Toll-like receptor 2-mediated induction of human beta-defensin 2 expression by Leptospira interrogans in human kidney cells.

BACKGROUND: Leptospirosis is a zoonotic disease caused by Leptospira interrogans. Severe leptospirosis is often accompanied by kidney dysfunction caused by chronic infection. The kidney pathology involves bacterial invasion and inflammation caused by pro-inflammatory cytokines. Human beta defensins (hBDs) are antimicrobial peptides induced by microbial infection and/or pro-inflammatory cytokines. One function of hBDs is the recruitment of immune cells that leads to inflammation. However, the expression of hBDs by kidney epithelium in response to pathogenic Leptospira has never been investigated. OBJECTIVE: To determine the expression of hBDs in human kidney epithelium responses to Leptospira. METHODS: Human kidney cells were infected with Leptospira interrogans serovar Autumnalis in the presence or absence of anti-TLR2 neutralizing antibody (Ab) for 6 hours. TLR2, hBDs and pro-inflammatory cytokines mRNA expressions were analyzed by quantitative polymerase chain reaction (qPCR). RESULTS: Pathogenic Leptospira upregulated the expressions of pro-inflammatory cytokines and hBD2, but not TLR2, hBD1 and hBD3 in kidney cells. The expressions of hBD2 and pro-inflammatory cytokines were inhibited in the presence of anti-hTLR2 neutralizing Ab. CONCLUSIONS: Our results provide the first evidence that pathogenic Leptospira induces hBD2 expression in kidney cells. The expressions of pro-inflammatory cytokines and hBD2 in the cells in response to pathogenic Leptospira are regulated by TLR2. Pro-inflammatory cytokines and hBD2 might be play role in recruitment of immune cells to the kidney and contribute to the development of inflammation-mediated tissue damage in the kidney. However, further study is needed to improve the understanding of the role of these molecules in immune response activation.

Endothelial Activation in Orientia tsutsugamushi Infection Is Mediated by Cytokine Secretion From Infected Monocytes.

Scrub typhus, caused by Orientia tsutsugamushi, is a common systemic infection in Asia. Delay in diagnosis and treatment can lead to vasculitis in the visceral organs and other complications. The mechanisms that drive endothelial activation and the inflammatory response in O. tsutsugamushi infection remain unknown. In addition, the interaction between monocytes and endothelial cells is still unclear. Here we demonstrate that O. tsutsugamushi-infected human dermal microvascular endothelial cells produced moderate levels of chemokines and low levels of IL-6 and IFN-ß, but not TNF or IL-1ß. Recombinant TNF and cytokine-rich supernatants from infected monocytes markedly enhanced chemokine production in infected endothelial cells. We also show that TNF and monocyte supernatants, but not O. tsutsugamushi infection of endothelial cells per se, upregulated the endothelial cell surface expression of ICAM-1, E-selectin, and tissue factor. This finding was consistent with the inability of O. tsutsugamushi to induce cytokine secretion from endothelial cells. The upregulation of surface molecules after stimulation with monocyte supernatants was significantly reduced by neutralizing anti-TNF antibodies. These results suggest that endothelial cell activation and response are mainly mediated by inflammatory cytokines secreted from monocytes.

Efficacy and outcome of simple limbal epithelial transplantation for limbal stem cell deficiency verified by epithelial phenotypes integrated with clinical evaluation.

PURPOSE: To evaluate the efficacy and outcome of simple limbal epithelial transplantation (SLET) for limbal stem cell deficiency (LSCD) using epithelial phenotype detection integrated with clinical manifestation. METHODS: This prospective multicenter study included patients with LSCD who underwent autologous SLET (autoSLET) and living-related allogenic SLET (Lr-alloSLET). All patients were assessed by slit-lamp biomicroscopy, in vivo confocal microscopy (IVCM), and impression cytology with immunofluorescence staining (ICIF) before and after surgery. The criteria for success were the presence of a clinically non-conjunctivalized cornea and corneal epithelium detected by IVCM or ICIF. Otherwise, the case would be considered a failure. Visual improvement and risk factors for SLET failure were analyzed. RESULTS: A total of 28 eyes of 26 patients (11 autoSLET and 17 Lr-alloSLET) were included. The median age was 53 years (range, 35-63), and the follow-up time was 29.5 months (range, 17.5-39.8). The overall survival rate was 89.3% at 2 years and 75.6% at 3 years with no difference between autoSLET and Lr-alloSLET (p = 0.24). Seven eyes subsequently underwent penetrating keratoplasty. Immunohistochemistry analysis showed that all corneal buttons had corneal epithelium and limbal stem cell markers. Visual improvement was achieved in both SLET groups (p < 0.001). Failed SLET developed between 5 and 32 months postoperatively. However, absolute risk factors for SLET failure were unidentified. CONCLUSION: The efficacy of autoSLET and Lr-alloSLET for LSCD was excellent. Limbal explants can regenerate and restore the corneal surface while maintaining the characteristics of limbal stem cells as shown by epithelial phenotype detection and immunohistochemistry integrated with clinical evaluation.

Phenotypic Characterization of Corneal Epithelium in Long-Term Follow-Up of Patients Post-Autologous Cultivated Oral Mucosal Epithelial Transplantation.

PURPOSE: To analyze the phenotype of the corneal epithelium in patients with long-term follow-up who underwent autologous cultivated oral mucosal epithelial transplantation (COMET) using in vivo confocal microscopy (IVCM) and impression cytology with immunofluorescence staining (ICIF). METHODS: Thirteen eyes from patients with severe limbal stem cell deficiency, who underwent COMET at least 48 months before, were recruited in this noncomparative cohort study. After eye examination, IVCM and ICIF were performed. Clinical manifestations of the cornea were evaluated and compared with epithelial findings detected by IVCM and ICIF [cytokeratin (CK) 3, CK7, and CK12]. Two corneal buttons derived from patients receiving the corneal transplantation post-COMET were sent for immunohistochemistry (CK3, CK6, CK7, CK12, paired box gene 6, p63, zonula occludens-1, and integrin ß -1). RESULTS: The mean age of patients was 51.2 ± 20.6 years, and the mean follow-up time since COMET was 78.7 ± 16.3 months. Six of 13 eyes showed clinically successful COMET. In these eyes, IVCM demonstrated predominant cornea-like epithelium and ICIF reported positivity for CK3 and CK12, confirming the presence of oral mucosal and corneal epithelium. Meanwhile, 7 eyes showed total conjunctivalization, corresponding with substantial conjunctival epithelium detected by IVCM and positivity for conjunctival (CK7) and oral mucosal epithelial (CK3) markers detected by ICIF. The immunohistochemistry of corneal buttons stained positive for oral mucosal, corneal epithelial, and stem cell markers (CK3, CK12, and p63). CONCLUSIONS: In long-term follow-up of COMET, epithelium of successful patients demonstrated cornea-like phenotype, whereas failed cases revealed mainly conjunctival phenotype. However, there were evidences that oral mucosal epithelial cells remained across the cornea in both successful and failed COMET as detected by IVCM and ICIF.

Effect of Gold Nanoparticles on the TLR2-Mediated Inflammatory Responses Induced by Leptospira in TLR2-Overexpressed HEK293 Cells.

Leptospira infection can cause potential hazards to human health by stimulating inflammation, which is mediated mainly through the Toll-like receptor 2 (TLR2) pathway. Gold nanoparticles (AuNPs) are promising for medical applications, as they display both bioinert and noncytotoxic characteristics. AuNPs have been shown to have the ability to modify immune responses. To understand the in vitro immunomodulatory effect of AuNPs in a Leptospira infection model, the activation of TLR2 expression was examined in HEK-Blue-hTLR2 cells treated with Leptospira serovars and/or AuNPs (10 and 20 nm). The ability of AuNPs to modulate an inflammatory response induced by Leptospira was examined in terms of transcript expression level modulation of three proinflammatory cytokines (tumor necrosis factor-α, interleukin (IL)-1ß and IL-6) using two-stage quantitative real-time reverse transcriptase PCR. The results revealed that the administration of 10 nm AuNPs could augment the Leptospira-induced TLR2 signaling response and upregulate the expression of all three cytokine gene transcripts, whereas the 20 nm AuNPs attenuated the TLR2 activation and expression of proinflammatory cytokines. This indicates that AuNPs can modulate inflammatory parameters in Leptospira infection and different-sized AuNPs had different immunomodulatory functions in this model.

Characterization of limbal explant sites: Optimization of stem cell outgrowth in in vitro culture.

Simple limbal epithelial transplantation (SLET) and cultivated limbal epithelial transplantation (CLET) are proven techniques for treating limbal stem cell deficiency (LSCD). However, the precise regions that are most suitable for preparing explants for transplantation have not been identified conclusively. Accordingly, this in vitro study aimed at determining ideal sites to be selected for tissue harvest for limbal stem cell culture and transplantation. We evaluated cell outgrowth potential and the expression of stem cell markers in cultures from 48 limbal explants from five cadaveric donors. The limbal explants were generated from the three specific sites: Lcor (located innermost and adjacent to the cornea), Lm (middle limbus), and Lconj (located outermost adjacent to the conjunctiva). We found that explants from the Lconj and Lm sites exhibited higher growth potential than those from the Lcor site. Transcript encoding the stem cell marker and p63 isoform, ΔNp63, was detected in cells from Lm and Lconj explants; expression levels were slightly, though significantly (p-value < 0.05), higher in Lm than in Lconj, although expression of ΔNp63α protein was similar in cells from all explants. Differential expression of ATP-Binding Cassette Subfamily G Member 2 (ABCG2) did not reach statistical significance. Immunohistochemistry by indirect immunofluorescence analysis of limbus tissue revealed that the basal layer in explant tissue from Lconj and Lm contained markedly more stem cells than found in Lcor explant tissue; these findings correlate with a higher capacity for growth. Collectively, our findings suggest that explants from the Lconj and Lm sites should be selected for limbal cell expansion for both CLET and SLET procedures. These new insights may guide surgeons toward specific limbal sites that are most suitable for stem cell culture and transplantation and may ultimately improve treatment outcomes in the patients with LSCD.

Epithelial analysis of simple limbal epithelial transplantation in limbal stem cell deficiency by in vivo confocal microscopy and impression cytology.

Simple limbal epithelial transplantation (SLET) is a relatively new treatment for severe limbal stem cell deficiency. Outcomes of treatment are typically determined based on clinical manifestations. In this prospective-multicenter study, we aimed to analyze the epithelial phenotypes of the corneas after SLET using IVCM and IC, and correlated them with clinical findings. Ten eyes of nine patients, who underwent SLET (five autologous SLET and five living-related SLET) were recruited. A set of examinations included slit-lamp biomicroscopy, corneal in vivo confocal microscopy (IVCM), and impression cytology (IC) was performed in all eyes at least twice (≥ 3-month interval) postoperatively. Then, a correlation between findings of the three examinations was analyzed. There were seven eyes with clinical success (no central neovascularization) showed pure corneal epithelial phenotype or mixed corneal-conjunctival phenotypes (mostly cornea) in either IVCM or IC. Three eyes with clinical failure, presented with peripheral and central neovascularization, showed total or predominant conjunctival phenotype in IVCM and sole conjunctival phenotype in IC. From a total of 22 sets of examinations, there was a high correlation between clinical manifestation vs. IC (κ = 0.844, observed agreement = 81.82%) and a substantial correlation between clinical manifestation vs. IVCM (κ = 0.727, observed agreement = 76.19%) and between IVCM versus IC (κ = 0.729, observed agreement = 76.19%). In conclusion, IVCM and IC facilitate determination of epithelial phenotype of the cornea after SLET. There was a substantial to high correlation between IVCM, IC and clinical presentations. Findings observed by IVCM and IC may allow early detection of epithelial alterations in eyes underwent SLET before clinical recognition.

Role of Toll-like receptor 2 in mediating the production of cytokines and human beta-defensins in oral mucosal epithelial cell response to Leptospiral infection.

BACKGROUND: Pathogenic Leptospira spp. is the causative agent of leptospirosis. Oral mucosal cavity is one of portal entry for this bacterium. Oral mucosal epithelium provides a physical barrier and secretes cytokines, chemokines and antimicrobial peptides (AMPs) in response to microbial infection. Human ß-defensins (hBDs); hBD1, hBD2, and hBD3 are predominantly AMPs expressed in the oral cavity. Toll-like receptors (TLRs) have been reported in hBD regulation. TLR2 recognizes leptospiral lipopolysaccharide, and plays a key role in the early control of leptospirosis. OBJECTIVE: The aim of this study is to investigate the role of TLR2 in mediating the production of cytokines and hBDs in oral mucosal epithelial cell response to leptospiral infection. METHODS: Cultivated oral mucosal epithelial cells were prepared, characterized, and compared with oral mucosal tissues. The TLR1-10 and hBD mRNA expressions were examined. Pro-inflammatory cytokine and hBD1-3 expressions in response to leptospires were determined by quantitative (q) RT-PCR. RESULTS: The cultivated oral epithelium expressed TLR2 and hBD1-3. The induction of IL-ßIL-8, TNF-α, and hBD2 were increased in response to Leptospira via TLR2 recognition. CONCLUSION: The characteristics of primary epithelial cells and tissue were similar in terms of TLR expression. All primary epithelial cells expressed TLR2 and hBD1-3. We used primary epithelial cells to study response to L. interrogans. Our results yielded the first evidence that human TLR2 regulates hBD2 expression in oral mucosa epithelial responded to L. interrogans. Expression of hBD2 may act to neutralize the virulence or prevent the invasion of L. interrogans at the portal of entry.

Cytoprotective effect of glutaraldehyde erythropoietin on HEK293 kidney cells after silver nanoparticle exposure.

The toxic effects from exposure to silver nanoparticles (AgNPs), which are broadly present in many consumer products, have long raised concerns. Many studies have focused on the mechanisms of nanosilver, which cause toxicity in human cells, but little is known about prevention of this type of injury. This study investigated the in vitro effects of glutaraldehyde erythropoietin (GEPO), a cytoprotective compound derived from erythropoietin, in terms of cell protection against AgNP-induced injury. HEK293 cells were pretreated with or without GEPO before administration of AgNPs. The protective effects of GEPO in this cell line were assessed by the percentage of viable cells, alterations of cell morphology, and the proliferative capability of the cells. In addition, we assessed the role of GEPO in lowering cellular oxidative stress and regulating expression of the anti-apoptotic protein Bcl2. The results showed rescue effects on the percentage of viable and proliferative cells among GEPO pretreated cells. Pretreatment with GEPO maintained the normal cell shape and ultrastructural morphology. Moreover, GEPO reduced the generation of reactive oxygen species in cells and activated expression of Bcl2, which are the major mechanisms in protection against cellular toxicity induced by AgNPs. In conclusion, our study showed that the cytotoxic effects from exposure to AgNPs can be prevented by GEPO.