Identification of antigenic epitopes in the haemagglutinin protein of H7 avian influenza virus

ABSTRACT

The H7 subtype avian influenza virus (AIV) has been reported to infect not only poultry but also humans. The haemagglutinin (HA) protein is the major surface antigen of AIV and plays an important role in viral infection. In this study, five monoclonal antibodies (mAbs, 2F8, 3F6, 5C11, 5E2 and 5C12) against the HA protein of H7 virus were produced and characterized. Epitope mapping indicated that ¹⁰³RESGSS¹⁰⁷ was the minimal linear epitope recognized by the mAbs 2F8/3F6/5C11, and mAbs 5E2/5C12 recognized the epitope 103-145aa. The protein sequence alignment of HA indicated that the two epitopes were not found in other subtypes of AIV, and none of the five mAbs cross-reacted with other subtypes, suggesting these mAbs are specific to H7 virus. The epitope ¹⁰³RESGSS¹⁰⁷ was highly conserved among Eurasian lineage strains of H7 AIV, whereas three amino acid substitutions (E104R, E104K and E104G) in the epitope occurred in 98.44% of North-American lineage strains. Any of these single mutations prevented the mutated epitope from being recognized by mAbs 2F8/3F6/5C11; thus, these mAbs can distinguish between Eurasian and North-American lineages of H7 strains. Furthermore, the mAbs 2F8, 3F6 and 5C11 could be highly blocked with H7-positive serum in blocking assays, revealing that ¹⁰³RESGSS¹⁰⁷ may be a dominant epitope stimulating the production of antibodies during viral infection. These results may facilitate future investigations into the structure and function of HA protein, as well as surveillance and detection of H7 virus.

RESEARCH HIGHLIGHTS

• Five mAbs against HA protein of H7 AIV were generated and characterized.

• Two novel epitopes ¹⁰³RESGSS¹⁰⁷ and 103-145aa were identified.

• The epitope ¹⁰³RESGSS¹⁰⁷ differs between Eurasian and North-American lineages.

• The mAbs 2F8, 3F6 and 5C11 could distinguish two lineages of H7 strains.

     

葡甘聚糖-胶原蛋白-壳聚糖共混膜(I)

用溶液共混法制备了葡甘聚糖-胶原蛋白-壳聚糖(KCCS)共混膜。并用FT-IR,X-RD,SEM及透光率表征了膜的结构,同时测试了膜的抗张强度、断裂伸长率、吸水率、透水汽性、渗透性和吸附性。结果表明:共混膜中葡甘聚糖、胶原蛋白及壳聚糖之间存在着强烈的相互作用和良好的相容性,三者共混明显改善了纯聚合物和二元膜的性能。以共混膜为载体培养内皮细胞,发现共混膜具有良好的细胞相容性,预示着共混膜可作为潜在的组织工程支架材料。     

Enhanced expression of transforming growth factor-beta type I and type II receptors in wound granulation tissue and hypertrophic scar.

In the present study we have analyzed and compared, by immunohistochemistry and in situ hybridization, the expression pattern of the R4/ALK5 transforming growth factor (TGF)-beta type I receptor (RI) and the TGF-beta type II receptor (RII) in normal human skin, in wounded skin at various stages during the transition of wound granulation tissue to scar, and in long-persisting post-burn hypertrophic scars. In normal human skin, expression of RI and RII was clearly visible in the epidermis, in epidermal appendages, and in vascular cells, although only a small number of dermal fibroblasts revealed detectable levels of TGF-beta receptor expression. In contrast, granulation tissue fibroblasts showed strong expression of both TGF-beta receptor types, although in normal-healing excisional wounds their density decreased during granulation tissue remodeling. However, in post-burn hypertrophic scars, RI- and RII-overexpressing fibroblasts were found in high densities up to 20 months after injury. From these findings we suggest that the repair process of deep wounds involves the transformation of a subset of fibroblastic cells toward an increased TGF-beta responsiveness and a transient accumulation of these cells at the wound site. In addition, our study provides evidence that excessive scarring is associated with a failure to eliminate TGF-beta receptor-overexpressing fibroblasts during granulation tissue remodeling, which leads to a persistent autocrine, positive feedback loop that results in over-production of matrix proteins and subsequent fibrosis.     

Correlation of AIB1 overexpression with advanced clinical stage of human colorectal carcinoma

AIB1, a member of the steroid receptor coactivator 1 family, has been cloned on 20q12 and is a candidate oncogene in human breast cancer. It is commonly amplified and overexpressed in several types of human cancers. In this study, we examined the expression of AIB1, as related to clinicopathologic features, in 85 human colorectal cancers (CRCs). The status of the number of AIB1 copies, p53 expression, and DNA ploidy was also analyzed. The overexpression of AIB1 was detected in 35% of CRCs. Amplification of AIB1 was observed in 10% of CRCs. In addition, the overexpression of AIB1 was observed more frequently in CRCs in later clinical stages (T3 N1 M0/T3 N0 2M1), compared with that in T3 N0 M0 stage (P < .05). These results suggest that overexpression of AIB1 might provide a selective advantage for the developmental growth and/or progression of subsets of CRCs. In addition, a significant correlation (P < .05) of overexpression of AIB1 with p53 overexpression as well as with aneuploid DNA content was observed in these CRCs. The overexpression of p53 was also correlated significantly with CRC DNA ploidy (P < .05). Furthermore, there was a substantial population of CRCs showing overexpression of both AIB1 and p53 protein and all had aneuploid DNA content; most of these were in the later clinical stage. These findings suggest a possible convergence of AIB1 with a pathway involving p53, which might induce chromosomal instability and affect the clinical phenotype of a subset of CRCs.     

穿戴式呼吸感应体积描记用于睡眠呼吸事件检测

可穿戴式呼吸感应体积描记(背心式RIP)系统是我们根据呼吸感应体积描记技术的基本原理研发的一种可穿戴、低负荷的呼吸监测系统.在实现通气量无创测量的基础上,我们将该系统用于睡眠期呼吸事件检测,将该系统与多导睡眠图仪(PSG)对9例疑似睡眠呼吸暂停低通气综合症(SAHS)病人和7名健康男性志愿者进行同步对照检测与分析.通过对比实验,根据背心式RIP系统发生呼吸事件的特征性变化,提出了背心式RIP系统判别呼吸事件的规则.依据该规则,所有经背心式RIP系统诊断为SAHS患者的结果与PSG的诊断结果完全一致,背心式RIP系统检测呼吸事件的敏感性为97.8%,特异性为95.8%,实验结果表明背心式RIP系统能够可靠地检测出睡眠呼吸事件.由于其低生理、心理负荷特性,不需要佩带口鼻气流传感器,可用于家庭环境下、自然睡眠过程的睡眠呼吸紊乱性疾病的诊断.     

Novel protein microarray for the detection of avian influenza virus antibodies and simultaneous distinction of antibodies against H5 and H7 subtypes

ABSTRACT

Avian influenza virus (AIV) can cause serious zoonotic disease, thereby threatening the poultry industry and human health. An efficient and rapid detection approach is crucial to prevent and control the spread of avian influenza. In this study, a novel protein microarray was developed. Haemagglutinin proteins of H5 and H7 subtypes and nucleoprotein (NP) were purified and spotted onto the initiator-integrated poly-(dimethylsiloxane) as antigens. Monoclonal antibodies with inhibition effect were screened and utilized for the synchronous detection of three avian influenza antibodies in different species. In the protein microarray, the cut-off values were 40%, 50% and 30% inhibition for H5 antibody detection; 50%, 50% and 20% for NP antibody detection; 40%, 50% and 40% for H7 antibody detection in chicken, peacock and duck sera, respectively. The 95 serum samples were detected by microarray, and results were compared with the findings of AIV antibody test enzyme-linked immunosorbent assay (ELISA) or haemagglutination inhibition (HI) test. NP antibody detection in the microarray showed 100% (55/55) agreement ratio in chicken using ELISA. Compared with HI, H5 antibody detection in the microarray showed 100% (95/95) agreement ratio in chicken, peacock and duck, whilst those of H7 displayed 98.18% (54/55) agreement in chicken, 100% (20/20) in peacock and 90% (18/20) in duck. In conclusion, this novel protein microarray is a high-throughput and specific method for the detection of AIV antibodies and simultaneous distinction of antibodies against H5 and H7 subtypes. It can be applied to the serological diagnosis and epidemiological investigation of AIV.

RESEARCH HIGHLIGHTS

• A novel protein microarray method has been developed.

• The microarray can detect AIV antibodies and distinguish between H5 and H7 subtypes.

• The study lays the foundation for simultaneous identification of multiple pathogens.

     

Fluorescence in situ hybridization and immunohistochemical assays for HER-2/neu status determination: application to node-negative breast cancer

BACKGROUND: HER-2/neu (ERBB2) gene amplification and/or overexpression is a major event in human breast tumorigenesis. HER-2/neu gene alterations have been the most frequently assessed prognostic factors during the last 10 years in breast cancer and have recently emerged as a management decision tool and a therapeutic target. There is still controversy over the best method to determine whether a tumor is HER-2/neu positive. Because of the increasing demand for HER-2/neu gene status determination in clinical practice, we compared HER-2/neu gene alterations at the DNA level (gene amplification) and the protein level (overexpression) in a panel of patients with lymph node-negative breast cancer who had received local radiotherapy alone, with no adjuvant therapy. METHODS: We tested 100 excised lymph node-negative breast tumors, using fluorescence in situ hybridization (FISH) with a biotinylated HER-2/neu DNA probe and immunohistochemical assays (IHC) with 2 different antibodies. RESULTS: The FISH frequency of HER-2/neu gene amplification was 15%, and the IHC frequency of overexpression was 21%. CONCLUSION: Although HER-2/neu amplification by FISH and HER-2/neu overexpression by IHC correlated well in this panel of lymph node-negative breast carcinomas, there were a number of discordant cases, pointing to the important need for determining HER-2/neu alteration for the future management of HER-2/neu-based clinical applications.     

经皮穿刺椎间盘髓核摘除术入路的研究

为提供椎间盘髓核摘除术形态学依据，在7具成人横断面标本上观察测量了L1～L5椎间盘平面经皮穿刺椎间盘髓核摘除术的进针点、角度和深度。发现在L1～L3、L4～L5椎间盘髓核的穿刺点以距后正中线8～10cm，9～11cm，穿刺角度以46．7°～59．5°，穿刺深度为110．2～128．9mm为最宜。     

Uptake and pathogenic effects of amyloid beta peptide 1-42 are enhanced by integrin antagonists and blocked by NMDA receptor antagonists

Many synapses contain two types of receptors - integrins and N-methyl-D-aspartate (NMDA) receptors - that have been implicated in peptide internalization. The present studies tested if either class is involved in the uptake of the 42-residue form of amyloid beta peptide (Abeta1-42), an event hypothesized to be of importance in the development of Alzheimer's disease. Cultured hippocampal slices were exposed to Abeta1-42 for 6 days in the presence or absence of soluble Gly-Arg-Gly-Asp-Ser-Pro, a peptide antagonist of Arg-Gly-Asp (RGD)-binding integrins, or the disintegrin echistatin. Abeta uptake, as assessed with immunocytochemistry, occurred in 42% of the slices incubated with Abeta peptide alone but in more than 80% of the slices co-treated with integrin antagonists. Uptake was also found in a broader range of hippocampal subfields in RGD-treated slices. Increased sequestration was accompanied by two characteristics of early stage Alzheimer's disease: elevated concentrations of cathepsin D immunoreactivity and activation of microglia. The selective NMDA receptor antagonist D-(-)-2-amino-5-phosphonovalerate completely blocked internalization of Abeta, up-regulation of cathepsin D, and activation of microglia. Our results identify two classes of receptors that cooperatively regulate the internalization of Abeta1-42 and support the hypothesis that characteristic pathologies of Alzheimer's disease occur once critical intraneuronal Abeta concentrations are reached.