Novel multi-probe RNase protection assay set for detection of endotoxin associated receptors gene expression

Objective: To construct the multi-probe ribonuclease protection assay (RPA) template set to be used for detecting expression patterns of MD-2, TLR4, CD14 mRNAs in human peripheral blood mononuclear cells. Methods : The designed cDNA fragments of the three genes were generated by polymerase chain reaction (PCR)using specific primers and directionally cloned into EcoR I and Hind III sites of expression plasmid pSP72 containing the T7/ promoter, the linearized plasmids was used as template to synthesize anti-sense RNA probes. Then we extracted total RNA from peripheral blood mononuclear cells (PBMC) and detected the dynamic expression patterns of the three genes with RPA method. Results: The proper sequence and orientation of the template set were confirmed by sequencing and the template set was successfully used to assay TLR4, MD-2 and CD14 mRNAs in human PBMC. The results showed that the three detected genes decreased transiently 1-3 hours after 100 ng/ml LPS stimulation. Conclusions: These new RPA multi-probe set provided valuable tool for the simultaneous quantitative determination of expression of TLR4, CD14 and MD-2 mRNAs in both constitutive and inducible types.     

协同刺激分子CD28在结核杆菌抗原体外激活人外周血γδT细胞中的作用

目的 :为了探讨CD2 8协同刺激分子在结核杆菌 (Mtb)低分子多肽抗原体外激活人外周血γδ T细胞中的作用。方法 :采用激发型抗CD2 8单抗模拟第二信号 ,Mtb低分子多肽抗原作为刺激原 ,对纯化的人外周血T细胞进行体外刺激和培养 ;用流式细胞仪检测γδ T细胞上CD2 8分子的表达、γδ T细胞的增殖效应及活化的γδ T细胞上CD6 9分子的表达。结果 :人外周血γδ T细胞中有 5 0 %左右表达CD2 8分子 ;抗CD2 8单抗协同Mtb抗原可刺激γδ T细胞的活化和增殖 ;但抗CD2 8单抗或Mtb抗原单独刺激则无作用。活化的γδ T细胞表面表达CD6 9分子。结论 :Mtb抗原在选择性活化人外周血γδ T细胞时需要第二信号的参与 ;CD2 8在Mtb抗原激活γδ T细胞时可提供协同刺激信号 ;CD6 9可作为γδ T细胞的早期活化标志。     

共刺激分子CD80和CD86在蠕虫保护性免疫及其Th2免疫应答中作用机制的研究

目的:探讨共刺激分子CD80和CD86在蠕虫保护性免疫及其Th2免疫应答中的作用机制。方法:采用巴西日圆线虫第3期幼虫感染BALB/c小鼠,在感染当天、感染后第3、7d分别腹腔注射大鼠抗CD80和/或抗CD86单克隆抗体,以阻断协同刺激信号。在感染当天,感染后第7、14d,计数小鼠外周血液的嗜酸性粒细胞,于感染后第14d计数小肠绒毛嗜酸性粒细胞;采用WatanabeN法测定IgE。结果:联合应用抗CD80和抗CD86两种单克隆抗体可导致成虫产卵量明显增加,成虫数量显著增多及排虫时间延迟。同时两种单克隆抗体也完全抑制了外周血液及小肠组织的嗜酸性粒细胞的增多并部分抑制了IgE水平的升高。而单独应用抗CD80或抗CD86McAb对保护性免疫及Th2型免疫应答反应均无明显影响。结论:在蠕虫保护性免疫和Th2型免疫应答反应中,T细胞的活化需要CD80和CD86两种共刺激分子的参与;而CD80或CD86单一刺激分子即可提供足够的共刺激信号。研究提示,人为调控CD80和CD86共刺激分子可能有助于对蠕虫病及Th2型免疫应答所介导的其它疾病的治疗。     

三七多糖对创伤大鼠CD+4/CD+8和IL-2水平的影响(摘要)

观察三七多糖对创伤大鼠免疫功能的影响.方法:40只SD大鼠,随机分为正常对照组(A)、创伤对照组(B)、创伤后三七多糖28.5 mg·kg-1.     

Expression of CD44V2 in transitional cell carcinoma of the urinary bladder and in urine

CD44 is the principal cell surface receptor for hyaluronate. Variant forms of the receptor, produced by alternative splicing, have been found to be associated with tumor progression in a variety of cancers. Based on investigations at the RNA level, it has recently been proposed that expression of CD44 variant V2 was present in urothelial cancer but not in normal urothelium. Since a distinctive marker for urothelial cancer would be extremely useful, frozen sections of normal urothelium and urothelial cancer were examined for expression of standard CD44 and CD44V2. Frozen sections of specimens of 35 patients with transitional cell carcinoma of the bladder, 16 specimens of normal bladder and 5 ureters were examined. Immunohistochemical staining was performed using a polyclonal antibody to CD44V2 (PAB CD44V2), a monoclonal antibody to CD44V2 (MAB CD44V2) and a monoclonal antibody to CD44S (MAB CD44S). CD44V2 and CD44S were also measured in lysates of urine sediments from 21 patients by enzyme-linked immunoabsorbent assay (ELISA). All investigated transitional cell carcinomas expressed CD44V2. There was no differentiation between invasive and noninvasive carcinoma. CD44V2 was also expressed in normal urothelium. Standard CD44 was expressed by the transitional cell carcinoma, normal urothelium, musculature and interstitial tissue. The amount of CD44V2 and CD44S in lysates of urine sediments is not correlated to diagnosis. In contrast to investigations at the RNA level, CD44V2 on the protein level seems not to be a distinctive marker for urothelial cancer. Therefore, CD44V2 will not be a useful diagnostic marker for detection of transitional cell carcinoma.     

Soluble CD14 but not interleukin-6 is a new marker for clinical activity in atopic dermatitis

Summary Levels of soluble IL-2 receptors, IL-6, soluble CD23, soluble CD14 and ECP (eosinophilic cationic protein) were measured as markers of T-cell, B-cell, monocyte and eosinophilic leucocyte activation in 26 patients with atopic dermatitis (AD) on admission to (A) and at discharge from (D) the Department of Dermatology in Zurich. The serum levels of sIL-2R, IL-6, sCD23, sCD14 and ECP were significantly elevated in AD patients in comparison with the normal values of healthy donors. A significant decrease in sIL-2R (p=0.0093) and in sCD14 (p=0.0134) levels was demonstrated between A and D, correlating with the improvement in the skin intensity score (SIS). In addition, a significant correlation of the sCD14 levels and the SIS at A was demonstrated (p=0.0415). These results also incriminate monocytes in the pathogenesis of AD, indicating that, besides sIL-2R and ECP, SCD14 could also be a possible marker for the disease activity.     

The expression of the B-cell marker mb-1 (CD79a) in Hodgkin's disease

Recent evidence indicates that membrane-bound immunoglobulin on B lymphocytes is associated with a molecule which comprises the products of the mb-1 and B29 genes. This molecule is a highly specific marker for B-cells, presumably because of its central functional role in antigen triggering, and has recently been clustered as CD79a at the 5th Leucocyte Workshop. Recently there has been controversy surrounding reports of B-cell antigen expression by Reed–Sternberg and related cells, and we have therefore studied 108 cases of Hodgkin's disease immunohistochemically using a novel antibody which detects mb-1 protein in paraffin sections. The results were compared with those achieved using antibody L26 to detect CD20. The mb-1 protein was present in the neoplastic cells in all 14 cases of lymphocyte predominance Hodgkin's disease studied, and CD20 immunoreactivity was also found in seven of the eight cases of this subtype studied. Of the non-lymphocyte predominance cases, 20% (19/94) expressed mb-1 and 30% (20/67) CD20 in the Reed–Sternberg cells, but the cells positive for either of these two markers usually constituted only a very small proportion of the neoplastic population. However, in occasional cases (one of 94 for mb-1 and five of 67 for CD20), more than 50% of the neoplastic cells expressed one or both B-cell antigens. These results confirm the B-cell origin of the neoplastic cells in lymphocyte predominance Hodgkin's disease, but they also indicate that, contrary to our previous study, mb-1 expression may occasionally be found in what appears, on histological grounds, to be other types of Hodgkin's disease.     

IgE and atopy in perinatally HIV-infected children

Elevated serum immunoglobulin E (IgE) and increased prevalence of atopy is reported in patients infected with human immunodeficiency virus (HIV). The elevated serum IgE may be attributed to polyclonal stimulation of B cells or IgE production against allergens, viruses, fungi and bacteria. This study investigates the prevalence of atopy in perinatally HIV-infected children, and the relationships between serum IgE (and other serum immunoglobulins) with atopy, CD4+ cell count and HIV-disease stage. Serum immunoglobulin levels, epicutaneous skin test for common aeroallergens, clinical Centers for Disease Control and Prevention (CDC) classification, CD4+ cell counts and allergy history were extracted from the charts of perinatally HIV-infected children on highly active antiretroviral therapy. The prevalence of atopy (52%) and the pattern of aeroallergen sensitivity were comparable with the US pediatric population. Serum IgE levels did not correlate with clinical disease stage. However, in non-atopic patients, serum IgE levels increased with disease progression (p = 0.02). There was an inverse relationship between the prevalence of elevated serum IgE levels and atopy with progression of disease (p = 0.019). Serum IgE did not correlate with atopy, CD4+ cell count, or duration of HIV infection or levels of serum immunoglobulins. This is the first study to show no increased prevalence of atopy in perinatally HIV-infected children compared with the general population. In advanced stages of HIV, elevated serum IgE may be specific for antigens other than those known as allergens.     

Production and characterization of a new monoclonal antibody effective in recognizing the CD3 T-cell associated antigen in formalin-fixed embedded tissue

Phenotypic analysis of lymphoproliferative disorders is now considered mandatory for accurate classification which is the basis for optimum patient management. This is presently carried out in most cases using a range of antibodies recognizing B and T-cell antigens effective in paraffin sections, and an antibody to CD3 is currently a key member of such panels, indicating T-cell phenotype. Current antibodies to CD3 are polyclonal with the inherent disadvantages of this type of reagent compared to monoclonal antibodies. In this study, we have used a recombinant fusion protein representing part of the epsilon subunit of the CD3 molecule to generate a novel monoclonal antibody (NCL-CD3-PS1) effective in paraffin sections. The antibody has been characterized biochemically and by immunohistochemistry using a wide range of normal and pathological tissues. Lineage and phenotype specificity have been supported in our study and results from other laboratories are awaited with interest.