP-A, PAPP-Ai, carrying a 29-residue highly basic insert. The point of insertion corresponds to a junction between two exons in the human PAPP-A gene. The human intron flanked by these exons does not encode a homologous corresponding insert, which is unique to the mouse. The overall sequence identity between murine and human PAPP-A is 91%, and murine PAPP-A contains sequence motifs previously described in the sequence of human PAPP-A. Through expression in mammalian cells, we show that murine PAPP-A and PAPP-Ai are active metalloproteinases, both capable of cleaving insulin-like growth factor binding protein (IGFBP)-4 and -5. Cleavage of IGFBP-4 is dramatically enhanced by the addition of IGF, whereas cleavage of IGFBP-5 is slightly inhibited by IGF, as previously established with human PAPP-A. Surprisingly, however, quantitative analyses demonstrate that the murine PAPP-Ai cleaves IGFBP-4 very slowly compared to PAPP-A, even though its ability to cleave IGFBP-5 is unaffected by the presence of the insert. By RT-PCR analysis, we find that both variants are expressed in several tissues. The level of mRNA in the murine placenta does not exceed the levels of other tissues analyzed. Furthermore, the IGFBP-4-proteolytic activity of murine pregnancy serum is not elevated. This is in striking contrast to the increase seen in human pregnancy serum, and the expression of PAPP-A in the human placenta, which exceeds other tissues at least 250-fold. Interestingly, the position of the insert of PAPP-Ai, within the proteolytic domain, lies in close proximity to the cysteine residue, which in human PAPP-A forms a disulfide bond with the proform of eosinophil major basic protein (proMBP). ProMBP functions as a proteinase inhibitor in the PAPP-A-proMBP complex, but whether any mechanistic parallel on regulation of proteolytic activity can be drawn between the insert of PAPP-Ai and the linkage to proMBP is not known. Importantly, these data support the development of the mouse as a model organism for the study of PAPP-A, which must take into account the differences between the mouse and the human. 相似文献
17.
18.
19.
There is growing evidence which suggests that dysregulation of apoptosis may lead to several disease states including cancer. To investigate the mechanism controlling the induction of cell death, apoptosis defective/resistant (Apt-) mutants were isolated and characterized in this study. FDC-P1, a mouse myeloid cell line that depends upon IL-3 for survival and growth but undergoes apoptosis when deprived of growth factor, was mutagenized by treatment with ethyl methane sulfonate. We selected cells that survived the growth factor deprivation but did not grow without the factor. Surviving cells were cloned by limiting dilution and four clones that showed the least morphological characteristics and biochemical changes of apoptosis were chosen. Unlike the parent FDC-P1, these mutants were cross resistant to apoptosis induced by a variety of antitumor drugs such as Adriamycin, Dexamethasone, VP-16, as well as reactive oxygen species (ROS) generated by xanthine/xanthine oxidase (X/XO). We used one of these Apt- mutant to test candidate death genes. Our findings suggest that the preferential increase in Bax/Bcl-2 ratio, p53, c-Myc, Caspase-3 and decrease in AP-1 on treatment with various anticancer drugs may contribute to the preferential apoptotic response in FDC-P1 cells but to varying degrees. Whereas, the higher constitutive level of antioxidant enzymes superoxide dismutase and catalase in the Apt- mutant may contribute at least in part to its resistance. 相似文献
20.
Immunological evidence for the presence of B protein (apoprotein of beta-lipoprotein) in normal and abetalipoproteinemic plasma 总被引:3,自引:0,他引:3
R S Lees 《Journal of lipid research》1967,8(4):396-405
The antigenicity of -lipoprotein that had been chemically altered by acetylation or arsanilation was compared with that of native beta-lipoprotein, of lymph chylomicrons, of plasma proteins with d > 1.21, and of plasma from patients with abetalipoproteinemia. Chemical alteration causes structural changes in beta-lipoprotein which render it immunologically identical with a protein that is present both in normal plasma and in plasma from patients with abetalipoproteinemia. This protein has been identified by immunoelectrophoresis as abeta -globulin which does not stain for lipid. It is presumed to be the lipid-free apoprotein of beta-lipoprotein (B protein). The findings suggest that abetalipoproteinemia is not due to inability to synthesize B protein, but might instead be due to a defect in the formation of the complete beta-lipoprotein macro-molecule. 相似文献