30 research outputs found
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Caveolin-1 Associated Adenovirus Entry into Human Corneal Cells
The cellular entry of viruses represents a critical area of study, not only for viral tropism, but also because viral entry dictates the nature of the immune response elicited upon infection. Epidemic keratoconjunctivitis (EKC), caused by viruses within human adenovirus species D (HAdV-D), is a severe, ocular surface infection associated with corneal inflammation. Clathrin-mediated endocytosis has previously been shown to play a critical role in entry of other HAdV species into many host cell types. However, HAdV-D endocytosis into corneal cells has not been extensively studied. Herein, we show an essential role for cholesterol rich, lipid raft microdomains and caveolin-1, in the entry of HAdV-D37 into primary human corneal fibroblasts. Cholesterol depletion using methyl-Ξ²-cyclodextrin (MΞ²CD) profoundly reduced viral infection. When replenished with soluble cholesterol, the effect of MΞ²CD was reversed, allowing productive viral infection. HAdV-D37 DNA was identified in caveolin-1 rich endosomal fractions after infection. Src kinase activity was also increased in caveolin-1 rich endosomal fractions after infection, and Src phosphorylation and CXCL1 induction were both decreased in caveolin-1-/- mice corneas compared to wild type mice. siRNA knock down of caveolin-1 in corneal cells reduced chemokine induction upon viral infection, and caveolin-1-/- mouse corneas showed reduced cellular entry of HAdV-D37. As a control, HAdV-C2, a non-corneal pathogen, appeared to utilize the caveolar pathway for entry into A549 cells, but failed to infect corneal cells entirely, indicating virus and cell specific tropism. Immuno-electron microscopy confirmed the presence of caveolin-1 in HAdV-D37-containing vesicles during the earliest stages of viral entry. Collectively, these experiments indicate for the first time that HAdV-D37 uses a lipid raft mediated caveolin-1 associated pathway for entry into corneal cells, and connects the processes of viral entry with downstream proinflammatory cell signaling
Viral Capsid Is a Pathogen-Associated Molecular Pattern in Adenovirus Keratitis
Human adenovirus (HAdV) infection of the human eye, in particular serotypes 8, 19 and 37, induces the formation of corneal subepithelial leukocytic infiltrates. Using a unique mouse model of adenovirus keratitis, we studied the role of various virus-associated molecular patterns in subsequent innate immune responses of resident corneal cells to HAdV-37 infection. We found that neither viral DNA, viral gene expression, or viral replication was necessary for the development of keratitis. In contrast, empty viral capsid induced keratitis and a chemokine profile similar to intact virus. Transfected viral DNA did not induce leukocyte infiltration despite CCL2 expression similar to levels in virus infected corneas. Mice without toll-like receptor 9 (Tlr9) signaling developed clinical keratitis upon HAdV-37 infection similar to wild type mice, although the absolute numbers of activated monocytes in the cornea were less in Tlr9β/β mice. Virus induced leukocytic infiltrates and chemokine expression in mouse cornea could be blocked by treatment with a peptide containing arginine glycine aspartic acid (RGD). These results demonstrate that adenovirus infection of the cornea induces chemokine expression and subsequent infiltration by leukocytes principally through RGD contact between viral capsid and the host cell, possibly through direct interaction between the viral capsid penton base and host cell integrins
High-grade neuroendocrine carcinoma of the gastrointestinal tract: A retrospective study.
Emergence of Resistant Clones in Medullary Thyroid Cancer May Not Be Rescued by Subsequent Salvage Highly Selective Rearranged During Transfection-Inhibitor Therapy
UV-inactivated adenovirus induces leukocyte infiltration and cytokine expression.
<p>(A) Representative dot plots of single cell suspensions prepared from corneas at 4 dpi stained with Gr1 and F4/80 and gated on CD45<sup>high</sup> labeled cells. Corneas were infected with virus free buffer (M), or intact (V), UV-inactivated (UV), and heat-inactivated (H) HAdV-37. (B) Quantification of average numbers of Gr1 and F4/80 stained corneal cells in intact (V), UV-inactivated (UV), or heat-inactivated (H) virus injected corneas at 4 dpi (nβ=β6 mice/group). Data is derived from three separate experiments, and error bars represent SD. (C) Myeloperoxidase (MPO) levels assessed 24 hours post injection with virus free buffer (M), intact virus (V), UV-inactivated virus (UV), or heat-inactivated virus (H) are shown (nβ=β9 mice/group). Data represents mean of three independent experiments Β± SD. (DβF) Cytokine expression in corneas after injection with virus free buffer (M), intact virus (V), UV-inactivated virus (UV), or heat-inactivated virus (H) as measured at 16 hpi by ELISA for CXCL1 (D), CCL2 (E), and IL-6 (F) protein (nβ=β9 mice/group). Data represents mean of three independent experiments Β± SD. * p<.05, ANOVA.</p
Viral gene expression is not essential for adenovirus keratitis.
<p>(A) Real-time PCR for the relative expression of viral transcript E1A10S at 4 hpi in mock (M), intact (V), UV-inactivated (UV), or heat-inactivated (H) HAdV-37 infected A549 cells. Data represents mean of three separate experiments Β± SD. (B) Mouse corneas injected with Cy3-labeled intact (V), UV- inactivated (UV), or heat-inactivated (H) virus were analyzed by confocal microscopy at 90 min pi (nβ=β5 corneas/group). Red: Cy3-labeled virus. Green: intracellular actin (phalloidin stain). Blue: nuclei (TO-PRO3 stain). Scale bar 20 Β΅M. (C) Representative photographs and (D) hematoxylin and eosin stained histopathological sections of mice corneas at 4 dpi, infected with virus free buffer (M), intact virus (V), UV-inactivated virus (UV), or heat-inactivated virus (H) (nβ=β5 mice/group).</p
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Role of MyD88 in Adenovirus Keratitis
Pattern recognition receptors (PRRs) are critical to the early detection and innate immune responses to pathogens. In particular, the TLR system and its associated adaptor proteins play essential roles in early host responses to infection. Epidemic keratoconjunctivitis, caused by the human adenovirus, is a severe ocular surface infection associated with corneal inflammation (stromal keratitis). We previously showed that adenovirus capsid was a key molecular pattern in adenovirus keratitis, with viral DNA playing a lesser role. We have now investigated the role of the adaptor molecule MyD88 in a mouse model of adenovirus keratitis in which there is no viral replication. In MyD88β/β mice infected with human adenovirus type 37, clinical keratitis was markedly reduced, along with infiltration of CD45+ cells, and expression of inflammatory cytokines. Reduction of inflammatory cytokines was also observed in infected primary human corneal fibroblasts pretreated with a MyD88 inhibitory peptide. Keratitis similar to wild type mice was observed in TLR2, TLR9, and IL-1R knockout mice, but was reduced in TLR2/9 double knockout mice, consistent with synergy of TLR2 and TLR9 in the response to adenovirus infection. MyD88 co-immunoprecipitated with Src kinase in mice corneas and in human corneal fibroblasts infected with adenovirus, and MyD88 inhibitory peptide reduced Src phosphorylation, linking MyD88 activation to inflammatory gene expression through a signaling cascade previously shown to be directed by Src. Our findings reveal a critical role for the PRRs TLR2 and 9, and their adaptor protein MyD88, in corneal inflammation upon adenovirus infection