Chuan-Hui Kuo

Clinical application of real-time polymerase chain reaction for diagnosis of herpetic diseases of the anterior segment of the eye

PurposeTo assess the value of quantification of herpes simplex virus (HSV) DNA for the differential diagnosis of herpetic diseases of the anterior segment of the eye.MethodsOne hundred forty-four samples from 90 patients with ocular inflammatory diseases were examined for HSV DNA by real-time polymerase chain reaction (PCR) with primers set for the consensus sequence of HSV-1/2 DNA polymerase. The samples included corneal epithelial scrapings, tear fluid (200μl of eye wash), and aqueous humor.ResultsIn cases of typical herpetic epithelial keratitis, a large number of copies of HSV DNA were detected (mean, 1.0 × 107 copies in epithelial scrapings and 3.5 × 105 copies in tear fluid). In atypical epithelial keratitis cases, a smaller number of HSV DNA copies were detected. In stromal keratitis cases, the number of copies of HSV DNA in the tear fluid (mean: 4.7 × 102 copies) was significantly smaller than in cases of epithelial keratitis. In the aqueous humor, the number of copies was small in endotheliitis cases (mean, 2.9 × 102 copies/μl), but the range was great, from (1.2–4.8) × 105/μl in herpetic iridocyclitis. Seventeen percent of cases in which HSV was not suspected to be involved showed a small number of copies of HSV DNA, indicating the unexpected involvement of HSV in these cases.ConclusionsReal-time PCR is an informative method of diagnosing herpetic eye diseases and evaluating the possible involvement of HSV in other inflammatory ocular diseases.

Evidence That Formation of Vimentin·Mitogen-activated Protein Kinase (MAPK) Complex Mediates Mast Cell Activation following FcϵRI/CC Chemokine Receptor 1 Cross-talk

Background: CC chemokine ligand 2 (CCL2) recruits leukocytes in inflammatory tissues. Results: Vimentin, a cytoskeletal protein, interacted with phosphorylated MAPKs, was critical for CCL2 production in mast cells activated via FϵcRI and a CC chemokine receptor. Conclusion: Vimentin was involved in optimal CCL2 production in mast cells. Significance: This work contributes to understanding of mechanisms for chemokine production in mast cells, which are therapeutic targets for allergic inflammation. Accumulating evidence points to cross-talk between FcϵRI and CC chemokine receptor (CCR)-mediated signaling pathways in mast cells. Here, we propose that vimentin, a protein comprising type III intermediate filament, participates in such cross-talk for CCL2/monocyte chemotactic protein 1 (MCP-1) production in mast cells, which is a mechanism for allergic inflammation. Co-stimulation via FcϵRI, using IgE/antigen, and CCR1, using recombinant CCL3/macrophage inflammatory protein-1α (MIP-1α), increased expression of phosphorylated, disassembled, and soluble vimentin in rat basophilic leukemia (RBL)-2H3 cells expressing human CCR1 (RBL-CCR1 cells) and bone marrow-derived murine mast cells, both models of mucosal type mast cells. Furthermore, co-stimulation enhanced production of CCL2 as well as phosphorylation of MAPK. Treating the cells with p38 MAPK inhibitor SB203580, but not with MEK inhibitor PD98058, reduced CCL2 production, suggesting that p38 MAPK, but not ERK1/2, plays a critical role in the chemokine production. Immunoprecipitation analysis showed that vimentin interacts with phosphorylated ERK1/2 and p38 MAPKs in the co-simulated cells. Preventing disassembly of the vimentin by aggregating vimentin filaments using β,β′-iminodipropionitrile reduced the interaction of vimentin with phosphorylated MAPKs as well as CCL2 production in the cells. Taken together, disassembled vimentin interacting with phosphorylated p38 MAPK could mediate CCL2 production in mast cells upon FcϵRI and CCR1 activation.

Pathogenesis of Herpetic Stromal Keratitis in CCR5- and/or CXCR3-Deficient Mice

Purpose: Herpetic stromal keratitis (HSK) is an immunopathological reaction to herpes simplex virus type 1 (HSV-1) corneal infection. It has been reported that CD4+ cells play the most important role in the pathogenesis of this disease. In this study, we have focused on two chemokine receptors, CCR5 and CXCR3, which are expressed on CD4+ Th1 cells in mice HSK model. Methods: CCR5-deficient (CCR5KO), CXCR3-deficient (CXCR3KO), CCR5/CXCR3 double-deficient (DKO), and wild type (WT) mice (C57/BL6 background) were infected intracorneally with HSV-1 (CHR3 strain). The corneas were examined biomicroscopically, and cryosections of the corneas were examined histologically and immunohistochemically. Real-time RT-PCR and RNase protection assay (RPA) were performed, and the virus titers were measured in excised eyes and trigeminal ganglia (TG). Results: The HSK clinical severity in DKO mice was significantly lower than that in WT mice, and this was reversed by transfer of cells from the spleen of WT mice to DKO mice. Histologically, the numbers of T cells (CD4+ and CD8+ cells) and neutrophils infiltrating the cornea were significantly fewer in CCR5KO, CXCR3KO, and DKO mice. Transcript levels of immune-related cell surface marker in the eye by RPA were reduced in DKO mice. The expression of I-TAC was significantly increased in the cornea of CCR5KO mice, and MIP-1α and MIP-1β were significantly lower in CXCR3KO mice than in WT mice by RT-PCR. There were no significant differences of virus titers in the eye and TG among any groups of mice except the increase in the TG of DKO mice on day 5 PI. Conclusions: The suppression of chemotaxis and activation of CD4+ Th1 cells by the lacking of CXCR3 and CCR5 causes a decrease of other infiltrating cells, resulting in a lower severity of HSK. These results suggest that targeting chemokine receptors is a promising way to treat HSK.

Transcriptional Analyses before and after Suppression of Immediate Hypersensitivity Reactions by CCR3 Blockade in Eyes with Experimental Allergic Conjunctivitis

PURPOSE To characterize the transcriptome of allergic conjunctivitis mediated by eosinophil-related chemokine receptor CCR3 and to identify a candidate for possible therapeutic intervention in eosinophilic inflammation of the eye. METHODS Mice were sensitized to ragweed pollen, and allergic conjunctivitis was induced by an allergen challenge. The induction of allergic conjunctivitis was used to determine whether an inhibition of CCR3 would suppress eosinophilic inflammation and the allergen-induced immediate hypersensitivity reaction. In addition, sensitized mice were treated with a CCR3 antagonist or an anti-CCR3 antibody before the allergen challenge. Eosinophilic inflammation was evaluated histologically at 24 hours after the allergen challenge. Transcriptional changes with or without a blockade of CCR3 were determined by microarray analyses. RESULTS Blockade of CCR3 significantly suppressed allergen-induced clinical signs, mast cell degranulation, and eosinophilic inflammation. Clustering analysis of the transcriptome during the early phase identified clusters of genes associated with distinct biological processes. A CCR2 ligand, monocyte chemoattractant protein (MCP)-1, was identified in the cluster of genes related to mast cell activation. MCP-1, an attractant of monocytes but not eosinophils, was in the top 10 transcripts among the genome and was suppressed by CCR3 blockade. Importantly, antibody blockade of MCP-1 suppressed the eosinophilic inflammation significantly. CONCLUSIONS CCR3 regulates not only the eosinophilic inflammation but also the clinical signs and mast cell degranulation. The CCR3-mediated transcriptome is characterized by many biological processes associated with mast cell activation. Among these CCR3-mediated processes, MCP-1 was found to be significantly involved in eosinophilic inflammation probably by an indirect pathway.

Role of periostin and interleukin-4 in recurrence of pterygia.

PURPOSE To identify the candidate genes for pterygia recurrence from a pterygia transcriptome and to analyze their transcriptional regulation and functional relationships. METHODS Transcriptional networks for pterygia recurrence were constructed using network analysis that was applied to 184 genes that showed a significant twofold change in the whole genome. Of the identified recurrence-related candidate genes in the major networks, periostin and IL-4 were analyzed for transcriptional relationships using pterygia-derived fibroblasts. Immunohistochemical analysis was used to study pterygia tissue. Effector candidate molecule for recurrence periostin was analyzed for cell adhesive function. RESULTS The pterygia transcriptome was divided into four major biological networks with high significance scores (P < 10(-17)). The classifier with the highest accuracy using the support vector machine algorithm was periostin, which was successfully linked to the network of cell cycle, connective tissue development and function, and cell morphology. Analyses using pterygia-derived fibroblasts showed that periostin was required for cell adhesion that was mediated by a presumed pterygia-related extracellular matrix protein, fibronectin. Periostin was found to be transcriptionally induced by IL-4. The IL-4-stimulated periostin induction was suppressed by MAP kinase/ERK kinase 1 inhibitor, indicating an involvement of the MAP kinase pathway. Pathologically, IL-4 was transcriptionally elevated in recurrent pterygia tissue and was localized to perivascular tissues and endothelial cells in the stroma of the subconjunctiva of pterygia. CONCLUSIONS Periostin is induced by IL-4 and is involved in the fibronectin-mediated pterygia fibroblast adhesion. These findings indicate that periostin probably promotes the recurrence of pterygia.

The role of TRB3 in mast cells sensitized with monomeric IgE

Mast cells play a key role in immunoglobulin E (IgE)-associated allergic disorders; however, the cellular effects of sensitization remain poorly understood. Using gene microarrays and the multiplexing ELISA method, we examined the effects of sensitization on RBL-CCR1 cell transcription and chemokine/cytokine secretion. Sensitization most prominently increased transcription of Trb3, the gene for tribbles homolog 3 (TRB3), and also increased the release of most of the cytokines and chemokines tested. Knockdown of TRB3 via RNAi significantly induced the production of tumor necrosis factor-α (TNF-α), interleukin-4 (IL-4), interleukin-6 (IL-6), and the chemokine mast cell protease-1 (MCP-1). TRB3 deficiency also induced IκBα phosphorylation. TRB3 may therefore serve as a negative regulator of pro-inflammatory cytokines and chemokines, controlling the extent of the inflammatory response.

Role of CCL7 in Type I Hypersensitivity Reactions in Murine Experimental Allergic Conjunctivitis

Molecules that are necessary for ocular hypersensitivity reactions include the receptors CCR1 and CCR3; CCL7 is a ligand for these receptors. Therefore, we explored the role of CCL7 in mast cell activity and motility in vitro and investigated the requirement for CCL7 in a murine model of IgE-mediated allergic conjunctivitis. For mast cells treated with IgE and Ag, the presence of CCL7 synergistically enhanced degranulation and calcium influx. CCL7 also induced chemotaxis in mast cells. CCL7-deficient bone marrow–derived mast cells showed decreased degranulation following IgE and Ag treatment compared with wild-type bone marrow–derived mast cells, but there was no difference in degranulation when cells were activated via an IgE-independent pathway. In vivo, CCL7 was upregulated in conjunctival tissue during an OVA-induced allergic response. Notably, the early-phase clinical symptoms in the conjunctiva after OVA challenge were significantly higher in OVA-sensitized wild-type mice than in control challenged wild-type mice; the increase was suppressed in CCL7-deficient mice. In the OVA-induced allergic response, the numbers of conjunctival mast cells were lower in CCL7-deficient mice than in wild-type mice. Our results demonstrate that CCL7 is required for maximal OVA-induced ocular anaphylaxis, mast cell recruitment in vivo, and maximal FcεRI-mediated mast cell activation in vitro. A better understanding of the role of CCL7 in mediating ocular hypersensitivity reactions will provide insights into mast cell function and novel treatments for allergic ocular diseases.