ELISA测定血清可溶性HLA—Ⅰ类抗原

目的　建立定量测定可溶性HLA I类抗原的ELISA方法。方法　测定上海地区汉族正常人群血清可溶性HLA I类抗原的含量 ,并进一步研究可能影响这一含量的因素。结果　测得 116例上海地区汉族正常个体血清可溶性HLA I类抗原的含量为 5 2 9 3 2±2 82 88ng/ml。结论　性别和常见HLA A位点抗原型别对血清可溶性HLA I类抗原的含量未造成显著影响。     

Association of I and II class HLA antigens with Cushing's syndrome]

Incidence of class I and II HLA antigens was studied in 69 patients suffering from Icenko--Cushing syndrome. Positive association was reported for antigens DRw53, DQw3, DR4, DR5, negative for antigen DR2. Antigen combinations DR5, DR7 and DR4, DR5 proved more prevalent. The risk to develop the disease rose 2-3-fold in case of HLA-DR5 phenotype occurrence with antigen DR4 or DR7. Association with DRw53 and DQw3 antigens showing wide specificity seemed most significant.     

No differences in colony formation of peripheral blood stem cells frozen with 5% or 10% dimethyl sulfoxide

High-dose chemotherapy with autologous stem cell rescue usually requires cryopreservation of the cells. For several years, 10% dimethyl sulfoxide (DMSO) has been used as the standard cryoprotectant. Because DMSO infusion can lead to toxic clinical complications in a dose-related manner, we wanted to evaluate if reduction to 5% DMSO would be possible. We have compared colony formation in the myeloid, erythropoietic, and megakaryocyte lineages in peripheral blood progenitor cell (PBPC) samples cryopreserved in parallel with 5% and 10% DMSO. Twenty-seven PBPC samples from patients with malignant diseases were investigated after 3 months of cryopreservation in liquid N(2), and samples from 14 of these patients were investigated after 1 year. A significantly higher colony formation was demonstrated for colony-forming units-erythrocyte (CFU-E) and CFU-granulocyte, erythrocyte, macrophage, megakaryocyte (GEMM) both at 3 months and at 1 year in the 5% samples. For CFU-granulocyte-macrophage (GM) and CFU-megakaryocyte (Mk) no significant difference was demonstrated neither at 3 months nor at 1 year in samples frozen with 5% and 10% DMSO. Also, there was a statistically significant correlation between the CFU-total and CFU-Mk-total, indicating that the CFU-total might be used as an evaluation of megakaryocyte progenitors. Viability testing with the Trypan Blue exclusion test showed that cells cryopreserved in 5% DMSO had significantly higher viability than the cells cryopreserved in 10% DMSO. We conclude that 5% DMSO is at least as good for cryopreservation of small-volume PBPC samples as the conventional 10% DMSO, and our results suggest that the possibility of using 5% DMSO for cryopreservation of autologous PBPC grafts should be further investigated in clinical studies.     

Quantification of soluble serum HLA class I antigens in healthy volunteers and AIDS patients

A solid-phase enzyme immunoassay (ELISA) has been used to quantify human soluble Class I histocompatibility antigens in serum samples from voluntary blood donors and AIDS patients. Statistical analysis of the results showed significantly raised levels (p less than 0.01) of free HLA Class I in sera from AIDS patients (2.95 +/- 1.80 micrograms/ml) when compared with the blood donors (1.06 +/- 0.6 micrograms/ml). The assay is specific, reproducible and easy to perform. Potential uses of this determination are discussed.     

人脐带间充质干细胞经5-氮胞苷诱导后心肌特异性肌钙蛋白I的表达

背景：人脐带间充质干细胞具有多向分化潜能,经诱导后是否可向心肌细胞分化？目前的报道尚不多。目的：观察人脐带间充质干细胞经5-氮胞苷诱导分化后肌钙蛋白I的表达情况。方法：取第2代人脐带间充质干细胞标本,经消化、离心、沉淀后,分为7份,制成细胞悬液,调整细胞浓度为2×10^7L^-1,分别加入浓度为80,40,20,10,5,2．5,0μmol／L5-氮胞苷于同样条件继续培养,作用24h后除去,继续培养4周。结果与结论：①人脐带间充质干细胞经5-氮胞苷处理24h之后,各组细胞都会出现部分死亡,失去典型的梭形形态,成为棍状或者柱状,40,80μmol／L组细胞形态变化明显。②免疫组化染色显示5,10,20,40,80μmol／L组诱导后2周心肌特异性肌钙蛋白I表达呈阳性,对照组与2．5μmol／L组心肌特异性肌钙蛋白I表达为阴性。选取5个高倍视野进行细胞计数,80,40,20,10μmol／L组间心肌样细胞的转化率差异无显著性意义,均明显高于5μmol／L组（P〈0．05）。⑨超微结构观察显示经5-氮胞苷诱导后4周时部分人脐带间充质干细胞内可见典型的肌小节及心房颗粒。而未经5-氮胞苷诱导的对照组无以上改变。结果提示经5-氮胞苷诱导后人脐带间充质干细胞能够向心肌样细胞转化。     

HLA antigens on colorectal adenoma and cancer cells

HLA-DR and -ABC antigens on adenoma and cancer cells of the colon and rectum were investigated. Fifteen specimens of adenomas from 15 patients with sporadic colorectal adenoma, seven specimens of adenomas from seven patients with familial polyposis coli (FPC), and 10 specimens of cancers from 10 patients with colorectal cancer were obtained. Normal colonic mucosa far from the lesions, taken from 15 patients with sporadic adenoma or cancer, served as normal control mucosa. HLA antigens were identified using an immunoperoxidase staining method. Epithelia of all normal control mucosa (n = 15) expressed HLA-ABC antigens, but not HLA-DR antigens. HLA-DR antigens were expressed on 47% (7/15) of sporadic adenomas, 71% (5/7) of adenomas in FPC, and 100% (10/10) of cancers. The extent of HLA-DR expression on adenoma and cancer cells became broader with more severe dysplasia in adenoma, and increased undifferentiation in cancer. It also became broader with increasing mononuclear cell infiltration in both adenomas and cancers. HLA-DR antigens on adenoma and cancer cells appeared to be related to the neoplastic transformation of the epithelia, and to the mononuclear cell infiltration. Partial disappearance of HLA-ABC antigens on adenoma and cancer cells was observed in a few specimens of both adenomas and cancers.     

Expression of HLA class I antigens in human tumors and their involvement in tumor growth

Summary A decreased expression of major histocompatibility complex (MHC) class I antigens is a common feature of many experimental and human tumors and can often be correlated with malignancy grade. In fact, reduction of class I antigens is associated in most tumors with an enhanced ability to elude immune surveillance. Loss of HLA-A,B,C antigens ranges from a decrease in the percentage of A,B,C-positive cells to selective loss of particular antigens and total loss of class I molecule expression. In man, this has been documented in melanomas, carcinomas, lymphomas, neuroblastoma and acute leukemias. The reduction in membrane antigens is generally associated with a parallel fall in immunoprecipitable intracellular proteins and the corresponding mRNAs in the absence of structural changes in the coding genes. The literature concerning the above mentioned topics is reviewed and discussed. This work was supported in part byConsiglio Nazionale delle Ricerche (CNR), Roma, Italy,Progetto Finalizzato ‘Biotecnologie e Biostrumentazione’. T. Crepaldi was supported by a fellowship of ‘Comitato Gigi Ghirotti’.     

Determinants recognized by human cytotoxic T cells on a natural hybrid class I HLA molecule

The major histocompatibility complex class I HLA molecules are the primary determinants recognized by allogeneic cytotoxic T lymphocytes (CTL), and serve as restricting elements for CTL recognition of viral, chemical, or minor histocompatibility antigens. HLA-Aw69 is a naturally occurring hybrid class I molecule that we have used to investigate the regions of class I antigens involved in human CTL recognition. HLA-Aw69 appears to have resulted from an exon shuffle between two closely related class I genes: the alpha 1 domain of HLA-Aw69 is identical to that of HLA-Aw68, while the alpha 2 and alpha 3 domains are identical to HLA-A2. The determinants recognized by human allogeneic CTL clones specific for HLA-A2, -Aw68, and/or -Aw69 fall into three patterns: (a) CTL determinants are located on both the alpha 1 and alpha 2 domains; (b) interaction of the alpha 1 and alpha 2 domains results in new combinatorial determinants; (c) interaction of the alpha 1 and alpha 2 domains in the hybrid molecule results in the loss of CTL determinants that are present on both parental molecules. Thus, using human CTL clones, target cells, and HLA molecules, we show that the interaction of the alpha 1 and alpha 2 domains alters CTL determinants in ways not directly predictable from primary structure.     

Autoreactive T cell clones specific for class I and class II HLA antigens isolated from a human chimera

T cell clones of donor origin that specifically react with recipient cells were obtained from a SCID patient successfully reconstituted by allogeneic fetal liver and thymus transplantation performed 10 yr ago. The majority of these clones displayed both cytotoxic and proliferative responses towards PBL and an EBV-transformed B cell line derived from the patient. In addition, these T cell clones had proliferative and cytotoxic responses towards the parental PBL, EBV cell lines, and PHA blasts. Blocking studies with anti-class I and anti-class II HLA mAbs indicated that the activity of the CD4+ T cell clones was specifically directed against class II HLA antigens of the recipient. On the other hand, the cytotoxic and proliferative responses of the CD8+ T cell clones were specific for class I HLA antigens which are ubiquitously expressed on the recipient cells. Thus, the establishment of transplantation tolerance observed in this stable human chimera is not due to the elimination of host-reactive T cells from the repertoire and suggests the presence of a peripheral autoregulatory suppressor mechanism.     

5-氮胞苷诱导兔脂肪干细胞分化为心肌样细胞的特定基因表达

背景:采用生物学技术将脂肪干细胞进行心肌内移植,修复损伤心肌组织是近年来心血管病研究领域的一大热点。脂肪干细胞经诱导分化后的特定心肌基因表达尚无全面的研究报道。目的:观察兔脂肪干细胞经5-氮胞苷诱导分化为心肌样细胞后心肌特异性基因的表达。方法:取第2代脂肪干细胞,加入2.5,5,10,20,40,80μmol/L5-氮胞苷经24h作用后,用完全培养液对其继续进行培养,4周后再观察细胞生长和形态学的改变。待诱导1,2,3,4周,利用RT-PCR技术检测心钠素,脑钠素,α-骨骼肌动蛋白的基因表达。结果与结论:5-氮胞苷诱导1周,其细胞大多呈紧密平行排列,其体积有所增大,梭形细胞比例相对有所下降,40,80μmol/L组细胞形态变化明显。诱导第4周时,40,80μmol/L组细胞折光性减弱,细胞活性减弱,细胞死亡数继续增加。RT-PCR检测心钠素,脑钠素,α-骨骼肌动蛋白的基因表达呈逐渐增加趋势;10μmol/L组各基因的表达明显高于其他各组(P<0.05)。结果提示兔脂肪干细胞经5-氮胞苷诱导分化的细胞具有心肌细胞的某些基因的特异性表达。     

Human histocompatibility determinants and virus antigens: effect of measles virus infection on HLA expression

Histocompatibility antigens on the surface of human lymphoblastoid cells were quantified by a microadsorption technique. During the course of measles virus infection, no quantitative or qualitations in surface HLA antigens were observed. In contrast, infection with poliovirus type 1 or vesicular stomatitis virus, or treatment with puromycin (50 microgram/ml) resulted in a significant decrease in surface HLA. These experiments suggest that an inhibition of host protein synthesis rather than the insertion of virus-specificied antigens into the membrane results in a net decrease in amounts of this cell surface antigen. The HLA antigens also appear to be both functionally and structurally distinct from measles virus surface antigens. Pretreatment of cells with HLA-directed antibody did not prevent the infection of these cells by measles virus, thus HLA antigens appear unrelated to the measles virus receptor site on the plasma membrane. Electron microscopic studies revealed that measles virus maturation occurs at membrane sites devoid of demonstrable HLA. Furthermore, HLA antigens could not be detected on the surfaces of mature infectious virions.     

Constitutive and inducible expression of HLA class II determinants by human osteoblast-like cells in vitro

Activated immune cells release cytokines which modulate the activity of bone cells in vitro. Expression of major histocompatibility complex (HLA in humans) class II determinants on bone surface cells may be important in local immune cell activation. In this study, expression of HLA-DR and DQ by cultured human bone cells (HBC) derived from normal trabecular bone surfaces was assessed by fluorescence-activated cell sorter (FACS) analysis and immunoperoxidase techniques using monoclonal antibodies. A subset of HBC (10-30%) expressed DR constitutively while 5-15% displayed DQ during long-term culture. HBC lacked a number of monocyte and lymphocyte markers. In addition, both DR+ and DR- HBC (FACS separated) produced osteocalcin stimulated by 1,25-dihydroxyvitamin D2 (1,25(OH)2D3). This suggests that both phenotypes belong to the osteoblast lineage. The number of DR+ HBC was increased by interferon-gamma (IFN gamma; 40-95% DR+ cells) whereas DQ+ HBC remained unchanged or was slightly increased (5-20% DQ+ cells). Moreover, 1,25(OH)2D3 enhanced IFN gamma-induced DR expression and at high concentration (10(-7) M) augmented DR expression by itself. Other major osteotropic factors, parathyroid hormone, interleukin 1, and calcitonin, did not affect HBC DR expression. The findings suggest that HBC may participate in activation of the immune system and that some osteotropic factors may regulate this function.     

Lack of HLA class I antigen expression by cultured melanoma cells FO-1 due to a defect in B2m gene expression

The melanoma cell line FO-1 does not express HLA class I antigens and does not acquire them on the cell surface after incubation with IFN-gamma. Immunochemical studies showed that FO-1 cells synthesize HLA class I heavy chain, but do not synthesize beta 2-microglobulin (beta 2-mu). The latter abnormality is associated with lack of beta 2-mu mRNA which remains undetectable in FO-1 cells incubated with IFN-gamma. The defect was identified as a genetic lesion in the B2m gene, since DNA hybridization analysis detected a deletion of the first exon of the 5'-flanking region, and of a segment of the first intron of the B2m gene. HLA class I antigen expression was reconstituted on melanoma cells FO-1 after transfection with the wild-type mouse B2m gene, thereby confirming the abnormality of the endogenous B2m gene. The defect identified in FO-1 cells is distinct from that underlying the lack of HLA class I antigen expression by lymphoblastoid cells Daudi, but is remarkably similar to that causing lack of H-2 class I antigen expression by mouse lymphoblastoid cells R1 (TL-). These results suggest that genetic recombination in the 5' region of the B2m gene is a recurrent mechanism in B2m gene defects. In addition to contributing to our understanding of molecular abnormalities in HLA class I antigen expression by melanoma cells, FO-1 cells represent a useful model for analyzing the role of HLA class I antigens in the biology of melanoma cells and in their interaction with cells of the immune system.     

Simple ELISA method for the evaluation of soluble HLA class I antigens in human serum

A simple sandwich ELISA method has been developed for the quantification of soluble HLA class I antigens (s-HLA) in human serum. The assay utilizes the monoclonal antibody Q6/64, directed to a monomorphic determinant of the HLA alpha-chain, to capture the antigen and the biotinylated NAMB1 monoclonal antibody, directed to beta 2-microglobulin, as the detection antibody. The extract of the LG-2 lymphoid cell line and pooled sera from 100 healthy subjects are utilized as standards. The arbitrary value of 100 s-HLA Relative Units/mL (RU/mL) is given to the 1:20 dilution of pooled human sera whose optical density value corresponds to the one of the extract of 1 x 10(6) LG-2 cells (6.25 micrograms/mL protein concentration). The assay is easy to perform, specific, reproducible (intra- and inter-assay variations ranging from 3.2% to 8.87%), sensitive (detection limit of 6 RU/mL), and needs a small amount of serum (0.1 mL). The mean serum levels of s-HLA found in 100 healthy normal subjects are 41.9 +/- 13.4 RU/mL. The potential uses of the method are discussed.     

Comparative analysis of HLA class I antigens in pulmonary sarcoidosis and tuberculosis in the same ethnic group

OBJECTIVE: To compare occurrences of respective HLA class I antigens in patients with sarcoidosis (SA), patients with tuberculosis (TB), and healthy controls in the same ethnic group in Poland. PATIENTS AND METHODS: HLA-A, -B, and -C antigens were determined from 1994 to 1997 by using the National Institutes of Health method in 100 patients with SA, 100 patients with TB, and 100 healthy controls. Frequencies of specific antigens were compared among the 3 groups. RESULTS: Our study showed that among SA patients, the HLA-B51(5) and HLA-B8 antigens tended to be more common and the HLA-B13, -B35, and -Cw4 antigens tended to be less common than in the controls. However, after Bonferroni correction, only the HLA-B35 antigen was found to be significantly different in SA patients and controls. In TB patients, the expression of HLA-B62(15) and HLA-Cw5 antigens tended to be more common compared with controls and HLA-A2 less common compared with controls, but only the differences in B62(15) and Cw5 were significant after Bonferroni correction. HLA-B51(5) and HLA-B8 antigens were statistically more frequent and B13, B62(15), and Cw4 less frequent in SA than in TB patients and remained significant after Bonferroni correction. The occurrence of other antigens studied in both populations was comparable. CONCLUSION: We identified associations of HLA class I antigens in patients with SA or TB, with an expression pattern specific and different for each group.     

Cytotoxic T cell recognition of an endogenous class I HLA peptide presented by a class II HLA molecule

Human leukocytes were stimulated in vitro with peptides corresponding in sequence to the highly variable helix of the alpha 1 domain of various HLA-B and -C molecules. A CD4+ CD8- cytotoxic T cell line, CTL-AV, that is specific for the HLA-B7 peptide presented by HLA-DR11.1 was obtained. The HLA-DR11.2 molecule, which only differs at three residues from HLA-DR11.1, did not present the HLA-B7 peptide to CTL-AV. Peptides from the alpha 1 domain helix of other HLA-A and HLA-B molecules, but not HLA-C molecules, competed with the HLA-B7 peptide for binding to HLA-DR11.1. A cell line (WT50) that coexpresses HLA-B7 and HLA-DR11.1 was killed by CTL-AV in the absence of any added HLA-B7 peptide. The processing and presentation of HLA-B7 in these cells appears to be through the endogenous, and not the exogenous, pathway of antigen presentation. Thus, Brefeldin A inhibits presentation and chloroquine does not. Furthermore, introduction of purified HLA-B7 molecules into HLA-DR11.1+, HLA-B7- cells by cytoplasmic loading via osmotic lysis of pinosomes, but not by simple incubation, rendered them susceptible to CTL-AV killing. These results provide an example of class II major histocompatibility complex (MHC) presentation of a constitutively synthesized self protein that uses the endogenous pathway of antigen presentation. They also emphasize the capacity for presentation of MHC peptides by MHC molecules.     

Cholesterol-induced atherosclerosis in the rabbit: effect of dimethyl sulfoxide on existing lesions

Earlier studies have established that the analgesic and anti-inflammatory compound, dimethyl sulfoxide (DMSO), was effective in preventing atherosclerosis in cholesterol-fed rabbits. In the present studies, the effect of DMSO on existing atherosclerotic lesions in cholesterol-fed rabbits was investigated. Rabbits were placed on an atherogenic diet containing 1% cholesterol for a period of 10 weeks. At the end of the 10-week period, the rabbits were randomly divided into two groups: one group was placed on a control diet consisting of regular rabbit chow for an additional 12-week period, whereas the remaining group was continued on the atherogenic diet. During this period half of the rabbits in each of these groups were treated with DMSO (approximately 5 g/kg) which was included in their drinking water. Food consumption and fluid intakes were monitored daily and body weights at weekly intervals. Total serum cholesterol levels were measured at periodic intervals. Lipid deposits in the eye which accompany atherosclerosis were examined before and at 12 weeks after institution of the new dietary regimens. At the end of 12 weeks, all rabbits were killed and the thoracic aortas were examined for changes in the extent of atherosclerosis. Food consumption and body weight increased in rabbits on the control diet and in those treated with DMSO. Those maintained on the atherogenic diet showed little change in food intake or body weight. Fluid intake was significantly elevated in all rabbits placed on DMSO. Serum cholesterol levels returned to normal in all rabbits on the control diet. Serum cholesterol levels remained unchanged in rabbits kept on the atherogenic diet alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

2%二甲亚砜在血细胞抗凝血保存中的实验探讨

目的探讨用2%二甲亚砜(DMSO)和乙二胺四乙酸二钾(EDTA-K2)组成复合型抗凝剂用于血细胞分析仪标本保存。方法制备含有2%DMSO复合抗凝剂的抗凝管,随机抽取患者血液及已知血小板小于50×109/L患者血液各45份,每份标本分别装于1号管(含EDTA-K2国产抗凝管)、2号管(制备含2%DMSO的复合抗凝管)、3号管(无抗凝剂采集管)。将3号管立即在美国雅培CD1800血细胞分析仪上测定,将1号管和2号管标本分别于室温下1、4、8、12 h测定,数据用x±s表示并采用配对t检验。结果 1号管标本,其平均血小板体积于8h,其值增大,与2、3号管比较,差异有统计学意义(P<0.05),已知血小板小于50×109/L患者的标本在4~8 h与之比较差异均有统计学意义(P<0.05)。2号管在12 h内,随机抽取患者血液及已知血小板小于50×109/L患者的标本各项参数与3号管比较,差异均无统计学意义(P>0.05)。结论初步试验探讨表明,制备2%DMSO和EDTA-K2组成的复合抗凝剂更适用于血细胞分析的血液标本保存,在常规医学检验中为医疗纠纷提供了有利的凭据。