Mink Lung Epithelial Cells: Unique Cell Line That Supports Influenza A and B Virus Replication

We have demonstrated for the first time that a mink lung epithelial cell line (Mv1Lu) supports the replication of influenza A and B viruses, including the recently isolated H5N1 avian and human Hong Kong strains, to titers comparable to those in MDCK cells. These results suggest that Mv1Lu cells might serve as an alternative system for the isolation and cultivation of influenza A and B viruses and may be useful for vaccine development.     

Adhesion of Tritrichomonas foetus to Bovine Vaginal Epithelial Cells

An in vitro culture system of bovine vaginal epithelial cells (BVECs) was developed to study the cytopathogenic effects of Tritrichomonas foetus and the role of lipophosphoglycan (LPG)-like cell surface glycoconjugates in adhesion of parasites to host cells. Exposure of BVEC monolayers to T. foetus resulted in extensive damage of monolayers. Host cell disruption was measured quantitatively by a trypan blue exclusion assay and by release of (3)H from [(3)H]thymidine-labeled host cells. Results indicated contact-dependent cytotoxicity of host cells by T. foetus. The cytopathogenic effect was a function of T. foetus density. Metronidazole- or periodate-treated T. foetus showed no damage to BVEC monolayers. A related human trichomonad, Trichomonas vaginalis, showed no cytotoxic effects, indicating species-specific host-parasite interactions. A direct binding assay was developed and used to investigate the role of a major cell surface LPG-like molecule in host-parasite adhesion. The results of competition experiments showed that the binding to BVECs was displaceable, was saturable, and yielded a typical binding curve, suggesting that specific receptor-ligand interactions mediate the attachment of T. foetus to BVECs. Progesterone-treated BVECs showed enhanced parasite binding. T. foetus LPG inhibited the binding of T. foetus to BVECs; the LPG from T. vaginalis and a variety of other glycoconjugates did not. These data imply specificity of LPG on host-parasite adhesion. Periodate-treated parasites showed no adherence to host cells, indicating the involvement of carbohydrate containing molecules in the adhesion process.     

PspK of Streptococcus pneumoniae Increases Adherence to Epithelial Cells and Enhances Nasopharyngeal Colonization

Streptococcus pneumoniae (the pneumococcus) colonizes the human nasopharynx and can cause invasive disease aided by the pneumococcal capsule. Group II nontypeable S. pneumoniae (NTSp) lacks a polysaccharide capsule, and a subgroup of NTSp carriage isolates has been found to have a novel gene, pneumococcal surface protein K (pspK), which replaces the capsule locus. A recent rise in the number of NTSp isolates colonizing the human nasopharynx has been observed, but the colonization factors of NTSp have not been well studied. PspK has been shown to play a role in mouse colonization. We therefore examined PspK-mediated immune evasion along with adherence to host cells and colonization. PspK bound human secretory immunoglobulin A (sIgA) but not the complement regulator factor H and did not decrease C3b deposition on the pneumococcal surface. PspK increased binding of pneumococci to epithelial cells and enhanced pneumococcal colonization independently of the genetic background. Understanding how NTSp colonizes and survives within the nasopharynx is important due to the increase in NTSp carriage. Our data suggest that PspK may aid in the persistence of NTSp within the nasopharynx but is not involved in invasion.     

Adherence of Human Vaginal Lactobacilli to Vaginal Epithelial Cells and Interaction with Uropathogens

Three strains of Lactobacillus, identified as Lactobacillus acidophilus, Lactobacillus gasseri, and Lactobacillus jensenii, were selected from among 70 isolates from the vaginas of healthy premenopausal women for properties relevant to mucosal colonization or antagonism. All three self-aggregated and adhered to epithelial vaginal cells, displacing well-known vaginal pathogens, such as G. vaginalis, and inhibiting the growth in vitro of Escherichia coli and Streptococcus agalactiae. The surface components involved in self-aggregation appeared to be proteins for L. gasseri and lipoproteins for L. acidophilus and L. jensenii, as judged by susceptibility to treatment with appropriate degrading enzymes. The factors responsible for adherence to epithelial vaginal cells seemed to be glycoproteins (L. acidophilus and L. gasseri) and carbohydrate (L. jensenii). The receptors of the vaginal cells were glycolipids, which presumably were the targets of the competition observed between the lactobacilli and the pathogenic microbes.     

Cytotoxic T Cells in the Lungs of Mice Infected with an Influenza A Virus

Cytotoxic T cells are present in the lungs and the bronchoalveolar washings of mice infected intravenously (i.v.) or intranasally (i.n.) with live influenza A/WSN virus. After i.v. injection, cytotoxic T cell activity in both spleens and lungs reaches a peak at 6 days when the level of infectious virus recovered from the lungs falls sharply and the mice do not die. If a lethal dose of virus is given intranasally, very high levels of virus appear rapidly in the lungs, and the development of lung consolidation follows slightly behind the appearance of cytotoxic T cells there. When a non-lethal dose of virus is given intranasally, lower levels of virus are found in the lung and the appearance of cytotoxic T cells is delayed. These results suggest that the cytotoxic T cells play a protective role if the level of virus in the lungs does not reach very high levels. After injection of antithymocyte serum, the subsequent level of cytotoxic T cell activity in the lungs was greatly reduced, suggesting that the T cells recovered in lungs had at an earlier stage been circulating cells. However, splenectomized mice develop high levels of cytotoxic T cell activity, after intranasal infection of mice, indicating that the spleen did not contribute substantially to the T cells recovered in the lungs.     

H1N2新亚型流感病毒神经氨酸酶基因来源的进一步研究

对流感病毒Ｈ１Ｎ２亚型重组株Ａ／哈防／１／８８、Ａ／哈防／１２／９２以及Ｈ３Ｎ２亚型病毒株Ａ／雅防／２／８７、Ａ／京防／５７／８９８和Ａ／粤防／１／９２神经氨酸酶（ＮＡ）基因核苷酸全序列的测定，进一步弄清了Ａ／哈防／１／８８病毒株的ＮＡ基因确来自当时人群中流行的Ｈ３Ｎ２亚型病毒株，很可能是来自Ａ／雅防／２／８７类病毒株；而Ａ／哈防／１２／９２病毒株的ＮＡ基因可能是与Ａ／哈防／１／８８病毒株的ＮＡ基     

Cells Mediating Delayed-Type Hypersensitivity in the Lungs of Mice Infected with an Influenza A Virus

Effector cells that demonstrate delayed-type hypersensitivity (DTH) on transfer with antigen to naive mice can he recovered from the lungs of mice inoculated intranasally 6 days earlier with a lethal dose (usually 5 × 10⁴EID₅₀) of influenza A virus. The activity recovered was proportional to the dose of virus instilled intranasally and the extent of lung consolidation observed. Active cells could also be recovered from the draining lymph nodes and from the peripheral blood. The effector cells were identified as T lymphocytes of Ly I phenotype and required I-region sharing between donor and recipient for activity to be elicited. They were cross-reactive within the A group of influenza viruses. Two experiments are reported in which immune cell preparations that expressed DTH activity but had very little cytotoxic T cell activity were transferred to mice inoculated 1 or 2 days earlier with a lethal dose of virus. The mice were not protected from death, and in both experiments, the recipient mice died more rapidly than the controls. These results contrast with earlier results in which cell preparations with high cytotoxic T-cell activity were shown to protect recipient infected mice from death.     

Interaction of Mycoplasma gallisepticum with Chicken Tracheal Epithelial Cells Contributes to Macrophage Chemotaxis and Activation

Mycoplasma gallisepticum colonizes the chicken respiratory mucosa and mediates a severe inflammatory response hallmarked by subepithelial leukocyte infiltration. We recently reported that the interaction of M. gallisepticum with chicken tracheal epithelial cells (TECs) mediated the upregulation of chemokine and inflammatory cytokine genes in these cells (S. Majumder, F. Zappulla, and L. K. Silbart, PLoS One 9:e112796, http://dx.doi.org/10.1371/journal.pone.0112796). The current study extends these observations and sheds light on how this initial interaction may give rise to subsequent inflammatory events. Conditioned medium from TECs exposed to the virulent R_low strain induced macrophage chemotaxis to a much higher degree than the nonvirulent R_high strain. Coculture of chicken macrophages (HD-11) with TECs exposed to live mycoplasma revealed the upregulation of several proinflammatory genes associated with macrophage activation, including interleukin-1β (IL-1β), IL-6, IL-8, CCL20, macrophage inflammatory protein 1β (MIP-1β), CXCL-13, and RANTES. The upregulation of these genes was similar to that observed upon direct contact of HD-11 cells with live M. gallisepticum. Coculture of macrophages with R_low-exposed TECs also resulted in prolonged expression of chemokine genes, such as those encoding CXCL-13, MIP-1β, RANTES, and IL-8. Taken together, these studies support the notion that the initial interaction of M. gallisepticum with host respiratory epithelial cells contributes to macrophage chemotaxis and activation by virtue of robust upregulation of inflammatory cytokine and chemokine genes, thereby setting the stage for chronic tissue inflammation.     

Identification of Campylobacter jejuni Genes Involved in Its Interaction with Epithelial Cells

Campylobacter jejuni is the leading cause of infectious gastroenteritis in industrialized nations. Its ability to enter and survive within nonphagocytic cells is thought to be very important for pathogenesis. However, little is known about the C. jejuni determinants that mediate these processes. Through an extensive transposon mutagenesis screen, we have identified several loci that are required for C. jejuni efficient entry and survival within epithelial cells. Among these loci, insertional mutations in aspA, aspB, and sodB resulted in drastic reduction in C. jejuni entry and/or survival within host cells and a severe defect in colonization in an animal model. The implications of these findings for the understanding of C. jejuni-host cell interactions are discussed.Campylobacter jejuni is one of the most important causes of food-borne illness in industrialized nations and diarrhea in children in developing countries (45, 67). Despite its importance as a pathogen, its virulence mechanisms are just beginning to be understood. The ability of C. jejuni to enter nonphagocytic cells is thought to be very important for its pathogenesis (77). Correlation between the cultured-cell invasiveness of Campylobacter strains and the severity of the disease outcome has been reported (10, 11, 33, 47), and studies have visualized C. jejuni inside intestinal epithelial cells during human infections (68). C. jejuni can enter and survive within a variety of cultured cell lines (37, 49, 71, 73). Studies have revealed unique aspects in the cell biology of C. jejuni entry. For example, it has been shown that C. jejuni entry does not require an intact actin cytoskeleton although it requires an intact microtubular network (49). Other studies have implicated Rho-family GTPases in C. jejuni entry (39). However, little is known about the bacterial determinants specifically involved in mediating entry and intracellular survival. Although several studies have identified C. jejuni mutants exhibiting various degrees of deficiency in their ability to enter cultured cells (1, 2, 5, 13, 17, 25, 27, 29-31, 52, 63), there is no evidence indicating that the identified gene products directly mediate the entry process. Nonmotile mutants exhibit a drastic decrease in their ability to invade cultured cells (17, 70, 75). However, it is unclear whether the flagellar structure is directly involved in triggering bacterial internalization or whether the severe entry defect of nonmotile mutants indicates that motility per se is required for entry. A mutation in the pflA gene, which results in paralyzed flagella, has been shown to be defective for entry (75), suggesting that motility and not the flagellar structure itself is required for entry. However, the actual role of PflA is unknown, and therefore it is still possible that the phenotype of the pflA mutation may be due to functions other than its putative role in motility. We have carried out a mutagenesis screen to identify C. jejuni genes that are required for entry and/or survival within host cells. Among the identified loci, insertional mutations in aspA, aspB, and sodB resulted in a drastic reduction in C. jejuni entry and/or survival within host cells and a severe colonization defect in an animal model. Although our studies did not provide evidence for a direct involvement of these loci in the cell entry process, these findings highlight the importance of C. jejuni basic metabolism in its ability to interact with host cells.     

Exosomes Transport Anti-Human Immunodeficiency Virus Factors from Human Cervical Epithelial Cells to Macrophages

The female reproductive tract (FRT) is a major site of HIV sexual transmission. As the outermost layer of cells in the FRT, the human cervical epithelial cells (HCEs) have direct contact with HIV or infected cells. Our early work showed that supernatant (SN) from TLR3-activated HCEs contain the antiviral factors that could potently inhibit HIV replication in macrophages. However, it remains to be determined how HCEs transport the anti-HIV factors to macrophages. This follow-up study examined the role of exosomes in HCE-mediated anti-HIV activity. We found that TLR3 activation of HCEs resulted in the release of exosomes that contained multiple IFN-stimulated genes (ISGs: ISG56, OAS1, MxA, and Mx2) and the HIV restriction microRNAs (miR-28, miR-29 family members, miR-125b, miR-150, miR-382, miR-223, miR-20a, and miR-198). The depletion of exosomes from SN of TLR3-activated HCEs diminished HCE-mediated anti-HIV activity in macrophages, indicating that HCE-derived exosomes are responsible for transporting the antiviral molecules to macrophages. These in vitro findings suggest a novel antiviral mechanism by which HCEs participate in the FRT innate immunity against HIV infection. Further in vivo studies are necessary in order to develop an exosome-based delivery system for prevention and treatment of HIV infection through sexual transmission.     

Leukocytoclastic Vasculitis Associated with Influenza A Virus Infection

Leukocytoclastic vasculitis (LCV) usually presents palpable purpura characterized by inflammation of vessel walls and fragmentation of nuclei. Various conditions can cause LCV, and it can be induced by influenza A virus infection. We report a 2-yr-old Korean girl who presented palpable purpuric and hemorrhagic lesions with fever. She was diagnosed as LCV by skin biopsy, and influenza A virus was isolated from nasopharyngeal swab. She was treated with oseltamivir (Tamiflu®) and prednisolone with dramatic effect of vasculitis and fever.     

Immunity to Escherichia coli in Pigs: Adhesion of Enteropathogenic Escherichia coli to Isolated Intestinal Epithelial Cells

A method was developed to test for the ability of Escherichia coli to adhere to isolated intestinal epithelial cells. Of the E. coli tested, those having either K88ac or K88ab antigens adhered to the cells, and those which did not have these antigens did not. Since some enteropathogenic E. coli did not have the ability to adhere, it is assumed that adherence is not an essential factor of pathogenesis but rather should be considered an enhancement to the pathogenicity of some E. coli. None of the E. coli enteropathogens of cattle tested adhered to either pig or cattle cells. Similarly, human strains did not adhere to pig cells. Although the test system may not have been ideal for human or bovine E. coli, the results reported here suggest that adhesiveness is a property limited to porcine enteropathogenic E. coli carrying one of the K88 antigens. Adhesiveness is associated with the K88c or K88b antigens, and their adhesive ability is only neutralizable by the homologous antisera.     

Immunity to Influenza in Ferrets X. Intranasal Immunization of Ferrets with Inactivated Influenza A Virus Vaccines

The response of ferrets after intranasal inoculation of inactivated A/Hong Kong/68 (H3N2) influenza virus vaccines is reported. Normal ferrets given either saline vaccine in drops or freeze-dried vaccine in an aerosol intranasally did not produce detectable serum or nasal hemagglutination inhibiting antibody and were found to be completely susceptible to challenge infection with A/Hong Kong/68 virus. Intranasal saline vaccine did not produce an additive effect on the response of ferrets simultaneously given the same vaccine intramuscularly with adjuvant. Ferrets primed by previous infection with A/PR/8/34 (H0N1) influenza virus, however, responded to intranasal immunization with saline A/Hong Kong/68 virus vaccine and produced serum and nasal antibody. These animals were found to be partially resistant to challenge infection, in contrast to similar animals given saline vaccine intramuscularly which were completely resistant to challenge infection. Primed ferrets did not respond after immunization with the freeze-dried aerosol vaccine, but this may have been due to a failure of the aerosol to be inhaled satisfactorily.     

Novel Laminin-Binding Protein of Streptococcus pyogenes, Lbp, Is Involved in Adhesion to Epithelial Cells

The lbp gene, which encodes a laminin-binding protein (Lbp) of Streptococcus pyogenes, was found in all S. pyogenes M types. An Lbp-deficient mutant showed a significantly lower efficiency of adhesion to HEp-2 cells than did the wild-type strain. These results indicate that Lbp is one of the important S. pyogenes adhesins.     

Reaction of Thymus Epithelial Cells with Antibodies to Group A Streptococcal Polysaccharide

Immunofluorescence studies show that thymus epithelial cells react with antibodies to group A streptococcal polysaccharide.     

Influenza Virus-Specific T Cells Fail to Reduce Lung Virus Titres in Cyclosporin-Treated, Infected Mice

Cyclosporin A (CsA) inhibited the function(s) of transferred influenza-specific K,D-restricted cytotoxic T cells, which led to clearance of virus in the lungs of influenza virus-infected mice. CsA had no effect on the migration of the transferred cells to the lungs. The pattern of migration and the number of cells recovered from the lungs were similar when cells were transferred into normal, untreated, infected or CsA-treated, infected mice. CsA had no effect on the in vitro expression of cytotoxic activity by the K,D-restricted cytotoxic T cells. These findings strongly suggest that the in vivo clearance of influenza virus by K,D-restricted cytotoxic T cells involves a lymphokine mechanism.     

The Interaction of Mitogen-Activated Protein Kinases to Epstein-Barr Virus Activation in Akata Cells

To understand the mechanism by which Epstein-Barr virus (EBV) is activated in Akata cells by cross-linking of surface immunoglobulin, the interaction between mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) and EBV activation was investigated. Immunoblotting using an anti-phosphoMAPK antibody (Ab) revealed that anti-IgG Ab induced rapid phosphorylation of MAPK in the cells. The phosphorylation was inhibited by MAPK/ERK kinase specific inhibitor, PD98059. The expressions of the EBV immediate early BZLF1 mRNA and its protein product ZEBRA, and early antigen were also inhibited by the inhibitor. These results indicate that MAPK is involved in the pathways of EBV activation.     

Attempts to Detect Homologous Autointerference In vivo with Influenza Virus and Vesicular Stomatitis Virus

Von Magnus particles of influenza virus and defective interfering T particles of vesicular stomatitis virus were unable to provide significant protection of mice from disease or death when inoculated intranasally or intracerebrally along with moderate or high doses of homologous infectious challenge virus. However, yields of infectious virus from the affected organs were reduced as compared to controls inoculated with infectious virus alone. Serial intracerebral passage of vesicular stomatitis virus in mouse brain at high doses failed to produce T particles detectable by in vitro autointerference assays on BHK₂₁ cells, whether or not T particles were introduced along with B virions at the first passage. When very low challenge doses of infectious B virions were inoculated intracerebrally along with high doses of homologous defective particles, there was significant prolongation of life, although most mice died eventually of slowly progressing disease. Also, the virus yields in the brains of these mice were significantly reduced, and virus was no longer detectable in the brains of “protected” mice surviving for 10 days or more. Our results suggest that although homologous autointerference does occur in vivo, it is a more complex phenomenon than in vitro cell culture experiments might indicate.