狼疮肾炎患者的sHLA—I类分子定量检测及mRNA表达水平的研究

目的定量检测狼疮肾炎(LN)患者的sHLA-Ⅰ类分子水平，并对其变化的可能机制从分子水平作初步探讨。方法ELISA双抗夹心法检测血浆的sHLA-Ⅰ类分子水平；RT-PCR检测PBMCHLA-Ⅰ类分子α链mRNA的表达水平。结果23例LN患者的血浆sHLA-Ⅰ类分子水平为(961.40±324.24)μg/L，LN患者与正常人相比明显增高(P＜0.01)；LN患者PBMCHLA-Ⅰ类分子α3结构域-C末端及变位剪接片段mRNA表达水平明显高于正常人(P＜0.01，P＜0.05)。结论LN患者血浆的sHLA-Ⅰ类分子水平明显增高，增高的可能机制之一是PBMCHLA-Ⅰ类分子α链变位剪接，使得39KD的sHLA-Ⅰ类分子增多。     

C-terminal epitope tagging facilitates comparative ligand mapping from MHC class I positive cells

Purification of specific class I molecules prior to peptide ligand characterization is complicated by the presence of multiple class I proteins in most cell lines. Immortalized B, T, and tumor cell lines typically express endogenous HLA-A, -B, and -C; and most individuals from which the cell lines are derived are heterozygous at these loci. Antibodies specific for a particular HLA molecule may be used for purification, but allele-specific antibodies can be biased by ligands occupying the peptide-binding groove. Through the use of C-terminal tagging, we have developed a method of soluble HLA production such that downstream purification does not skew the peptide analysis of the examined molecule. Comparison of peptides eluted from HLA class I molecules with and without C-terminal tags demonstrates that addition of a tag does not abrogate the peptide binding specificity of the original molecule. Both pooled Edman sequencing and mass spectrometric sequencing identified no substantial differences in peptides bound by untailed, 6-HIS-tailed, and FLAG-tailed class I molecules, demonstrating that the peptide specificity of a given molecule is not distorted by either tag. This production methodology bypasses problems with isolation of specific molecules and permits ligand mapping and epitope discovery in a variety of pathogen-infected and tumor cell lines.     

类风湿关节炎患者血浆sHLA-G表达及其与Treg细胞的相关性研究

目的探讨类风湿关节炎（rheumatoidarthritis,RA）患者血浆sHLA-G表达水平及其与Treg细胞水平和RA临床特征的相关性。方法选择40例RA患者和40例健康对照者,采用酶联免疫吸附试验（ELISA）法检测血浆sHLA-G表达水平,流式细胞术检测外周血CD3＋HLA-G＋T细胞、Treg细胞（CD4＋CD25＋Foxp3＋Treg细胞）含量。结果RA患者血浆sHLA-G水平显著低于健康对照组（P〈0．001）;RA患者外周血CD3＋HLA-G＋T细胞百分比明显低于健康对照组（P〈0．001）;Treg细胞百分比也较对照组明显降低（P〈0．01）。相关性分析显示,RA患者Treg细胞百分比与sHLA-G水平存在明显正相关性（P〈0．01）,而与CD3＋HLA-G＋T细胞百分比无显著相关（P〉0．05）;RA患者sHLA-G水平及CD3＋HLA-G＋T细胞百分比均与患者DAS28评分显著负相关（P〈0．05）,而与患者RF、ESR、CRP、抗CCP抗体和ANA水平等均无明显相关性（P〉0．05）。结论RA患者血浆sHLA-G表达异常降低,其可能参与了RA的发生和疾病进展;血浆sHLA-G水平与外周血Treg细胞百分比呈明显正相关,二者之间可能存在相互诱导和调节的信号途径,在诱导和维持机体自身免疫耐受中起着重要的协同作用。     

Soluble Human Leukocyte Antigens,Interleukin-6, and Interferon-γ During Pregnancy

PROBLEM: Soluble human leukocyte antigens (sHLA), interferon-γ (IFN-γ), and interleukin-6 (IL-6) were studied during human pregnancy to test the hypothesis that sHLA concentrations are regulated by these specific cytokines. METHOD OF STUDY: Enzyme-linked immunoadsorbent assays (ELISA) were used to measure sHLA I and II in maternal circulation, cord blood, and placenta effluents of pregnant and nonpregnant women; maternal serum cytokines were also determined. RESULTS: sHLA in maternal and cord blood were equivalent to that in the placenta. By the third trimester, sHLA I concentrations in maternal plasma were significantly reduced compared to the first or second trimesters. sHLA II was increased during the second trimester relative to that postpartum. Maternal IL-6 and IFN-γ concentrations were not statistically different throughout gestation or postpartum. CONCLUSIONS: These data do not suggest a role for maternal plasma IL-6 or IFN-γ in regulation of systemic sHLA class I during pregnancy, but they do not address whether such events take place in local tissues of the maternal-fetal unit.