Cross-species comparison illuminates the importance of iron homeostasis for splenic anti-immunosenescence.

Although immunosenescence may result in increased morbidity and mortality, many mammals have evolved effective immune coping strategies to extend their lifespans. Thus, the immune systems of long-lived mammals present unique models to study healthy longevity. To identify the molecular clues of anti-immunosenescence, we first built high-quality reference genome for a long-lived myotis bat, and then compared three long-lived mammals (i.e., bat, naked mole rat, and human) versus the short-lived mammal, mouse, in splenic immune cells at single-cell resolution. A close relationship between B:T cell ratio and immunosenescence was detected, as B:T cell ratio was much higher in mouse than long-lived mammals and significantly increased during aging. Importantly, we identified several iron-related genes that could resist immunosenescence changes, especially the iron chaperon, PCBP1, which was upregulated in long-lived mammals but dramatically downregulated during aging in all splenic immune cell types. Supportively, immune cells of mouse spleens contained more free iron than those of bat spleens, suggesting higher level of ROS-induced damage in mouse. PCBP1 downregulation during aging was also detected in hepatic but not pulmonary immune cells, which is consistent with the crucial roles of spleen and liver in organismal iron recycling. Furthermore, PCBP1 perturbation in immune cell lines would result in cellular iron dyshomeostasis and senescence. Finally, we identified two transcription factors that could regulate PCBP1 during aging. Together, our findings highlight the importance of iron homeostasis in splenic anti-immunosenescence, and provide unique insight for improving human healthspan.

A Sensitive and High-Throughput Flow Cytometry-Based Assay for Measuring Antibody Neutralization of Human Adenovirus Type 3.

The assessment of neutralization activity is an important step in the evaluation of neutralizing antibodies (NAbs). The traditional methods for measuring the antibody neutralization of human adenovirus type 3 (HAdV-3) are the microneutralization (MN) assay, which has insufficient sensitivity, and the plaque reduction neutralization test (PRNT), which is not suitable for high-throughput screening. Herein, we describe the development of a flow cytometry-based neutralization (FCN) assay for measuring the neutralization of sera, cell culture supernatants, and chimeric antibodies against HAdV-3 on the basis of a recombinant HAdV-3 (rHAdV-3) construct expressing the enhanced green fluorescent protein (EGFP). For flow cytometry-based assays, the optimal cell confluence was determined as 90%, and the virus was titrated using the assay. The established FCN assay follows the percentage law and an optimal MOI of not less than 5 × 10-4 was determined by using a purified chimeric antibody. In addition, comparison of the anti-HAdV-3 NAb titers of 72 human serum samples by the MN and FCN assays, showed that both assays correlated strongly with each other. Our FCN assay was an improvement over the MN assay because the observation period was reduced from 3 to 1 days and data analysis could be performed objectively and robotically. Importantly, the newly established FCN assay allows measurement of the neutralization activity of chimeric antibodies expressed in cell culture supernatants. Thus, this sensitive and high-throughput FCN assay is a useful alternative to the MN assay for measuring the antibody neutralization of HAdV-3 and for screening anti-HAdV-3 NAbs in cell culture supernatants.

Development of two antigen-binding fragments to a conserved linear epitope of human adenovirus and their application in immunofluorescence.

Detection of human adenoviruses (HAdVs) in nasopharyngeal swab samples by immunofluorescence assay (IFA) will be valuable for diagnosing HAdV infection, which is a leading cause of severe respiratory tract disease, and will help in curbing the spread of HAdV. Monoclonal antibodies employed in IFA for HAdV detection should ideally target highly conserved epitope types. Here, we describe the development of two antigen-binding fragments (Fabs) with specific reactivity to HAdV using phage antibody library technology. When tested with IFA, both Fabs recognized cells infected with several types of HAdV, some of which have been identified in epidemics globally, or associated with outbreaks of severe or fatal acute respiratory diseases. The specificity and cross-reactivity of both Fabs to HAdVs indicated that the generated Fabs could be applied in the development of IFAs to detect HAdVs. Both Fabs bound to the knob proteins, as shown by chemiluminescence enzyme immunoassay and western blot. In addition, epitope mapping showed that both Fabs recognized a conserved linear epitope among several types of HAdV. Two different Fabs recognized the same epitope, suggesting that the epitope triggered the production of at least two kinds of antibodies in the body. The generated Fabs exerted no neutralization against HAdVs. The results demonstrate that both Fabs bind to an epitope that plays no role in neutralization of HAdV.