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41.
TH17 cells mediate steroid-resistant airway inflammation and airway hyperresponsiveness in mice 总被引:1,自引:0,他引:1
McKinley L Alcorn JF Peterson A Dupont RB Kapadia S Logar A Henry A Irvin CG Piganelli JD Ray A Kolls JK 《Journal of immunology (Baltimore, Md. : 1950)》2008,181(6):4089-4097
Steroid-resistant asthma comprises an important source of morbidity in patient populations. T(H)17 cells represent a distinct population of CD4(+) Th cells that mediate neutrophilic inflammation and are characterized by the production of IL-17, IL-22, and IL-6. To investigate the function of T(H)17 cells in the context of Ag-induced airway inflammation, we polarized naive CD4(+) T cells from DO11.10 OVA-specific TCR-transgenic mice to a T(H)2 or T(H)17 phenotype by culturing in conditioned medium. In addition, we also tested the steroid responsiveness of T(H)2 and T(H)17 cells. In vitro, T(H)17 cytokine responses were not sensitive to dexamethasone (DEX) treatment despite immunocytochemistry confirming glucocorticoid receptor translocation to the nucleus following treatment. Transfer of T(H)2 cells to mice challenged with OVA protein resulted in lymphocyte and eosinophil emigration into the lung that was markedly reduced by DEX treatment, whereas T(H)17 transfer resulted in increased CXC chemokine secretion and neutrophil influx that was not attenuated by DEX. Transfer of T(H)17 or T(H)2 cells was sufficient to induce airway hyperresponsiveness (AHR) to methacholine. Interestingly, AHR was not attenuated by DEX in the T(H)17 group. These data demonstrate that polarized Ag-specific T cells result in specific lung pathologies. Both T(H)2 and T(H)17 cells are able to induce AHR, whereas T(H)17 cell-mediated airway inflammation and AHR are steroid resistant, indicating a potential role for T(H)17 cells in steroid-resistant asthma. 相似文献
42.
Screening random peptide libraries with subacute sclerosing panencephalitis brain-derived recombinant antibodies identifies multiple epitopes in the C-terminal region of the measles virus nucleocapsid protein 下载免费PDF全文
Owens GP Shearer AJ Yu X Ritchie AM Keays KM Bennett JL Gilden DH Burgoon MP 《Journal of virology》2006,80(24):12121-12130
Infectious and inflammatory diseases of the CNS are often characterized by a robust B-cell response that manifests as increased intrathecal immunoglobulin G (IgG) synthesis and the presence of oligoclonal bands. We previously used laser capture microdissection and single-cell PCR to analyze the IgG variable regions of plasma cells from the brain of a patient with subacute sclerosing panencephalitis (SSPE). Five of eight human IgG1 recombinant antibodies (rAbs) derived from SSPE brain plasma cell clones recognized the measles virus (MV) nucleocapsid protein, confirming that the antibody response in SSPE targets primarily the agent causing disease. In this study, as part of our work on antigen identification, we used four rAbs to probe a random phage-displayed peptide library to determine if epitopes within the MV nucleocapsid protein could be identified with SSPE brain rAbs. All four of the SSPE rAbs enriched phage-displayed peptide sequences that reacted specifically to their panning rAb by enzyme-linked immunosorbent assay. BLASTP searches of the NCBI protein database revealed clear homologies in three peptides and different amino acid stretches within the 65 C-terminal amino acids of the MV nucleocapsid protein. The specificities of SSPE rAbs to these regions of the MV nucleocapsid protein were confirmed by binding to synthetic peptides or to short cDNA expression products. These results indicate the feasibility of using peptide screening for antigen discovery in central nervous system inflammatory diseases of unknown etiology, such as multiple sclerosis, neurosarcoidosis, or Behcet's syndrome. 相似文献
43.
Differential Selection of Cells with Proviral c-myc and c-erbB Integrations after Avian Leukosis Virus Infection 下载免费PDF全文
Min Gong Helen L. Semus Kelly J. Bird Brian J. Stramer Alanna Ruddell 《Journal of virology》1998,72(7):5517-5525
Avian leukosis virus (ALV) infection induces bursal lymphomas in chickens after proviral integration within the c-myc proto-oncogene and induces erythroblastosis after integration within the c-erbB proto-oncogene. A nested PCR assay was used to analyze the appearance of these integrations at an early stage of tumor induction after infection of embryos. Five to eight distinct proviral c-myc integration events were amplified from bursas of infected 35-day-old birds, in good agreement with the number of transformed bursal follicles arising with these integrations. Cells containing these integrations are remarkably common, with an estimated 1 in 350 bursal cells having proviral c-myc integrations. These integrations were clustered within the 3′ half of c-myc intron 1, in a pattern similar to that observed in bursal lymphomas. Bone marrow and spleen showed a similar number and pattern of integrations clustered within 3′ c-myc intron 1, indicating that this region is a common integration target whether or not that tissue undergoes tumor induction. While all tissues showed equivalent levels of viral infection, cells with c-myc integrations were much more abundant in the bursa than in other tissues, indicating that cells with proviral c-myc integrations are preferentially expanded within the bursal environment. Proviral integration within the c-erbB gene was also analyzed, to detect clustered c-erbB intron 14 integrations associated with erythroblastosis. Proviral c-erbB integrations were equally abundant in the bone marrow, spleen, and bursa. These integrations were randomly situated upstream of c-erbB exon 15, indicating that cells carrying 3′ intron 14 integrations must be selected during induction of erythroblastosis. 相似文献
44.
The gene for autosomal dominant spinocerebellar ataxia (SCA1) maps telomeric to the HLA complex and is closely linked to the D6S89 locus in three large kindreds 总被引:16,自引:12,他引:4 下载免费PDF全文
Huda Y. Zoghbi Carla Jodice Lodewijk A. Sandkuijl Thomas J. Kwiatkowski Alanna E. McCall Sally A. Huntoon Patrizia Lulli Maria Spadaro Michael Litt Howard M. Cann Marina Frontali Luciano Terrenato 《American journal of human genetics》1991,49(1):23-30
We studied three large kindreds with the HLA-linked form of spinocerebellar ataxia (SCA1) in order to localize the SCA1 locus on the short arm of chromosome 6 (6p). Two loci containing highly informative dinucleotide repeat sequences were used for linkage analysis. These two loci are D6S89, which is telomeric to the HLA region, and T complex-associated testes-expressed 1 (TCTE1), centromeric to HLA. Pairwise linkage analysis of SCA1 and D6S89 revealed a maximum lod score of 5.86 in the Houston SCA1 (HSCA1) kindred and of 8.08 in the Calabrian SCA1 (SCA1) kindreds, at recombination fractions of .050 and .022, respectively. A maximum pairwise lod score of 4.54 at a recombination frequency of .100 was obtained for SCA1 and TCTE1 in the HSCA1 kindred. No evidence for linkage was detected between TCTE1 and SCA1 in the CSCA1 kindreds. Multilocus linkage analysis of SCA1, HLA, and D6S89 in all three kindreds provided strong evidence for localization of the SCA1 locus telomeric to the HLA regions. However, multilocus linkage analysis of SCA1, HLA, and TCTE1 with HSCA1 family genotypes indicated the possibility of a location of the SCA1 locus centromeric to HLA. An analysis of HSCA1 recombinants in this region of chromosome 6 revealed relatively high recombination frequencies between HLA and each of the other two markers and relatively low frequencies between the latter and SCA1, predicting that the SCA1 locus would tend to segregate away from HLA together with D6S89 or TCTE1, as found with the three-point linkage analyses for this family.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
45.
Wexler DS Gao L Anderson F Ow A Nadasdi L McAlorum A Urfer R Huang SG 《Journal of biomolecular screening》2005,10(4):383-390
Solubility and permeability are intimately linked in drug absorption processes. They have, however, been traditionally assayed separately. To support this linkage, a combined solubility/permeability assay was developed for determining absorption properties of chemical entities. First, solubility is determined at 4 pH values by comparing the concentration of a saturated compound solution to its dilute, known concentration. The filtered, saturated solution from the solubility assay is then used as input material for the membrane permeability determination. The permeability assay is a parallel artificial membrane technique whereby a membrane is created on a solid support parallel artificial membrane permeation assay (PAMPA). The 2 artificial membranes presented here model the gastrointestinal tract and the blood-brain barrier (BBB). Data are presented for control compounds, which are well documented in the literature and exemplify a range of solubility and membrane permeability. The advantages of the combination method are 1) reduction of sample usage and preparation time, 2) elimination of interference from compound precipitation in membrane permeability determination, 3) maximization of input concentration to permeability assay for improved reproducibility, and 4) optimization of sample tracking by streamlining data entry and calculations. BBB permeability ranking of compounds correlates well with literature CNS activity. 相似文献
46.
Previously, we have described a miniature protein-based approach to the design of molecules that bind DNA or protein surfaces with high affinity and specificity. In this approach, the small, well-folded protein avian pancreatic polypeptide acts as a scaffold to present and stabilize an alpha-helical or PPII-helical recognition epitope. The first miniature protein designed in this way, a molecule called p007, presents the alpha-helical recognition epitope found on the bZIP protein GCN4 and binds DNA with nanomolar affinity and exceptional specificity. In this work we use alanine-scanning mutagenesis to explore the contributions of 29 p007 residues to DNA affinity, specificity, and secondary structure. Virtually every residue within the p007 alpha-helix, and most residues within the p007 PPII helix, contribute to both DNA affinity and specificity. These residues include those introduced to make specific and nonspecific DNA contacts, as well as those that complete the miniature protein core. Moreover, there exists a direct correlation between the affinity of a p007 variant for specific DNA and the ability of that variant to select for specific DNA over nonspecific DNA. Although we observe no correlation between alpha-helicity and affinity, we observe a limited correlation between alpha-helicity and sequence specificity that emphasizes the role of coupled binding/folding in the function of p007. Our results imply that formation of a highly evolved set of protein.DNA contacts in the context of a well-packed hydrophobic core, and not the extent of intrinsic alpha-helical structure, is the primary determinant of p007 function. 相似文献
47.
48.
Simon Houston Shannon Russell Rebecca Hof Alanna K. Roberts Paul Cullen Kyle Irvine Derek S. Smith Christoph H. Borchers Michelle L. Tonkin Martin J. Boulanger Caroline E. Cameron 《Molecular microbiology》2014,91(3):618-634
The mechanisms that facilitate dissemination of the highly invasive spirochaete, Treponema pallidum, are incompletely understood. Previous studies showed the treponemal metalloprotease pallilysin (Tp0751) possesses fibrin clot degradation capability, suggesting a role in treponemal dissemination. In the current study we report characterization of the functionally linked protein Tp0750. Structural modelling predicts Tp0750 contains a von Willebrand factor type A (vWFA) domain, a protein‐protein interaction domain commonly observed in extracellular matrix (ECM)‐binding proteins. We report Tp0750 is a serine protease that degrades the major clot components fibrinogen and fibronectin. We also demonstrate Tp0750 cleaves a matrix metalloprotease (MMP) peptide substrate that is targeted by several MMPs, enzymes central to ECM remodelling. Through proteomic analyses we show Tp0750 binds the endothelial fibrinolytic receptor, annexin A2, in a specific and dose‐dependent manner. These results suggest Tp0750 constitutes a multifunctional protein that is able to (1) degrade infection‐limiting clots by both inhibiting clot formation through degradation of host coagulation cascade proteins and promoting clot dissolution by complexing with host proteins involved in the fibrinolytic cascade and (2) facilitate ECM degradation via MMP‐like proteolysis of host components. We propose that through these activities Tp0750 functions in concert with pallilysin to enable T. pallidum dissemination. 相似文献
49.
Trisha M. Finlay Samar Abdulkhalek Alanna Gilmour Christina Guzzo Preethi Jayanth Schammim Ray Amith Katrina Gee Rudi Beyaert Myron R. Szewczuk 《Glycoconjugate journal》2010,27(6):583-600
Thymoquinone (TQ) derived from the nutraceutical black cumin oil has been reported to be a novel agonist of Neu4 sialidase
activity in live cells (Glycoconj J DOI 10.1007/s10719-010-9281-6). The activation of Neu4 sialidase on the cell surface by TQ was found to involve GPCR-signaling
via membrane targeting of Gαi subunit proteins and matrix metalloproteinase-9 activation. Contrary to other reports, TQ had
no anti-inflammatory effects in vitro. Here, we show that MyD88/TLR4 complex formation and subsequent NFκB activation are induced by the Neu4 activity associated
with TQ-stimulated live primary bone marrow (BM) macrophage cells from WT and Neu1-deficient mice, HEK-TLR4/MD2 cells and
BMC-2 macrophage cell line but not with primary macrophage cells from Neu4-knockout mice. Tamiflu (oseltamivir phosphate),
pertussis toxin (PTX), a specific inhibitor of Gαi proteins of G-protein coupled receptor (GPCR) and the broad range inhibitor
of matrix metalloproteinase (MMP) galardin applied to live primary BM macrophage cells completely block TQ-induced MyD88/TLR4
complex formation. Using immunocytochemistry and western blot analyses, Tamiflu, galardin and PTX inhibit NFκB activation
induced by Neu4 activity associated with TQ-stimulated BMC-2 cells, HEK-TLR4/MD2 cells and primary BM macrophages from WT
mice. EMSA analyses on HEK-TLR4/MD2 nuclear cell extracts confirm the nuclear localization and DNA binding of TQ-induced NFκB
activation in a biphasic manner within 30 min. Co-immunoprecipitation experiments reveal for the first time that MMP-9 may
be an important intermediate link in the TQ-induced Neu4 activity circuitously targeting TLR4 receptors. Central to this process
is that Neu4 forms a complex with MMP-9, which is already bound to TLR4 receptors. Fluorescence spectrophotometer analyses
of live CD14-THP1 cells treated with TQ show Neu4 sialidase activity over 5 min. Using flow cytometry analyses, CD14-THP1
cells treated with TQ express stable protein levels of Neu4, TLR4 and MMP9 on the cell surface over 30 min except for a marked
diminution of MMP9 at 15 min. Using cytokine array profiling analyses of serum, Neu4-knockout mice respond poorly to TQ in
producing pro-inflammatory cytokines and chemokines after 5-h treatment compared to the wild-type or hypomorphic cathepsin
A mice with a secondary 90% Neu1 deficient mice. Our findings establish an unprecedented signaling paradigm for TQ-induced
Neu4 sialidase activity. It signifies that MMP-9 forms an important molecular signaling platform in complex with TLR4 receptors
at the ectodomain and acts as the intermediate link for TQ-induced Neu4 sialidase in generating a functional receptor with
subsequent NFκB activation and pro-inflammatory cytokine production in vivo. 相似文献
50.
Furuya M Kirschbaum SB Paulovich A Pauli BU Zhang H Alexander JS Farr AG Ruddell A 《Journal of immunology (Baltimore, Md. : 1950)》2010,185(10):5769-5777
The lymphatic circulation mediates drainage of fluid and cells from the periphery through lymph nodes, facilitating immune detection of lymph-borne foreign Ags. The 10.1.1 mAb recognizes a lymphatic endothelial Ag, in this study purified by Ab-affinity chromatography. SDS-PAGE and mass spectrometry identified murine chloride channel calcium-activated 1 (mCLCA1) as the 10.1.1 Ag, a 90-kDa cell-surface protein expressed in lymphatic endothelium and stromal cells of spleen and thymus. The 10.1.1 Ab-affinity chromatography also purified LFA-1, an integrin that mediates leukocyte adhesion to endothelium. This mCLCA1-LFA-1 interaction has functional consequences, as lymphocyte adhesion to lymphatic endothelium was blocked by 10.1.1 Ab bound to endotheliumor by LFA-1 Ab bound to lymphocytes. Lymphocyte adhesion was increased by cytokine treatment of lymphatic endothelium in association with increased expression of ICAM-1, an endothelial surface protein that is also a ligand for LFA-1. By contrast, mCLCA1 expression and the relative contribution of mCLCA1 to lymphocyte adhesion were unaffected by cytokine activation, demonstrating that mCLCA1 and ICAM-1 interactions with LFA-1 are differentially regulated. mCLCA1 also bound to the LFA-1-related Mac-1 integrin that is preferentially expressed on leukocytes. mCLCA1-mediated adhesion of Mac-1- or LFA-1-expressing leukocytes to lymphatic vessels and lymph node lymphatic sinuses provides a target for investigation of lymphatic involvement in leukocyte adhesion and trafficking during the immune response. 相似文献