John M. Nicholls

Identification of oxidative stress and toll-like receptor 4 signaling as a key pathway of acute lung injury

Summary Multiple lung pathogens such as chemical agents, H5N1 avian flu, or SARS cause high lethality due to acute respiratory distress syndrome. Here we report that Toll-like receptor 4 (TLR4) mutant mice display natural resistance to acid-induced acute lung injury (ALI). We show that TLR4-TRIF-TRAF6 signaling is a key disease pathway that controls the severity of ALI. The oxidized phospholipid (OxPL) OxPAPC was identified to induce lung injury and cytokine production by lung macrophages via TLR4-TRIF. We observed OxPL production in the lungs of humans and animals infected with SARS, Anthrax, or H5N1. Pulmonary challenge with an inactivated H5N1 avian influenza virus rapidly induces ALI and OxPL formation in mice. Loss of TLR4 or TRIF expression protects mice from H5N1-induced ALI. Moreover, deletion of ncf1, which controls ROS production, improves the severity of H5N1-mediated ALI. Our data identify oxidative stress and innate immunity as key lung injury pathways that control the severity of ALI.

Re-emergence of fatal human influenza A subtype H5N1 disease

Summary Human disease associated with influenza A subtype H5N1 reemerged in January, 2003, for the first time since an outbreak in Hong Kong in 1997. Patients with H5N1 disease had unusually high serum concentrations of chemokines (eg, interferon induced protein-10 [IP-10] and monokine induced by interferon γ [MIG]). Taken together with a previous report that H5N1 influenza viruses induce large amounts of proinflam-matory cytokines from macrophage cultures in vitro, our findings suggest that cytokine dysfunction contributes to the pathogenesis of H5N1 disease. Development of vaccines against influenza A (H5N1) virus should be made a priority.

Lung pathology of fatal severe acute respiratory syndrome

Summary Background Severe acute respiratory syndrome (SARS) is a novel infectious disease with global impact. A virus from the family Coronaviridae has been identified as the cause, but the pathogenesis is still unclear. Methods Post-mortem tissue samples from six patients who died from SARS in February and March, 2003, and an open lung biopsy from one of these patients were studied by histology and virology. Only one full autopsy was done. Evidence of infection with the SARS-associated coronavirus (SARS-CoV) and human metapneumovirus was sought by reverse-transcriptase PCR and serology. Pathological samples were examined by light and electron microscopy and immunohistochemistry. Findings All six patients had serological evidence of recent infection with SARS-CoV. Diffuse alveolar damage was common but not universal. Morphological changes identified were bronchial epithelial denudation, loss of cilia, and squamous metaplasia. Secondary bacterial pneumonia was present in one case. A giant-cell infiltrate was seen in four patients, with a pronounced increase in macrophages in the alveoli and the interstitium of the lung. Haemophagocytosis was present in two patients. The alveolar pneumocytes also showed cytomegaly with granular amphophilic cytoplasm. The patient for whom full autopsy was done had atrophy of the white pulp of the spleen. Electron microscopy revealed viral particles in the cytoplasm of epithelial cells corresponding to coronavirus. Interpretation SARS is associated with epithelial-cell proliferation and an increase in macrophages in the lung. The presence of haemophagocytosis supports the contention that cytokine dysregulation may account, at least partly, for the severity of the clinical disease. The case definition of SARS should acknowledge the range of lung pathology associated with this disease. Published online May 16, 2003 http://image.thelancet.com/extras/03art4347web.pdf

Proinflammatory cytokine responses induced by influenza A (H5N1) viruses in primary human alveolar and bronchial epithelial cells.

BackgroundFatal human respiratory disease associated with influenza A subtype H5N1 has been documented in Hong Kong, and more recently in Vietnam, Thailand and Cambodia. We previously demonstrated that patients with H5N1 disease had unusually high serum levels of IP-10 (interferon-gamma-inducible protein-10). Furthermore, when compared with human influenza virus subtype H1N1, the H5N1 viruses in 1997 (A/Hong Kong/483/97) (H5N1/97) were more potent inducers of pro-inflammatory cytokines (e.g. tumor necrosis factor-a) and chemokines (e.g. IP-10) from primary human macrophages in vitro, which suggests that cytokines dysregulation may play a role in pathogenesis of H5N1 disease. Since respiratory epithelial cells are the primary target cell for replication of influenza viruses, it is pertinent to investigate the cytokine induction profile of H5N1 viruses in these cells.MethodsWe used quantitative RT-PCR and ELISA to compare the profile of cytokine and chemokine gene expression induced by H5N1 viruses A/HK/483/97 (H5N1/97), A/Vietnam/1194/04 and A/Vietnam/3046/04 (both H5N1/04) with that of human H1N1 virus in human primary alveolar and bronchial epithelial cells in vitro.ResultsWe demonstrated that in comparison to human H1N1 viruses, H5N1/97 and H5N1/04 viruses were more potent inducers of IP-10, interferon beta, RANTES (regulated on activation, normal T cell expressed and secreted) and interleukin 6 (IL-6) in primary human alveolar and bronchial epithelial cells in vitro. Recent H5N1 viruses from Vietnam (H5N1/04) appeared to be even more potent at inducing IP-10 than H5N1/97 virus.ConclusionThe H5N1/97 and H5N1/04 subtype influenza A viruses are more potent inducers of proinflammatory cytokines and chemokines in primary human respiratory epithelial cells than subtype H1N1 virus. We suggest that this hyper-induction of cytokines may be relevant to the pathogenesis of human H5N1 disease.

Tropism of avian influenza A (H5N1) in the upper and lower respiratory tract

Poor human-to-human transmission of influenza A H5N1 virus has been attributed to the paucity of putative sialic acid α2-3 virus receptors in the epithelium of the human upper respiratory tract, and thus to the presumed inability of the virus to replicate efficiently at this site. We now demonstrate that ex vivo cultures of human nasopharyngeal, adenoid and tonsillar tissues can be infected with H5N1 viruses in spite of an apparent lack of these receptors.

Sialic acid receptor detection in the human respiratory tract: evidence for widespread distribution of potential binding sites for human and avian influenza viruses

BackgroundInfluenza virus binds to cell receptors via sialic acid (SA) linked glycoproteins. They recognize SA on host cells through their haemagglutinins (H). The distribution of SA on cell surfaces is one determinant of host tropism and understanding its expression on human cells and tissues is important for understanding influenza pathogenesis. The objective of this study therefore was to optimize the detection of α2,3-linked and α2,6-linked SA by lectin histochemistry by investigating the binding of Sambucus nigra agglutinin (SNA) for SAα2,6Gal and Maackia amurensis agglutinin (MAA) for SAα2,3Gal in the respiratory tract of normal adults and children.MethodsWe used fluorescent and biotinylated SNA and MAA from different suppliers on archived and prospectively collected biopsy and autopsy specimens from the nasopharynx, trachea, bronchus and lungs of fetuses, infants and adults. We compared different methods of unmasking for tissue sections to determine if these would affect lectin binding. Using serial sections we then compared the lectin binding of MAA from different suppliers.ResultsWe found that unmasking using microwave treatment in citrate buffer produced increased lectin binding to the ciliated and glandular epithelium of the respiratory tract. In addition we found that there were differences in tissue distribution of the α2,3 linked SA when 2 different isoforms of MAA (MAA1 and MAA2) lectin were used. MAA1 had widespread binding throughout the upper and lower respiratory tract and showed more binding to the respiratory epithelium of children than in adults. By comparison, MAA2 binding was mainly restricted to the alveolar epithelial cells of the lung with weak binding to goblet cells. SNA binding was detected in bronchial and alveolar epithelial cells and binding of this lectin was stronger to the paediatric epithelium compared to adult epithelium. Furthermore, the MAA lectins from 2 suppliers (Roche and EY Labs) tended to only bind in a pattern similar to MAA1 (Vector Labs) and produced a different binding pattern to MAA2 from Vector Labs.ConclusionThe lectin binding pattern of MAA may vary depending on the supplier and the different isoforms of MAA show a different tissue distribution in the respiratory tract. This finding is important if conclusions about the potential binding sites of SAα2,3 binding viruses, such as influenza or human parainfluenza are to be made.

Identification of Novel Epstein-Barr Virus MicroRNA Genes from Nasopharyngeal Carcinomas

ABSTRACT MicroRNAs (miRNAs) represent a conserved class of small noncoding RNAs that are found in all higher eukaryotes as well as some DNA viruses. miRNAs are 20 to 25 nucleotides in length and have important regulatory functions in biological processes such as embryonic development, cell differentiation, hormone secretion, and metabolism. Furthermore, miRNAs have been implicated in the pathology of various diseases, including cancer. miRNA expression profiles not only classify different types of cancer but also may even help to characterize distinct tumor stages, therefore constituting a valuable tool for prognosis. Here we report the miRNA profile of Epstein-Barr virus (EBV)-positive nasopharyngeal carcinoma (NPC) tissue samples characterized by cloning and sequencing. We found that all EBV miRNAs from the BART region are expressed in NPC tissues, whereas EBV miRNAs from the BHRF1 region are not found. Moreover, we identified two novel EBV miRNA genes originating from the BART region that have not been found in other tissues or cell lines before. We also identified three new human miRNAs which might be specific for nasopharyngeal tissues. We further show that a number of different cellular miRNAs, including miR-15a and miR-16, are up- or downregulated in NPC tissues compared to control tissues. We found that the tumor suppressor BRCA-1 is a target of miR-15a as well as miR-16, suggesting a miRNA role in NPC pathogenesis.

Identification of cytotoxic T cell epitopes within Epstein-Barr virus (EBV) oncogene latent membrane protein 1 (LMP1): evidence for HLA A2 supertype-restricted immune recognition of EBV-infected cells by LMP1-specific cytotoxic T lymphocytes

Epstein‐Barr virus (EBV) nuclear antigen 1 (EBNA1) and latent membrane proteins (LMP) are the only antigens consistently expressed in malignancies such as nasopharyngeal carcinoma (NPC) and Hodgkins disease (HD). Since EBNA1 is not recognized by EBV‐specific cytotoxic T lymphocytes (CTL), there is increasing interest in the identification of the potential target epitopes within LMP1. Although LMP1‐specific CTL have been isolated from seropositive individuals, earlier attempts to identify the peptide epitopes recognized by these T cells have been unsuccessful. In the present report we used a novel protocol to identify CTL epitopes within LMP1 which can be recognized by both polyclonal and clonal CTL. Firstly, a computer‐based program was employed to identify the potential HLA‐binding peptides within LMP1. Polyclonal CD8+ CTL were then isolated from seropositive donors that recognized the peptide epitopes YLLEMLWRL and YLQQNWWTL from LMP1 in association with HLA A2. Limiting dilution analysis of the memory CTL response revealed that the LMP1‐specific CTL response constitutes a minor component of the CTL response in healthy virus carriers. Interestingly, analysis of YLLEMLWRL‐specific CTL revealed that these CTL were able to lyse EBV‐infected B cells expressing different HLA A2 supertype alleles including A*0201, A*0202, A*0203, A*0204, A*0206, A*6802 and A*6901. These data strongly support the notion that HLA class I supertype‐restricted CTL may be of significant use in the development of peptide‐based immunotherapeutics against EBV‐associated malignancies in different ethnic populations.

Evolving complexities of influenza virus and its receptors

Sialic acids (Sias) are regarded as receptors for influenza viruses and are usually bound to galactose (Gal) in an alpha2-3 or alpha2-6 configuration. The detection of these Sia configurations in tissues has commonly been through the use of plant lectins that are able to identify which cells contain Siaalpha2-3- and Siaalpha2-6-linked glycans, although other techniques for receptor distribution have been used. Initial experiments indicated that avian versus human influenza virus binding was determined by either Siaalpha2-6 or Siaalpha2-3 expression. In this review, we suggest that the distribution and detection of these terminal Siaalpha2-3- and Siaalpha2-6-linked receptors within the respiratory tract might not be as clear cut as has been reported. We will also review how other viral and receptor components might act as determinants for successful viral replication and transmission. Understanding these additional components is important in comprehending the infection and the transmission of both existing human influenza viruses and newly emerging avian influenza viruses.

Innate immune responses to influenza A H5N1: friend or foe?

Avian influenza A H5N1 remains unusual in its virulence for humans. Although infection of humans remains inefficient, many of those with H5N1 disease have a rapidly progressing viral pneumonia that leads to acute respiratory distress syndrome and death, but its pathogenesis remains an enigma. Comparison of the virology and pathogenesis of human seasonal influenza viruses (H3N2 and H1N1) and H5N1 in patients, animal models and relevant primary human cell cultures is instructive. Although the direct effects of viral replication and differences in the tropism of the virus for cells in the lower respiratory tract clearly contribute to pathogenesis, we focus here on the possible contribution of the host innate immune response in the pathogenesis of this disease.