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1.
目的为了进一步探索VGF的生物学功能方法分别构建了痘苗病毒天坛株生长因子(Vacciniagrowthfactor,VGF)基因细胞膜外结构域(VGFsl)的真核及原核表达系统,在相应的细胞中获得了单独表达(真核)和融合表达(原核)。结果初步纯化的VGFsl表达产物能够与上皮生长因子(EGF)的受体结合,并刺激受体中酪氨酸残基发生磷酸化反应。与EGF相比,VGFsl表达产物刺激EGF受体自身磷酸化的能力更强。结论上述结果为VGF在病毒感染过程中的作用以及VGF作为新型多肽类药物的研究提供了有价值的线索。 相似文献
2.
da Fonseca Fla′vio Guimaraes Alves Silva Ricardo Lui′s Marques Joao Trindade Peregrino Ferreira Paulo Ce′sar Kroon Erna Geessien 《Virus genes》1999,18(2):151-160
Cowpox virus (CPV) is a member of the Orthopoxvirus genus and has the genetic capacity to encode a multitude of genes that interfere with the host inflammatory and immune response or modulate the physiological state of infected and non-infected cells. Among these CPV factors are receptors homologous to interferon and tumor necrosis factor receptors and also a viral cellular serine-proteinase analog. Here we describe the detection of a CPV gene that encodes a protein homologous to epidermal growth factor, transforming growth factor alpha and poxvirus growth factors, such as the vaccinia growth factor (VGF). The VGF and other poxvirus growth factors are produced early in the infection and are secreted into the medium where they bind to the EGF receptors, generating mytotic responses. The cowpox growth factor (CGF) gene was detected in three copies on the virus genome by PCR, and by northern and southern blot hybridization using VGF nucleotide sequences as primers and probes. The CPV gene has a strong nucleotide and predicted amino acid similarity with VGF, and is also produced early in the infection. 相似文献
3.
To determine if equine monocyte-derived dendritic cells (DC) were susceptible to equine infectious anemia virus (EIAV) infection, ex vivo-generated DC were infected with virus in vitro. EIAV antigen was detected by immunofluorescence 3 days post-infection with maximum antigen being detected on day 4, whereas there was no antigen detected in DC incubated with the same amount of heat-inactivated EIAV. No cytolytic activity was observed after EIAV(WSU5) infection of DC. These monocyte-derived DC were more effective than macrophages and B cells in stimulating allogenic T lymphocytes. Both infected macrophages and DC stimulated similar levels of memory CTL responses in mixtures of CD8+ and CD4+ cells as detected with (51)Cr-release assays indicating that EIAV infection of DC did not alter antigen presentation. However, EIAV-infected DC were more effective than infected macrophages when used to stimulate memory CTL in isolated CD8+ cells. The maintenance of antigen processing and presenting function by EIAV-infected DC in vitro suggests that this function is maintained during in vivo infection. 相似文献
4.
Clonal analysis of T-cell responses to herpes simplex virus: isolation, characterization and antiviral properties of an antigen-specific helper T-cell clone. 总被引:3,自引:0,他引:3
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A herpes simplex virus (HSV)-specific long-term T-cell clone has been established from the draining lymph node cells of BALB/c mice; the cells required repeated in vitro restimulation with UV-irradiated virus. The established T-cell clone expresses the Thy-1 and Lyt-1+2,3- surface antigens. For optimal proliferation of the cloned cells, both the presence of specific antigen and an exogenous source of T-cell growth factor are required. The proliferative response of the cloned T cells was found to be virus-specific but it did not distinguish between HSV-1 and HSV-2. Adoptive cell transfer of the cloned T cells helped primed B cells to produce anti-herpes antibodies: the response was antigen-specific and cell dose-dependent. The clone failed to produce a significant DTH reaction in vivo, but did produce high levels of macrophage-activating factor. Furthermore, the T-cell clone could protect from HSV infection, as measured by a reduction in local virus growth, and by enhanced survival following the challenge of mice with a lethal dose of virus. The mechanism(s) whereby this clone protects in vivo is discussed. 相似文献
5.
6.
Chronic systemic treatment with epidermal growth factor in pigs causes pronounced urothelial growth with accumulation of glycoconjugates. 总被引:1,自引:1,他引:0
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L. Vinter-Jensen C. O. Juhl J. C. Djurhuus S. S. Poulsen E. Z. Dajani K. D. Brown T. F. Orntoft P. S. Teglbjaerg E. Nex 《The American journal of pathology》1995,147(5):1330-1338
Epidermal growth factor (EGF) is present in large amounts in the urine, but the effects of systemically administered EGF on the urinary tract have not been described previously. In the present paper, we describe a potent growth induction of EGF on the urinary tract. Goettingen minipigs were treated with solvent (n = 5), EGF 30 micrograms/kg/day (n = 6) for 4 weeks, or EGF 30 micrograms/kg/day for 5 weeks followed by 3 weeks of recovery (n = 5). The ureters and bladders were examined by routine histology and electron microscopy and were immunostained for proliferating cell nuclear antigen. Four weeks of EGF treatment increased the median cross sectional area of the ureter fourfold with growth of all wall layers. The urothelium was widened from 5 cell layers in the controls to 10 in the EGF-treated animals. Proliferating cell nuclear antigen immunostaining revealed an increased mitotic activity in the basal zone of the urothelium. In the luminal zone, glycoconjugates accumulated in goblet cells, in cells with intracytoplasmic lumina, and beneath the luminal cell membrane in the umbrella cells. Our studies present a new experimental approach to growth induction of the urinary tract. The findings implicate the EGF system in regulating urothelial growth and glycoconjugate biosynthesis. 相似文献
7.
Döpper S Wilflingseder D Prodinger WM Stiegler G Speth C Dierich MP Stoiber H 《European journal of immunology》2003,33(8):2098-2107
Resting CD4(+) T cells in the lymphoid tissue (LT) are essential producers of virions at the beginning of HIV infection in vivo. We previously developed a model that allowed in vitro infection of non-prestimulated T lymphocytes in the presence of autologous B lymphocytes and complement. In this study, we try to clarify the mechanism(s) responsible for virus transmission in unstimulated autologous B cell/T cell co-cultures. Ex vivo analyses of patient plasma samples revealed that HIV was opsonized. Flow cytometry showed that opsonized virus preferentially bound to complement receptor (CR)-2 on B lymphocytes in primary B cell/T cell co-cultures. As indicated by cytokine measurements and transwell experiments, soluble factors seemed to play a minor role in enabling infection. Rather, direct interaction between B and T lymphocytes and direct binding of opsonized virus to CR2 on B cells turned out to be essential for productive infection. Antibodies blocking cell-cell adhesion inhibited p24 antigen production. An anti-CR2 antibody blocking C3d-CR2 binding also significantly reduced viral replication. Since the infection of unstimulated T cells by opsonized primary HIV isolates in the presence of B cells was highly efficient independent of the tropism of the virus, this mechanism may be critical in the pathogenesis of HIV. 相似文献
8.
The effects of growth factors on human normal placental cytotrophoblast cell proliferation 总被引:7,自引:1,他引:6
The effects of growth factors were investigated on the proliferation of a
normal placental cytotrophoblast cell line (NPC). Epidermal growth factor
(EGF), transforming growth factor alpha (TGF alpha) and insulin- like
growth factor-I (IGF-I) stimulated NPC cell proliferation. In contrast,
TGFbeta1 was found to be a negative regulator, inhibiting EGF- induced cell
proliferation. When EGF/TGF alpha receptor was analysed by radio-ligand
binding, two binding sites of different affinities were revealed in the
proliferating NPC cells but only the low affinity binding site was detected
in the non-proliferating cytotrophoblast cells in primary cultures. The
results suggest that EGF stimulates cytotrophoblast proliferation through
high affinity binding sites.
相似文献
9.
Pituitary carcinomas are only defined by their metastatic growth, which may be intracranial or systemic. To establish further morphological and immunohistochemical differences between pituitary carcinomas and adenomas, 19 ACTH-secreting adenomas (10 non invasive and 9 invasive) and 2 ACTH-secreting carcinomas with their metastases were studied for expression of the intermediate filaments keratin and vimentin and the tumor-associated antigens Ki67, proliferating cell nuclear antigen (PCNA), epidermal growth factor (EGF), cathepsin D, p53, and carcinoembryonic antigen (CEA). Immunohistochemistry was performed using avidin-biotin techniques on formalin-fixed, paraffin-embedded tissue. With the exception of one noninvasive pituitary adenoma, one carcinoma, and the metastases, all tumors contained keratin; none contained vimentin. All tumors stained negative for CEA and p53. Eleven (58.5%) adenomas and both pituitary carcinomas contained Ki67-positive nuclei; 14 (74%) adenomas and one carcinoma revealed PCNA. No correlation was found between the two markers. Seven (38%) adenomas showed a labeling index <1 % for cathepsin D, whereas none of the carcinomas or metastases did so. EGF was found in 7 (38%) adenomas and in both carcinomas. A tendency to a higher rate of EGF positivity in the invasive adenomas was observed. The metastases showed a higher labeling index, and far more intense staining results for Ki67, PCNA, and EGF than the primary tumor. The metastases also had a higher proliferation rate and growth factor content than the carcinoma itself. 相似文献
10.
In vivo expression of major histocompatibility complex molecules on oligodendrocytes and neurons during viral infection
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Demyelination in multiple sclerosis and in animal models is associated with infiltrating CD8+ and CD4+ T cells. Although oligodendrocytes and axons are damaged in these diseases, the roles T cells play in the demyelination process are not completely understood. Antigen-specific CD8+ T cell lysis of target cells is dependent on interactions between the T cell receptor and major histocompatibility complex (MHC) class I-peptide complexes on the target cell. In the normal central nervous system, expression of MHC molecules is very low but often increases during inflammation. We set out to precisely define which central nervous system cells express MHC molecules in vivo during infection with a strain of murine hepatitis virus that causes a chronic, inflammatory demyelinating disease. Using double immunofluorescence labeling, we show that during acute infection with murine hepatitis virus, MHC class I is expressed in vivo by oligodendrocytes, neurons, microglia, and endothelia, and MHC class II is expressed only by microglia. These data indicate that oligodendrocytes and neurons have the potential to present antigen to T cells and thus be damaged by direct antigen-specific interactions with CD8+ T lymphocytes. 相似文献
11.
IL-12 enhances the generation of tumour antigen-specific Th1 CD4 T cells during ex vivo expansion
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CD4+ T cells are essential for the immune response against cancer. Vaccination against cancer will likely only be effective at preventing growth of micrometastatic disease while adoptive T cell therapy will be better suited for eradication of bulky pre-existing disease (Knutson et al. Expert Opin Biol Ther 2002; 2:55-66). Problems with the use of adoptive T cell therapy include lack of CD4+ T cell help, low frequency of antigen-specific T cells, and lack of effective ex vivo expansion techniques. In this study, we focused on improving ex vivo expansion of CD4+ T helper cells. The effects of IL-12, along with IL-2, on the ex vivo generation of HER-2/neu antigen-specific T cells were examined. Patients were immunized with a peptide-based vaccine that contained a helper epitope, p776-790, derived from the intracellular domain of HER-2/neu. While T cell immunity to p776-790, assessed by proliferation assays, could be readily measured in short-term cultures, cell line generation by multiple in vitro stimulation with peptide and IL-2 as the only added cytokine resulted in loss of antigen-specific proliferation. The inclusion of IL-12, along with IL-2, restored antigen-specific proliferation in a dose-dependent fashion. The resulting p776-790-specific T cells responded readily to antigen by proliferating and producing type I cytokines (IFN-gamma and TNF-alpha). The increased proliferative response of the cultures was due in part to an increase in the number of HER-2/neu-specific T cells. These results suggest that IL-12 is an important cytokine for ex vivo recovery and maintenance of antigen-specific CD4+ T lymphocytes that would otherwise be lost by using IL-2 alone in combination with antigen. Furthermore, these results have important implications for ex vivo expansion of CD4+ T cell for use in anti-tumour adoptive immunotherapy. 相似文献
12.
Simian immunodeficiency virus (SIV)/immunoglobulin G immune complexes in SIV-infected macaques block detection of CD16 but not cytolytic activity of natural killer cells
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Wei Q Stallworth JW Vance PJ Hoxie JA Fultz PN 《Clinical and Vaccine Immunology : CVI》2006,13(7):768-778
Natural killer cells are components of the innate immune system that play an important role in eliminating viruses and malignant cells. Using simian immunodeficiency virus (SIV) infection of macaques as a model, flow cytometry revealed a gradual loss of CD16+ NK cell numbers that was associated with disease progression. Of note, the apparent loss of NK cells was detected in whole-blood samples but not in isolated peripheral blood mononuclear cells (PBMC), suggesting that an inhibitor(s) of the antibody used to detect CD16, the low-affinity immunoglobulin G (IgG) receptor, was present in blood but was removed during PBMC isolation. (Actual decreases in CD16+ cell numbers in PBMC generally were not detected until animals became lymphopenic.) The putative decrease in CD16+ cell numbers in whole blood correlated with increasing SIV-specific antibody titers and levels of plasma virion RNA. With the addition of increasing amounts of plasma from progressor, but not nonprogressor, macaques to PBMC from an uninfected animal, the apparent percentage of CD16+ cells and the mean fluorescence intensity of antibodies binding to CD16 declined proportionately. A similar decrease was observed with the addition of monomeric IgG (mIgG) and IgG immune complexes (IgG-ICs) purified from the inhibitory plasma samples; some of the ICs contained SIV p27(gag) antigen and/or virions. Of interest, addition of purified IgG/IgG-ICs to NK cell lytic assays did not inhibit killing of K562 cells. These results indicate that during progressive SIV and, by inference, human immunodeficiency virus disease, CD16+ NK cell numbers can be underestimated, or the cells not detected at all, when one is using a whole-blood fluorescence-activated cell sorter assay and a fluorochrome-labeled antibody that can be blocked by mIgG or IgG-ICs. Although this blocking had no apparent effect on NK cell activity in vitro, the in vivo effects are unknown. 相似文献
13.
A 34-kd protein with strong homology to ras-like proteins inhibits epidermal growth factor activity.
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Epidermal growth factor (EGF) and its analog, transforming growth factor-alpha, are felt to be important in oncogenesis. When malignant rabbit fibroma virus infects RK-13 rabbit kidney cells, a 34-kd protein that inhibits the effects of EGF on certain target cell lines is produced. We have purified this protein using high-pressure liquid chromatography and gel electrophoresis. This purified protein abolishes EGF-induced cellular proliferation. It also causes the EGF receptor-bearing A431 carcinoma cell line to stop proliferating in vitro. This purified 34-kd EGF inhibitor (EGFI) redirects cellular protein phosphorylation in the presence or absence of EGF. Whereas EGF increases phosphorylation of cellular proteins in normal rat kidney cells, clone 49F, and A431 EGFI generally decreases it. Both EGF and EGFI cause increased protein production in A431 and normal rat kidney cells. The major species of protein synthesized by cells seem invariant to EGFI, with or without EGF. The partial protein sequence of two fragments of EGFI shows striking similarity to two ras like proteins. Possible means by which such a ras-like protein might inhibit EGF-induced cellular proliferation are discussed. Therefore, a purified 34-kd ras-like protein inhibits EGF-induced cellular proliferation and changes the targets for cellular protein phosphorylation. Studies are in progress to characterize this protein further, both structurally and functionally. 相似文献
14.
Salmonella typhimurium displays normal invasion of mice with defective epidermal growth factor receptors. 总被引:2,自引:0,他引:2
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The role of the epidermal growth factor (EGF) receptor in cell invasion by Salmonella typhimurium was examined in vitro and in vivo by using waved-2 mice which express an EGF receptor with reduced kinase activity. S. typhimurium invaded fibroblasts from waved-2 mice as efficiently as fibroblasts from wild-type control animals. In vivo, S. typhimurium both invaded the gastrointestinal tract and penetrated through to the spleen of waved-2 mice. Our studies suggest that the EGF receptor has only a limited role, if any, in cell invasion by S. typhimurium. 相似文献
15.
The IA antigen is not the major receptor for lactate dehydrogenase-elevating virus on macrophages from CBA and BALB/c mice 总被引:2,自引:0,他引:2
Double staining and labeling procedures were employed to simultaneously identify IA+ cells and cells permissive for the replication of the lactate dehydrogenase-elevating virus (LDV) in populations of peritoneal and spleen macrophages from BALB/c and CBA/J mice. No correlation between the expression of IA antigen and LDV permissiveness was observed. Only a low proportion of resident peritoneal macrophages expressed IA antigen and the antigen was lost within 1-2 days in culture whether or not L cell-conditioned medium was present, whereas the cells retained undiminished LDV permissiveness for 4 days and longer. Induction of IA expression on macrophages by injection of mice with concanavalin A, starch or indomethacin (up to 50% of the total macrophages became IA+), or elimination of IA+ macrophages by treatment with anti-IA monoclonal antibodies plus complement had little or no effect on the ability of the cells to support LDV replication in vivo or in vitro. LDV infection of untreated or concanavalin A-treated or starch-treated mice caused a drastic decline in IA+ peritoneal macrophages within 1 day, but the number of IA+ macrophages returned to pre-infection levels by 7 days post-infection without rendering the cells LDV permissive. Treatment of macrophages with trypsin destroyed the LDV receptor on macrophages with minimal loss of IA antigen from the cells. We conclude that the IA antigen is not the major receptor for infection of macrophages from BALB/c or CBA mice by LDV. 相似文献
16.
This study reports the examination of in vivo and in vitro properties of an antigen-dependent murine cytotoxic T cell (Tc) clone T5/5 specific for type A influenza virus. This clone differs morphologically and in its migratory pattern and biological properties from a previously examined anti-influenza Tc clone L4 which grew in T cell growth factor independently of antigen. Unlike Tc clone L4, T5/5 cells do not release significant amounts of immune interferon on contact with influenza-infected target cells nor do they limit virus replication in vivo, although they efficiently lyse influenza-infected target cells and can release interferon in the presence of concanavalin A. Thus individual Tc clones vary in function and further work is required to establish the properties of Tc that are associated with host protection against virus infection. 相似文献
17.
18.
M Ito G G Fredrickson F Celada J L Schulman C A Bona T M Moran 《Journal of immunological methods》1987,103(2):229-237
Murine cytolytic T lymphocyte (CTL) clones specific for type A influenza virus antigens were generated by in vitro stimulation with syngeneic virus-infected cells in the presence of T cell growth factor (TCGF). All CTL clones recognize viral determinants shared by PR8 and X31 influenza viruses in association with a class I antigen, coded either by the H-2K or H-2D end of the appropriate haplotype. All clones express the Lyt2 antigen marker. Two of five clones also express an antigenic determinant of the V beta chain of the T cell receptor (TCR) identified by F23.1 monoclonal antibody. To effectively generate F23.1+ and antigen-specific CTL clones, heterogenous CTL lines were expanded with F23.1 coated Sepharose beads in the presence of TCGF and then stimulated with PR8 virus-infected cells. Thus, both the proliferative activity to PR8 and the expression of the F23.1 marker was increased significantly. Alternatively, F23.1+ T cells were sorted from in vivo primed mice and expanded with PR8 virus-infected stimulator cells in the presence of TCFG. This F23.1+ T cell line exhibited antigen-specific cytotoxicity for PR8 virus-infected target cells. Additionally, in an 'FcR-focused killing' assay only the F23.1+ CTL line and F23.1+ clones lysed Fc receptor bearing target cells in the presence of F23.1 antibody. These findings indicate that antigen-specific and F23.1+ clones can be selected with high efficiency by alternating stimulation with influenza virus-infected cells and with F23.1-coated Sepharose beads or through the use of a cytofluorograph. The usefulness of antigen-specific and F23.1+ CTL clones and other possible strategies for their selection are discussed. 相似文献
19.
Epidermal growth factor as a candidate for ex vivo expansion of bone marrow-derived mesenchymal stem cells 总被引:12,自引:0,他引:12
Bone marrow mesenchymal stem cells (BMMSCs) are pluripotent cells capable of differentiating into several cell types and are thus an attractive cell source for connective tissue engineering. A challenge in such a use is expansion and directed seeding in vitro, requiring proliferation and survival, and directed migration, respectively, prior to functional differentiation. The epidermal growth factor (EGF) receptor (EGFR) is the prototypal growth factor receptor and elicits these responses from a wide variety of stromal, epithelial, and endothelial cells. Ligands for this receptor are appealing for use in tissue engineering because they are relatively resistant to biological extremes and amenable to high-volume production. Therefore, we determined whether an EGFR ligand, EGF, could be used for ex vivo expansion of BMMSCs. EGF stimulated motility in rat and immortalized human BMMSCs. EGF-induced proliferation was observed in immortalized human BMMSCs but was not apparent in rat BMMSCs under our experimental conditions. EGF did not, however, rescue either type of BMMSC from apoptosis due to lack of serum. During our examination of key signaling intermediaries, EGF caused robust phosphorylation of extracellular signal-regulated protein kinase (ERK) and protein kinase B/akt (AKT) but only minimal phosphorylation of EGFR and phospholipase C-gamma in rat BMMSCs, whereas in the human BMMSCs these intermediaries were all strongly activated. EGF also induced robust ERK activation in primary porcine mesenchymal stem cells. EGF pretreatment or cotreatment did not interfere with secondarily induced differentiation of either type of BMMSC into adipogenic or osteogenic lineages. Platelet-derived growth factor (PDGF) effects were similar to but not additive with those elicited by EGF, with some quantitative differences; however, PDGF did interfere with the differentiation of these BMMSCs. These findings suggest that EGFR ligands could be used for ex vivo expansion and direction of BMMSCs. 相似文献
20.
Dendritic cells and viruses 总被引:3,自引:0,他引:3
Dendritic cells (DC) are potent at presenting viral antigens in the initiation of both primary and secondary responses. DC in the lymph nodes draining the site of infection with HSV express surface antigen and can stimulate proliferation of sensitised lymphocytes. In secondary stimulation, nonresponsiveness in cytotoxic T cell assays of cells from mice primed to Moloney virus was also overcome by stimulation in vitro with virus-pulsed DC. Marked primary proliferative and cytotoxic T cell responses were previously found only to alloantigens and to haptens, both presented on the surface of DC However, DC exposed in vitro or in vivo to influenza virus stimulated primary proliferative and cytotoxic T cell responses in normal syngeneic lymphocytes in 20-microliters hanging drop cultures (Macatonia, Taylor, Knight and Askonas, in press). This provides a method for analysing primary responses to viruses in vitro without the necessity of using pre-sensitised donors. Although DC may present HIV antigens to lymphocytes the DC are also susceptible to infection with HIV. This occurs in vivo as evidenced by the infection of Langerhans cells in AIDS patients. This infection of DC may not only compromise their function in antigen presentation but also act as a reservoir of virus which is handed on to T cells during the close clustering of the presenting cells with the T cells during the initiation of the immune response.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献