Efficacy of recombinant Marek’s disease virus vectored vaccines with computationally optimized broadly reactive antigen (COBRA) hemagglutinin insert against genetically diverse H5 high pathogenicity avian influenza viruses

The genetic and antigenic drift associated with the high pathogenicity avian influenza (HPAI) viruses of Goose/Guangdong (Gs/GD) lineage and the emergence of vaccine-resistant field viruses underscores the need for a broadly protective H5 influenza A vaccine. Here, we tested experimental vector herpesvirus of turkey (vHVT)-H5 vaccines containing either wild-type clade 2.3.4.4A-derived H5 inserts or computationally optimized broadly reactive antigen (COBRA) inserts with challenge by homologous and genetically divergent H5 HPAI Gs/GD lineage viruses in chickens. Direct assessment of protection was confirmed for all the tested constructs, which provided clinical protection against the homologous and heterologous H5 HPAI Gs/GD challenge viruses and significantly decreased oropharyngeal shedding titers compared to the sham vaccine. The cross reactivity was assessed by hemagglutinin inhibition (HI) and focus reduction assay against a panel of phylogenetically and antigenically diverse H5 strains. The COBRA-derived H5 inserts elicited antibody responses against antigenically diverse strains, while the wild-type-derived H5 vaccines elicited protection mostly against close antigenically related clades 2.3.4.4A and 2.3.4.4D viruses. In conclusion, the HVT vector, a widely used replicating vaccine platform in poultry, with H5 insert provides clinical protection and significant reduction of viral shedding against homologous and heterologous challenge. In addition, the COBRA-derived inserts have the potential to be used against antigenically distinct co-circulating viruses and future drift variants.     

一种对混养细胞基于形态的细胞分选方法

目的通过对采集的细胞图像的定量识别,并结合基于机器学习的聚类分析,实现对混合培养的多种细胞基于形态的快速识别分选。方法对体外混合培养的A549和3T3两种细胞进行免疫荧光染色以表征其形态轮廓,利用CellProfiler对采集的荧光图片进行细胞形态特征的提取,再通过CellProfiler Analyst对提取的数据进行机器学习,训练出一种规则,形成一种泛化能力,以达到对混合培养的两种细胞进行识别分选的目的。结果训练分类器准确率为81.24%,可以实现A549和3T3细胞的二分类。结论机器学习有助于提升数据聚类分析的准确率,将其应用于细胞图像的识别,可为临床对组织切片进行快速病理检测提供预判断,从而减轻医生的工作量,提高诊断的准确率。     

Demonstration of Bacteroides intermedius in periapical tissue using indirect immunofluorescence microscopy

Abstract The presence of bacteria in periapical lesions of teeth with necrotic pulp has been demonstrated by microbiological sampling of periapical lesions during endodontic surgery. The purpose of this study was to confirm the presence of Bacteroides intermedius in a periapical granuloma using an indirect immunofluorescence technique. Histologic sections of a periapical granuloma were incubated with a rabbit antiserum to B. intermedius. After incubation with secondary antibody (fluorescein-conjugated goat anti-rabbit IgG), an intense fluorescent staining was observed in areas of the tissue sections. Control tissue sections that were incubated without the antibody remained unstained. The results obtained with the indirect immunofluorescence technique supported our cultural findings that microorganisms, e.g. B. intermedius, were present in the tissue of the periapical granuloma.     

量子点荧光探针在人舌鳞状细胞癌组织中的应用研究

目的 通过免疫荧光技术,利用量子点荧光探针在人的舌鳞状细胞癌组织中检测特定蛋白,探讨量子点荧光探针在人舌鳞状细胞癌组织中的应用.方法 相同粒径的量子点通过问接免疫荧光技术分别对人舌鳞状细胞癌组织中的P53及Bcl-2蛋白进行特异荧光标记,荧光显微镜观察蛋白定位表达;不同粒径的量子点通过间接免疫荧光技术,在同一张舌鳞状细...     

Target-mediated clearance and bio-distribution of a monoclonal antibody against the Kunitz–type protease inhibitor 2 domain of Tissue Factor Pathway Inhibitor

Introduction

A humanised monoclonal antibody, concizumab, that binds with high affinity to the Kunitz-type protease inhibitor (KPI) 2 domain of human tissue factor pathway inhibitor (TFPI) is in clinical development. It promotes coagulation by neutralising the inhibitory function of TFPI and may provide a subcutaneous prophylaxis option for patients with haemophilia. We aimed to study biodistribution and pharmacokinetics (PK) of concizumab.

Materials and Methods

Blockage of cellular TFPI by concizumab was measured by tissue factor/Factor VIIa-mediated Factor X activation on human EA.hy926 cells. Biodistribution of concizumab was analysed in rabbits by immunohistology, and the PK was measured in rabbits and rats.

Results and Conclusions

Concizumab bound to cell surface TFPI on EA.hy926 cells and neutralised TFPI inhibition of Factor X activation. The antibody cross-reacted with rabbit TFPI, but not with rat TFPI, allowing for comparative PK studies. PK data in rats described a log-linear profile typical for a non-binding antibody, whereas PK data in rabbits revealed a non-linear, dose-dependent profile, consistent with a target-mediated clearance mechanism. Immunohistology in rabbits during target-saturation showed localisation of the antibody on the endothelium of the microvasculature in several organs. We observed a marked co-localisation with endogenous rabbit TFPI, but a negligible sub-endothelial build-up. Concizumab binds and neutralises the inhibitory effect of cell surface-bound TFPI. The PK profile observed in rabbits is consistent with a TFPI-mediated drug disposition. Double immunofluorescence shows co-localisation of the antibody with TFPI on the endothelium of the microvasculature and points to this TFPI as a putative target involved in the clearance mechanism.     

Antinuclear antibodies (ANA) screening by enzyme immunoassay with nuclear HEp-2 cell extract and recombinant antigens: analytical and clinical evaluation

OBJECTIVES: Immunofluorescence assay (IFA) has been the standard method for antinuclear antibodies (ANA). To simplify and standardize the ANA test, generic ANA solid phase enzyme immunoassay has been promoted. The objective of the present work has been to study the relationship with IFA and the clinical usefulness of a generic EIA for ANA (COBAS Core HEp-2 ANA EIA, Roche Diagnostics). DESIGNS AND METHODS: We studied 74 healthy individuals, 119 patients with defined systemic autoimmune diseases, 26 patients with other autoimmune diseases, and 490 routine samples sent to laboratory for ANA analysis. RESULTS: Precision study showed intra-assay coefficient of variations (CVs) below 8% and inter-assay CVs below 10%. In relation to IFA, a 0.6 kappa index of agreement was obtained. COBAS-ANA concentrations increased according to IFA titer and greatest COBAS-ANA responses were obtained with pure or mixed homogeneous patterns and centromeric patterns. Analysis of COBAS-ANA response to particular antigenic specificities showed that SS-B, Scl-70 and U1sn-RNP specificities were saturating at high concentrations, whereas Jo-1, SS-A and nuclear and centromeric specificities exhibited lower responses. Elevated serum concentrations of IgG and IgM did not interfere COBAS-ANA, but high serum rheumatoid factor (RF) concentrations produced a decrease of ANA. For systemic lupus erythematosus (SLE) patients, the COBAS-ANA best efficiency was obtained with a cut-off of 0.9, with a sensitivity of 97% and a specificity of 88%, whereas the best IFA-ANA efficiency was obtained with a 1:80 dilution, giving a sensitivity of 90% and a specificity of 99%. There were no differences between areas under ROC curves for COBAS-ANA and IFA-ANA. For other systemic and nonsystemic autoimmune diseases sensitivity and specificity of COBAS-ANA were similar or higher than that of 1:160 IFA-ANA titer. CONCLUSION: Sensitivity and specificity of COBAS Core ANA-EIA for SLE and other systemic and nonsystemic autoimmune diseases, together with performance characteristics make it an adequate automated system for ANA screening.