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.

Background: Pythium insidiosum , a pathogenic oomycete, is a common causative organism of infectious corneal ulcer. Studying the innate immune response at the ocular surface is important for better understanding of the underlying pathogenesis and host defense against P. insidiosum infection. Objective: The present study aims to investigate the role of Toll-like receptor (TLR)2 on human corneal epithelial cells (HCECs) in P. insidiosum infection. Methods: Human embryonic kidney (HEK) cells were stimulated with either P. insidiosum zoospores or hyphae. NF-kappa B activation was determined by spectrophotometric measurement of secreted embryonic alkaline phosphatase (SEAP) levels. The role of TLR2 in P. insidiosum infection was studied in HCECs and monocyte derived macrophages (MDMs) using anti-TLR2 neutralizing antibody. The expression levels of pro-inflammatory cytokines were determined. Results: Both P. insidiosum hypha and zoospore stimulated TLR2-dependent NF-kappa B activation in HEK-BlueTM-hTLR2 cells in dose-dependent manner. IL-6 and IL-8 , but not IL-1 beta, were upregulated in HCECs after stimulation with P. in-sidiosum . Blockade of TLR2 on HCECs altered neither IL-6 nor IL-8 expressions. In contrast, the 3 cytokines were upregulated in the stimulated MDMs and the expression levels of IL-1 beta and IL-8 but not IL-6 were attenuated in TLR2 blockade MDMs. Conclusion: P. insidiosum was recognized by human TLR2 on HEK cells. The mRNA expression levels of certain cyto-kines were dependent of TLR2 in P. insidiosum infected MDMs but not HCECs at early stage of infection.

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)-1β 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.

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 beta-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 TLRI-1O 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-1 beta, IL-8, TNF-alpha, 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. interrogansat the portal of entry.

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.

We have computationally identified a conserved region of flagellin B (FlaB) protein that is present only in pathogenic Leptospira but absent in non-pathogenic Leptospira or other flagellated bacteria. The predicted FlaB subunit protein (sFlaB) comprised a sequence of 50-amino acids: ¹⁴⁰ FARGSRVASMWFHMGPNQNQRERFYIGTMTSKALKLVKADGRPIAISSPG ¹⁸⁹ . The nucleotide sequence encoding sFlaB was amplified by polymerase chain reaction from Leptospira genomic DNA and cloned into a pET100 expression vector. The recombinant sFlaB was expressed in Escherichia coli BL21 and affinity purified to serve as an antigen to the immunochromatographic test (ICT) designed for specific IgM (ICT-IgM) and IgG (ICT-IgG) antibody detections. We evaluated 109 serum samples, including 46 from leptospirosis patients (29 acute sera and 17 convalescent sera) and 63 from patients with other acute febrile illnesses (46 acute sera and 17 convalescent sera). Compared to the paired serum results of the indirect immunofluorescent assay, the sensitivity of ICT-IgM and ICT-IgG was 80.40% and 71.73%, with specificities of 84.13% and 73.01%, respectively. For acute leptospirosis sera, ICT-IgM showed a sensitivity of 75.86% and a specificity of 89.13%, while ICT-IgG had a sensitivity of 68.97% and a specificity of 71.94%. IMPORTANCE: Leptospirosis is a zoonotic disease caused by pathogenic leptospires. The infected patient presents with a mild to severe febrile illness and may die while receiving inappropriate treatment. The microscopic agglutination test, the current gold standard method, is laborious and requires the use of live panel leptospires, which should only be done in a reference laboratory. In addition, the results of the paired serum samples are required for an accurate interpretation. Polymerase chain reaction (PCR) was used instead for diagnosis in the acute phase of infection. However, PCR requires an expensive machine and a specialist to analyze the results. Therefore, a simple and rapid test is needed for the early diagnosis of leptospirosis.