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Arvind K. Virmani Bashoo Naziruddin Vinay C. Desai Jon P. Lowry Donald C. Graves Goverdhan P. Sachdev 《In vitro cellular & developmental biology. Animal》1992,28(2):120-127
Summary The purpose of this investigation was to provide evidence for the secretion of high molecular weight mucins, CTM-A and CTM-B,
in primary culture of canine tracheal epithelial (CTE) cells. The cells were isolated from tracheas of mongrel dogs by pronase
treatment. Primary cultures of the epithelial cells were established using ICN cellagen inserts in Dulbecco’s modified Eagle’s/F12
medium supplemented with growth factors and could be maintained for up to 23 days. The evidence for the mucin secretion in
culture medium and their localization in the cells was established by a) positive immunocytochemical staining using specific
antibodies developed against purified native as well as deglycosylated CTM-A and CTM-B; b) incorporation of labeled amino
acids, followed by electrophoresis and autoradiography detection of glycoconjugates purified from the culture medium; c) comparison
of the amino acid compositions of mucin purified from canine tracheal pouch secretions and that purified from the culture
medium; and d) Western blot analyses using specific polyclonal antibodies directed against deglycosylated CTM-A and CTM-B.
Immunoaffinity purified secreted labeled glycoconjugates were resistant to hyaluronidase treatment. The effects of cyclic
AMP (1 × 10−5
M), dibutyryl cyclic AMP (1 × 10−5
M), 8-bromocyclic AMP (1 × 10−5
M), and prostaglandin E1 (1 × 10−6
M) on mucin secretion by CTE cells were also investigated. Secretion of mucins by CTE cells in culture was considerably more
enhanced by 8-bromocyclic AMP than that observed for other secretagogues used in this study. 相似文献
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The in vitro and in vivo effects of anti-galactose antibodies on endothelial cell activation and xenograft rejection 总被引:5,自引:0,他引:5
Xu H Yin D Naziruddin B Chen L Stark A Wei Y Lei Y Shen J Logan JS Byrne GW Chong AS 《Journal of immunology (Baltimore, Md. : 1950)》2003,170(3):1531-1539
We have previously produced a series of antigalactose (anti-Gal) hybridomas and characterized their heavy chain gene usage. Here we have quantified the affinity of these Abs for the alpha-Gal epitope and characterized their in vitro effects on endothelial cell activation and apoptosis. We report that anti-Gal mAbs derived from Gal(-/-) mice show a range of affinity for the alpha-Gal epitope, and that affinity was generally increased as the V(H) gene usage transitioned from germline sequences to sequences exhibiting somatic maturation. Despite an 85-fold range in affinity, all the anti-Gal mAbs examined induced alpha-Gal-specific endothelial cell activation, and after prolonged exposure induced endothelial cell apoptosis in a complement-independent manner. Only murine anti-Gal mAbs of the IgM or IgG3 subclass, but not IgG1, were effective at initiating complement-dependent cell lysis. Using a novel rat to mouse xenograft model, we examined the in vivo ability of these mAbs to induce xenograft rejection and characterized the rejection using histology and immunohistochemistry. Infusion of complement-fixing IgG3 mAbs resulted in either hyperacute rejection or acute vascular rejection of the xenograft. Surprisingly, infusion of an equal amount of a high affinity anti-Gal IgG1 mAb, that fixed complement poorly also induced a rapid xenograft rejection, which we have labeled very acute rejection. These studies emphasize the importance of in vivo assays, in addition to in vitro assays, in understanding the role of anti-Gal IgG-mediated tissue injury and xenograft rejection. 相似文献
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B Naziruddin B F Duffy J Tucker T Mohanakumar 《Journal of immunology (Baltimore, Md. : 1950)》1992,149(11):3702-3709
Human polymorphonuclear neutrophils (PMN) express the low affinity receptors for the Fc domain of IgG (Fc gamma R), Fc gamma RII (CD32), and the glycosyl phosphatidylinositol-linked isoform of Fc gamma RIII (Fc gamma RIIIB, CD16) on their cell surface. Both of these receptors have been shown to be signal-transducing molecules. However, the mechanisms involved in such signaling are not clearly understood. In this report, we investigated intracellular Ca2+ ([Ca2+]i) signals triggered in PMN by both the receptors using aggregated human IgG (AggIgG) and specific mAb to Fc gamma RII (KuFc79) and Fc gamma RIII (3G8) as ligands. Addition of AggIgG as well as cross-linking of mAb KuFc79 and 3G8 bound to PMN induced [Ca2+]i flux. However, preincubation of PMN with mAb KuFc79 (whole Ig or Fab fragments) in the absence of cross-linking abrogated the [Ca2+]i flux induced by AggIgG and mAb 3G8, indicating that Fc gamma RII receptor occupancy by mAb KuFc79 can block signals mediated by Fc gamma RIIIB. KuFc79-isotype-matched control mAb (MOPC 195) did not abolish the signals generated by AggIgG and mAb 3G8. In addition, mAb KuFc79 did not abrogate [Ca2+]i responses elicited by the receptor for the chemotactic peptide FMLP indicating that modulation of signal transduction by Fc gamma RII-bound KuFc79 is selective for certain receptors. Immunofluorescence analysis of PMN initially treated with mAb KuFc79 followed by AggIgG showed that KuFc79 did not block the binding of AggIgG to PMN. Similarly, competitive binding studies revealed no stearic hindrance between mAb KuFc79 bound to Fc gamma RII and mAb 3G8 bound to Fc gamma RIIIB. Thus, the ability of mAb KuFc79 to modulate signals induced by AggIgG and 3G8 strongly suggests that Fc gamma RII may regulate Fc gamma RIIIB signaling. While previous studies on Fc gamma RII revealed a requirement for cross-linking of the receptor to induce its effector functions, the present study shows that binding of mAb KuFc79 to Fc gamma RII itself, even in a univalent form, results in cross-regulation of Fc gamma RIIIB-triggered signals. Treatment of PMN with protein tyrosine kinase inhibitors, genistein and herbimycin A, abrogated the [Ca2+]i signals elicited by both mAb KuFc79 and 3G8. These results suggest that tyrosine kinase enzyme(s) associated with these receptors may be crucial for positive/negative signals triggered by Fc gamma RII and Fc gamma RIIIB. 相似文献
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