Biosynthesis and function of membrane bound and secreted forms of recombinant CD11b/CD18 (Mac-1)

Full-length (membrane bound) and truncated (secreted) forms of the beta 2 integrin heterodimer, CD11b/CD18 (Mac-1), were expressed in a human kidney cell line (293) that normally does not express leukocyte adhesion molecules (Leu-CAMs). The biosynthesis of recombinant Mac-1 in 293 cells differed from that reported for leukocytes in that heterodimer formation was not required for CD11b to be exported to the cell surface. A stable cell line was constructed that constitutively secreted the recombinant, truncated Mac-1 heterodimer into growth conditioned cell culture medium. A novel monoclonal antibody that enabled an immunoaffinity method for the selective purification of recombinant Mac-1 heterodimers was identified. Sufficient protein was purified to allow the first measurement of the 50% inhibitory concentration (IC₅₀) for CD11b/CD18 and for the direct comparison of the inhibitory activity of recombinant soluble Mac-1 with that of various CD18 and CD11b specific monoclonal antibodies. Purified recombinant soluble Mac-1 inhibited the binding of neutrophils, activated by opsonized zymosan or fMet-Leu-Phe peptide, to human umbilical vein endothelial cells. Similarly, the recombinant integrin was effective in inhibiting the binding of unactivated neutrophils to tumor necrosis factor (TNF-alpha) activated endothelial cells. The availability of an abundant source of purified, biologically active Mac-1 will enable direct physical and chemical investigations into the relationship between the structure and function of this leukocyte adhesion molecule.     

抑制CD36与Nogo-B表达抑制三阴性乳腺癌细胞增殖与迁移

分化聚类36（cluster of differentiation 36,CD36）是一种位于细胞表面的膜蛋白受体,可以结合并转运脂肪酸。内质网膜蛋白4B （Nogo-B）在肝脏中调控脂肪酸代谢而影响肝癌的发展。目前并不清楚CD36和Nogo-B的相互作用是否能够影响乳腺癌细胞的增殖和迁移。本研究在三阴性乳腺癌（triple-negative breast cancer,TNBC）细胞中同时干预CD36与Nogo-B的表达来探索它们对细胞增殖与迁移的影响。结果表明在三阴性乳腺癌细胞中,单独抑制CD36或Nogo-B的表达都能够抑制细胞的增殖与迁移;同时抑制CD36与Nogo-B的表达时,这种抑制效果更加明显,且Vimentin、B细胞淋巴瘤-2（B-cell lympoma-2,BCL2）和增殖细胞核抗原（proliferating cell nuclear antigen,PCNA）的表达受到抑制。在小鼠移植瘤模型中,E0771细胞转染CD36或Nogo-B的siRNA后成瘤能力降低;同时敲减CD36和Nogo-B时,肿瘤生长速度显著减慢。机制研究发现,抑制CD36和Nogo-B表达能够抑制脂肪酸结合蛋白4（fatty acid binding protein 4,FABP4）和脂肪酸转运蛋白4（fatty acid transport protein 4,FATP4） mRNA的含量,同时CD36和Nogo-B过表达刺激的细胞增殖被FABP4的siRNA降低,预示着抑制乳腺癌细胞中CD36与Nogo-B的表达可能通过抑制脂肪酸的吸收和转运而抑制细胞的生长和迁移。此外,抑制CD36与Nogo-B的表达可激活P53-P21-Rb信号通路,参与抑制CD36与Nogo-B表达而抑制的细胞增殖与迁移。本研究证明同时抑制CD36和Nogo-B的表达能够协同抑制三阴性乳腺癌细胞的增殖和迁移,为临床抗三阴性乳腺癌药物的开发提供了新的靶点。     

传染性单核细胞增多症患儿NLR、CD4+/CD8+比值、ADA与EBV-DNA载量的相关性分析及对肝损害的影响

摘要 目的：探讨传染性单核细胞增多症（IM）患儿外周血中性粒细胞/淋巴细胞比值（NLR）、CD4⁺/CD8⁺比值、腺苷脱氨酶（ADA）与EB病毒（EBV）-脱氧核糖核酸（DNA）载量的相关性,分析其对IM患儿肝损害的影响。方法：选择2019年1月至2022年4月我院儿科收治的102例IM患儿（IM组）,另选择同期我科收治的95例EB病毒检测阴性的发热患儿（非IM组）和体检健康的73例健康儿童（对照组）。根据是否发生肝损害将IM患儿分为肝损害组（61例）和非肝损害组（41例）。比较外周血NLR、CD4⁺/CD8⁺比值、ADA与EBV-DNA载量,Pearson法分析NLR、CD4⁺/CD8⁺比值、ADA与EBV-DNA载量的相关性。多因素Logistic回归分析IM患儿发生肝损害的影响因素。结果：IM组ADA高于非IM组和对照组（P＜0.05）,且非IM组高于对照组（P＜0.05）,NLR、CD4⁺/CD8⁺比值低于非IM组和对照组（P＜0.05）,且非IM组低于对照组（P＜0.05）,IM组EBV-DNA载量高于非IM组（P＜0.05）。IM患儿ADA与EBV-DNA载量呈正相关（r=0.493,P＜0.05）,NLR、CD4⁺/CD8⁺比值与EBV-DNA载量呈负相关（r=-0.419、-472,P＜0.05）。肝损害组ADA、EBV-DNA载量高于非肝损害组（P＜0.05）,NLR、CD4⁺/CD8+⁺比值低于非肝损害组（P＜0.05）。肝脏肿大、高EBV-DNA载量、高ADA是IM患儿肝损害的危险因素（P＜0.05）,高NLR、高CD4⁺/ CD8⁺比值是保护因素（P＜0.05）。结论：IM患儿ADA增高,NLR、CD4⁺/CD8⁺比值降低,与EBV-DNA载量增加以及肝损害有关。     

The CD95 (Fas/APO-1) receptor is phosphorylatedin vitro andin vivo and constitutively associates with several cellular proteins

Different CD95 (Fas/APO-1) isoforms and phosphory lated CD95 species were identified in human T and B cell lines. We had shown previously that the CD95 intracellular domain (IC), expressed as a glutathione S-transferase (GST) fusion protein in murine L929 fibroblasts, was phosphorylatedin vivo. GST-CD95IC was phosphorylatedin vitro by a kinase present in extracts from the human lymphocytic cell lines Jurkat and MP-1 and from murine L929 cells. Phosphoamino acid analysis indicated that phosphorylation occurred at multiple threonine residues and also at tyrosine (Tyr232 and Tyr291) and serine. Amino acids 191 to 275 of CD95 were sufficient for phosphorylation at threonine, tyrosine and serine and also mediated interaction with a 35 kDa cellular protein. Immuno-precipitation of CD95 and chemical cross-linking revealed CD95-associated proteins of approximately 35, 45 and 75 kDa. GST-CD95IC affinity chromatography detected binding of the 35 and 75 kDa protein species. The 75 kDa species may correspond to the CD95-associated proteins RIP or FAF1 and the 35 kDa protein may represent a TRADD analogue. These data indicate that several cellular proteins interact with CD95, possibly in a multi-protein complex, and that a kinase activity is associated with CD95 not onlyin vitro but alsoin vivo. Therefore, receptor phosphorylation may play a role in CD95 signal transduction. This work was in part supported by a grant from the Health Research Council of New Zealand (to JW).     

利用IL－3／GM－CSF融合蛋白（PIXY－321）扩增脐血造血细胞方法的初步建立

利用单克隆抗体免疫磁珠吸附方法分离脐血ＣＤ３４＋细胞,并观察了ＩＬ３／ＧＭＣＳＦ融合蛋白（ＰＩＸＹ３２１）对脐血ＣＤ３４＋细胞的刺激作用。ＰＩＸＹ３２１对脐血ＣＤ３４＋细胞扩增作用大于ＩＬ３和ＧＭＣＳＦ单独及联合应用。在液体培养条件下,每毫升２０ｎｇＰＩＸＹ３２１可有效地扩增脐血造血祖细胞,适宜扩增时间为５－８天,扩增后造血祖细胞的数量可达扩增前的８－１０倍,从而初步建立了一种简单可行的脐血造血细胞扩增方法。     

CD23McAb抑制活化B细胞增殖机制的初步探讨

本研究用ＣＤ２３单克隆抗体交联活化的人扁桃体Ｂ细胞,证明ＣＤ２３ＭｃＡｂ对Ｂ细胞呈双向调节效应,即：高浓度区抑制Ｂ细胞增殖,低浓度区促进Ｂ细胞增殖,继而,用对Ｂ细胞有抑制效应浓度的ＣＤ２３ＭｃＡｂ交联Ｂ细胞膜ＣＤ２３分子,通过研究抑制效应恢复条件,探讨了ＣＤ２３ＭｃＡｂ产生抑制效应的作用机制。结果显示：去除ＣＤ２３ＭｃＡｂ后,抑制作用不能恢复,用ＳＡＣ再次活化,使Ｂ细胞恢复增殖状态,用促Ｂ细胞增殖     

Oral administration of antigen does not influence the proliferation and IFN-γ production of responsive CD8+ T cells but enables to establish T cell clones with different lymphokine production profile.

Feeding of a whole casein diet, which abolished the α_s1-casein-specific proliferation and IFN-γ productivity of CD⁴⁺ T cells, did not affect the proliferative response of CD8⁺ T cells with regard to the antigen dose response, cell dose response, kinetics of the proliferation and epitope specificity, as well as IFN-γ production. To assess the characteristics of the CD8⁺ T cells, we established α_s1-casein-specific CD8⁺ T cell clones from both casein-fed and control mice. The established clones produced different amount of IFN-γ and IL-10, and one clone derived from the casein-fed mice produced a remarkable amount of IL-10. The clones from casein-fed mice produced considerable amounts of TGF-β, while those from control mice produced only small amounts. The possible role of CD8⁺ T cells in oral tolerance is discussed. This revised version was published online in June 2006 with corrections to the Cover Date.