樟脑气味嗅觉条件刺激诱发小鼠条件反射性抗体增强的实验研究

目的 对流感疫苗(流感病毒血凝素表面抗原)的接种,观察条件反射性免疫反应.方法 采用36只BALB/c小鼠肌肉注射流感疫苗抗原(3 μg/每只动物)为非条件性刺激,与樟脑气味嗅觉条件刺激一次性结合.第6周末再次给予条件刺激,观察条件反射性流感疫苗的抗体反应.结果 条件刺激组在一次性条件刺激后,抗流感疫苗的抗体水平OD值增高(第9周0.68±0.06;第10周0.60±0.06),与非条件刺激组(第9周0.53±0.06;第10周0.48±0.04)比较差异有显著性( P <0.01).与条件训练对照组和非条件训练对照组比较差异有显著性( P <0.05).结论 经过一次樟脑气味嗅觉条件刺激与流感疫苗非条件刺激的结合训练后,单独条件刺激能够诱导动物出现条件反射性抗体增强反应.     

Ethanol selectively impairs clathrin-mediated internalization in polarized hepatic cells

Although alcoholic liver disease is clinically well-described, the molecular basis for alcohol-induced hepatotoxicity is not well understood. Previously, we determined that the clathrin-mediated internalization of asialoglycoprotein receptor was impaired in ethanol-treated WIF-B cells whereas the internalization of a glycophosphatidylinositol-anchored protein thought to be endocytosed via a caveolae/raft-mediated pathway was not changed suggesting that clathrin-mediated endocytosis is selectively impaired by ethanol. To test this possibility, we examined the internalization of a panel of proteins and compounds internalized by different mechanisms in control and ethanol-treated WIF-B cells. We determined that the internalization of markers known to be internalized via clathrin-mediated mechanisms was impaired. In contrast, the internalization of markers for caveolae/raft-mediated endocytosis, fluid phase internalization or non-vesicle-mediated uptake was not impaired in ethanol-treated cells. We further determined that clathrin heavy chain accumulated at the basolateral surface in small puncta in ethanol-treated cells while there was decreased dynamin-2 membrane association. Interestingly, the internalization of resident apical proteins that lack any known internalization signals was also disrupted by ethanol suggesting that these proteins are internalized via clathrin-mediated mechanisms. This conclusion is consistent with our findings that dominant negative dynamin-2 overexpression impaired internalization of known clathrin markers and single spanning apical residents, but not of markers of fluid phase or raft-mediated internalization. Together these results indicate that ethanol exposure selectively impairs hepatic clathrin-mediated internalization by preventing vesicle fission from the plasma membrane.     

A179L, a viral Bcl-2 homologue, targets the core Bcl-2 apoptotic machinery and its upstream BH3 activators with selective binding restrictions for Bid and Noxa

Several large DNA viruses encode Bcl-2 protein homologues involved in the regulation of the cellular apoptosis cascade. This regulation often involves the interaction of these viral proteins with diverse cellular Bcl-2 family members. We have identified the specific interactions of A179L, an African swine fever virus (ASFV) Bcl-2 homologue, with the active forms of the porcine BH3-only Bid protein (truncated Bid p13 and p15). Transient expression of ASFV A179L gene in Vero cells prevented apoptosis induced by these active forms of Bid protein. Interestingly, A179L protein was able to interact, also with the main core Bcl-2 proapoptotic proteins Bax and Bak, and with several BH3-only proteins with selective binding restrictions for full length Bid and Noxa. These results suggest a fine regulation for A179L action in the suppression of apoptosis in infected cells which is essential for efficient virus replication.     

Characterizing immunodominant and protective influenza hemagglutinin epitopes by functional activity and relative binding to major histocompatibility complex class II sites

In the present study the analysis of functional activity and major histocompatibility complex (MHC) binding of two adjacent MHC class II-restricted epitopes, located in the C-terminal 306–329 region of human influenza A virus hemagglutinin 1 subunit (HA1) conserved with subtype sequences and not affected by antigenic drift, was undertaken to explore the hierarchy of local immnodominance. The functional activity of two T cell hybridomas of the memory/effector Th1 phenotype in combination with in vivo immunization studies provided a good tool for investigating the functional characteristics of the T cell resonse. The in vitro binding assays performed with a series of overlapping, N-terminal biotinylated peptides covering the 306–341 sequence enabled us to compare the relative binding efficiency of peptides, comprising two distinct epitopes of this region, to I-E^d expressed on living antigen-presenting cells. Our studies revealed that (i) immunization of BALB/c mice with the 306–329 H1 or H2 peptides resulted in the activation and proliferation of T cells recognizing both the 306–318 and the 317–329 epitopes, while the 306–329 H3 peptide elicits predomonantly 306–318-specific T cells, (ii) the 317–329 HA1 epitope of the H1 and H2 but not the H3 sequence is recognized by T cells and is available for recognition not only in the 317–329 peptide but also in the extended 306–329 or 306–341 peptides, (iii) the 306–318 and the 317–329 hemagglutinin peptides encompassing the H1, H2 but not the H3 sequence bind with an apparently similar affinity to and therefore compete for I-E^d binding sites, and (iv) the 317–341, the 317–329 peptides and their truncated analogs show subtype-dependent differences in MHC binding and those with lower binding capacity represent the H3 subtype sequences. These results demonstrate that differences in the binding capacity of peptides comprising two non-overlapping epitopes located in the C-terminal 306–329 region of HA1 of all three subtype-specific sequences to MHC class II provide a rationale for the local and also for the previously observed in vivo immunodominance of the 306–318 region over the 317–329 epitope in the H3 but not in the H1 or H2 sequences. In good correlation with the results of the binding and functional inhibition assays, these data demonstrate that in the H1 and H2 subtypes both regions are available for T cell recognition, they compete for the same restriction element with an appearently similar binding efficiency and, therefore, function as co-dominant epitopes. Due to the stabilizing effect of the fusion peptide, peptides comprising the 306–341 or 317–341 H1 sequences are highly immunogenic and elicit a protective immune response which involves the production of antibodies and interleukin-2 and tumor necrosis factor producing effector Th1 cells both directed against the 317–329 region. Based on the similarity of the I-E^d and HLA-DR1 peptide binding grooves and motifs, these results suggest that amino acid substitutions inserted to the H3 subtype sequence during viral evolution can modify the relative MHC binding capacity and invert the local hierarchy of immunodominance of two closely situated epitopes that are able to bind to the same MHC class II molecule.     

Characterization of glycan binding specificities of influenza B viruses with correlation with hemagglutinin genotypes and clinical features

The carbohydrate binding specificities are different among avian and human influenza A viruses and may affect the tissue tropism and transmission of these viruses. The glycan binding biology for influenza B, however, has not been systematically characterized. Glycan binding specificities of influenza B viral isolates were analyzed and correlated to hemagglutinin (HA) genotypes and clinical manifestations. A newly developed solution glycan array was applied to characterize the receptor binding specificities of influenza B virus clinical isolates from 2001 to 2007 in Taiwan. Thirty oligosaccharides which include α‐2,3 and α‐2,6 linkage glycans were subjected to analysis. The glycan binding patterns of 53 influenza B isolates could be categorized into three groups and were well correlated to their HA genotypes. The Yamagata‐like strains predominantly bound to α‐2,6‐linkage glycan (24:29, 83%) while Victoria‐like strains preferentially bound to both α‐2,3‐ and α‐2,6‐linkage glycans (13:24, 54%). A third group of viruses bound to sulfated glycans and these all belonged to Victoria‐like strains. Based on the HA sequences, Asn‐163, Glu‐198, Ala‐202, and Lys‐203 were conserved among Victoria‐like strains which may influence their carbohydrate recognition. The viruses bound to dual type glycans were more likely to be associated with the development of bronchopneumonia and gastrointestinal illness than those bound only to α‐2,6 sialyl glycans (P < 0.05). Glycan binding analyses provide additional information to monitor the antigenic shift, tissue tropism, and transmission capability of influenza B viruses, and will contribute to virus surveillance and vaccine strain selection. J. Med. Virol. 84:679–685, 2012. © 2011 Wiley Periodicals, Inc.     

Structural insights into key sites of vulnerability on HIV-1 Env and influenza HA

Human immunodeficiency virus-1 (HIV-1) envelope protein (Env) and influenza hemagglutinin (HA) are the surface glycoproteins responsible for viral entry into host cells, the first step in the virus life cycle necessary to initiate infection. These glycoproteins exhibit a high degree of sequence variability and glycosylation, which are used as strategies to escape host immune responses. Nonetheless, antibodies with broadly neutralizing activity against these viruses have been isolated that have managed to overcome these barriers. Here, we review recent advances in the structural characterization of these antibodies with their viral antigens that defines a few sites of vulnerability on these viral spikes. These broadly neutralizing antibodies tend to focus their recognition on the sites of similar function between the two viruses: the receptor-binding site and membrane fusion machinery. However, some sites of recognition are unique to the virus neutralized, such as the dense shield of oligomannose carbohydrates on HIV-1 Env. These observations are discussed in the context of structure-based design strategies to aid in vaccine design or development of antivirals.     

Carbohydrate–protein interactions and multivalency: implications for the inhibition of influenza A virus infections

Introduction: Protein–carbohydrate interactions play a very important role in many biological processes. A single interaction between a protein and a carbohydrate is usually weak, but multivalent ligands can compensate for this deficiency by binding multiple binding sites to one biological entity simultaneously. Over the past few years, numerous efforts have been made for the design and synthesis of carbohydrate-based multivalent ligands thereby serving as potent inhibitors for pathogens such as the influenza A virus.

Areas covered: In this review, the authors cover a variety of multivalent systems from small to large molecules which showed a potent inhibitory effect against several pathogens.

Expert opinion: Scaffold structure, linker type, and ligand density are important parameters that need to be optimized for potent multivalent inhibitors. The challenges of multivalent glycodrugs include issues such as bioavailability, pharmacokinetics, and immunogenicity which greatly depend on where the compounds are used in the body. Anti-flu (influenza) applications in the lungs using multivalent carbohydrates particularly has potential because of the high binding affinities. With much more research focusing on Influenza A virus inhibition, therapeutic applications may be achieved in the near future.     

Mitigation strategies to reduce the generation and transmission of airborne highly pathogenic avian influenza virus particles during processing of infected poultry

Airborne transmission of H5N1 highly pathogenic avian influenza (HPAI) viruses has occurred among poultry and from poultry to humans during home or live-poultry market slaughter of infected poultry, and such transmission has been experimentally reproduced. In this study, we investigated simple, practical changes in the processing of H5N1 virus-infected chickens to reduce infectious airborne particles and their transmission. Our findings suggest that containing the birds during the killing and bleeding first step by using a disposable plastic bag, a commonly available cooking pot widely used in Egypt (halla), or a bucket significantly reduces generation of infectious airborne particles and transmission to ferrets. Similarly, lack of infectious airborne particles was observed when processing vaccinated chickens that had been challenged with HPAI virus. Moreover, the use of a mechanical defeatherer significantly increased total number of particles in the air compared to manual defeathering. This study confirms that simple changes in poultry processing can efficiently mitigate generation of infectious airborne particles and their transmission to humans.     

Influenza vaccine viruses and the development of seasonal vaccines: A Japanese perspective

In Japan, the Ministry of Health, Labour and Welfare (MHLW) designates one specific virus strain for each component of the quadrivalent seasonal influenza vaccine, and four domestic manufacturers produce egg-based influenza vaccines with the same formulation (inactivated, split-virus) using uniform vaccine strains. Thus, discussions of the development of effective seasonal influenza vaccines so far has focused solely on the antigenic match between the vaccine strains and epidemic viruses. However, in 2017, the Japanese selection system of vaccine viruses demonstrated that even a candidate vaccine virus that is antigenically similar to the predicted circulating viruses is not necessarily suitable for vaccine production, given lower productivity of the vaccine. Taking this experience into account, the MHLW reformed the scheme of vaccine strain selection in 2018, and instructed the Vaccine Epidemiology Research Group created by the MHLW to probe how the virus strains for the seasonal influenza vaccine should be selected in Japan. In this context, a symposium, entitled “Issues of the Present Seasonal Influenza Vaccines and Future Prospects”, was held as part of the 22nd Annual Meeting of the Japanese Society for Vaccinology in 2018, and subjects related to the influenza vaccine viruses were discussed among relevant administrators, manufacturers, and researchers. This report summarizes the presentations given at that symposium in order to convey the present scheme of vaccine virus selection, the evaluation of the resulting vaccines, and the efforts at new vaccine formulation in Japan. Notably, from March 2022, the MHLW has launched a discussion of the merits of the seasonal influenza vaccines produced by foreign manufacturers.     

聚桂醇和血凝酶局部注射联合密集套扎治疗肝硬化伴食管静脉曲张破裂出血的临床研究

目的 探讨聚桂醇和血凝酶局部注射并联合密集套扎法治疗肝硬化伴食管静脉曲张破裂出血(EVB)的临床疗效.方法 选取2016年2月-2019年12月在该科住院的肝硬化伴EVB患者45例,随机分为两组.对照组(n=21)采用密集套扎治疗,治疗组(n=24)在密集套扎治疗的基础上,再在每两个结扎点曲张静脉内注射混合液(聚桂醇1...