CD20scFv-IgGFc T 淋巴细胞的建立及其对B细胞淋巴瘤靶向杀伤作用的比较

目的：研究CD20scFv嵌合T淋巴细胞靶向杀伤Daudi细胞时杀伤的效果和T细胞活化情况。方法：将两种质粒转染至PA317细胞中，用转染成功的PA317上清液感染外周血T淋巴细胞后经800 mg/L的G418筛选1周后去杀伤Daudi、K562细胞，分别在不同时点用流式细胞仪检测Daudi细胞AnnexinⅤ的阳性率 ，用ELISA检测细胞因子IL-2、IFNγ。结果：Daudi细胞AnnexinⅤ的阳性率在24 h内两实验组与K562组相比明显增高，而实验组之间没有显著差异。72 h时CD20scFv-IgGFc-CD28-ζ组比CD20scFv-IgGFc组IL-2（1 509.00 ng/L比220.54 ng/L）和IFNγ（912.16 ng/L比251.42 ng/L）的分泌量有显著增高。结论：①两种CD20scFv特异的T细胞在引起Daudi细胞早期凋亡无显著差异，说明在CD20scFv的靶向杀伤过程中CD28-ζ基因可能不主导Daudi细胞的早期凋亡。②CD20scFv-IgGFc-CD28-ζ组IL-2、IFNγ增幅更加明显，说明CD3ζ和CD28嵌合的T细胞可以自身活化而不受MHC的限制，从而增强了T细胞的活化和杀伤等功能。     

Molecular cloning of a novel human gene (SIRP-B2) which encodes a new member of the SIRP/SHPS-1 protein family

A full-length cDNA encoding a novel protein was isolated and sequenced from a human placental cDNA library. This cDNA consists of 1735 base pairs and has a predicted open reading frame (ORF) encoding 354 amino acids. It possesses a putative signal sequence, a long extracellular domain, a transmembrane region, a short intracellular domain, and no catalytic domain, which is highly homologous to signal-regulatory protein (SIRP)-β, suggesting that it seems to be a new member of the SIRP family. Polymerase chain reaction (PCR)-based mapping with both a monochromosomal hybrid panel and radiation hybrid cell panels placed the gene to human chromosome 20p13 near the marker D20S906. Received: August 11, 2000 / Accepted: September 21, 2000     

High incidence of horse serum protein allergy in various autoimmune disorders.

In 186 persons (68 patients suffering from different so-called autoimmune diseases, 30 kidney recipients, 38 control patients from a surgical ward, and 50 healthy volunteers) the immune response to horse IgG was examined. The lowest rate of sensitization was found in kidney transplant recipients (3%) and the highest in autoimmune patients (33%). After excluding 39 patient who had received horse serum treatment prior to the examination, it was found that without previous injection of horse serum, 27% of the patients with autoimmune disease were sensitized to horse IgG. Compared to the other groups (kidney transplant recipients, 4%: surgical controls, 0%; healthy volunteers, 3%), this difference was statistically significant (p is less than 0.01).     

Brain prostaglandin formation is increased by α-synuclein gene-ablation during global ischemia

We have previously demonstrated that α-synuclein (Snca) gene ablation reduces brain arachidonic acid (20:4n − 6) turnover rate in phospholipids through modulation of endoplasmic reticulum-localized acyl-CoA synthetase activity. Although 20:4n − 6 is a precursor for prostaglandin (PG), Snca effect on PG levels is unknown. In the present study, we examined the effect of Snca ablation on brain PG level at basal conditions and following 30 s of global ischemia. Brain PG were extracted with methanol, purified on C₁₈ cartridges, and analyzed by LC–MS/MS. We demonstrate, for the first time, that Snca gene ablation did not affect brain PG mass under normal physiological conditions. However, total PG mass and masses of individual PG were elevated ∼2-fold upon global ischemia in the absence of Snca. These data are consistent with our previously observed reduction in 20:4n − 6 recycling through endoplasmic reticulum-localized acyl-CoA synthetase in the absence of Snca, which may result in the increased 20:4n − 6 availability for PG production in the absence of Snca during global ischemia and suggest a role for Snca in brain inflammatory response.     

Increased lymphocyte trafficking to colonic microvessels is dependent on MAdCAM-1 and C-C chemokine mLARC/CCL20 in DSS-induced mice colitis

Although enhanced lymphocyte trafficking is associated with colitis formation, little information about its regulation is available. The aim of this study was to examine how the murine liver and activation-regulated chemokine (mLARC/CCL20) contributes to lymphocyte recruitment in concert with vascular adhesion molecules in murine chronic experimental colitis. T and B lymphocytes isolated from the spleen were fluorescence-labelled and administered to recipient mice. Lymphocyte adhesion to microvessels of the colonic mucosa and submucosa was observed with an intravital microscope. To induce colitis, the mice received two cycles of treatment with 2% dextran sodium sulphate (DSS). In some of the experiments antibodies against the adhesion molecules or anti-mLARC/CCL20 were administered, or CC chemokine receptor 6 (CCR6) of the lymphocytes was desensitized with excess amounts of mLARC/CCL20. Significant increases in T and B cell adhesion to the microvessels of the DSS-treated mucosa and submucosa were observed. In chronic colitis, the accumulation of lymphocytes was significantly inhibited by anti-mucosal addressin cell adhesion molecule (MAdCAM)-1 mAb, but not by anti-vascular cell adhesion molecule-1. In DSS-treated colonic tissue, the expression of mLARC/CCL20 was significantly increased, the blocking of mLARC/CCL20 by monoclonal antibody or the desensitization of CCR6 with mLARC/CCL20 significantly attenuated the DSS-induced T and B cell accumulation. However, the combination of blocking CCR6 with MAdCAM-1 did not further inhibit these accumulations. These results suggest that in chronic DSS-induced colitis, both MAdCAM-1 and mLARC/CCL20 may play important roles in T and B lymphocyte adhesion in the inflamed colon under flow conditions.     

Increased CD8+ T Cell Apoptosis in Scleroderma Is Associated with Low Levels of NF-κB

Our objectives were (1) to compare lymphocyte subpopulation apoptosis rates in SSc patients versus healthy controls and (2) to compare Bcl-2 and NF-kappa B expression in cultured CD8 lymphocytes of SSc patients versus controls. Peripheral blood samples were obtained from 27 SSc patients meeting the American College of Rheumatology criteria for SSc and 28 healthy individuals. Mononuclear cells were isolated by Ficoll-Hypaque density gradient separation and cultured for 48 hr. For determination of apoptosis within specific cell populations, samples were labeled with PE-conjugated monoclonal antibody to CD8, CD4, and a FITC-conjugated monoclonal antibody to Annexin V. Flow cytometry was carried out with a FACS operating with Cellquest software. CD8+ lymphocytes were positively selected with magnetic microbeads conjugated to antihuman CD8. CD8 T cells were separated, then incubated with activation for 48 hr, and NF-kappa B and Bcl-2 analysis was carried out using Western immunoblotting. The CD4:CD8 ratio was increased in SSc compared to controls (2.6 +/- 1.13 vs.1.87 +/- 0.76; P = 0.018). The spontaneous apoptosis rate of SSc CD8 lymphocytes was increased compared to that of controls of (21.9 +/- 13.7 vs. 13.3 +/- 9.9; P = 0.019). No difference was found in the rate of CD4 apoptosis of SSc patients versus controls (9.8 +/- 5.2 vs. 7.18 +/- 4.89%; P = ns). The expression of NF-kappa B in SSc CD8 lymphocytes was decreased compared with that of CD8 lymphocytes from healthy controls (144 +/- 13 vs. 188 +/- 11; P = 0.018). Whereas expression of Bcl-2 was similar in activated CD8+ T cells of SSc patients and healthy controls, CD8+ T cell apoptosis rate was found to be in reverse correlation with expression of NF-kappa B in these cells ( r = - 0.53, P = 0.029). The increased CD4:CD8 ratio in SSC may result from increased CD8+ T cell apoptosis. Increased SSc CD8 T cell apoptosis is associated with low levels of NF-kappa B.