Tespa1 deficiency reduces the antitumour immune response by decreasing CD8+T cell activity in a mouse Lewis lung cancer model.

Thymocyte-expressed, positive selection-associated 1 (Tespa1) is a key molecule in T-cell development and has been linked to immune diseases. However, its role in antitumour CD8+T cell immunity remains unclear. Here, we demonstrated that Tespa1 plays an important role in antitumour CD8+T cell immunity. First, compared with wild-type (WT) mice, Lewis lung cancer cells grew faster in Tespa1 knockout (Tespa1-/-) mice, with reduced apoptosis, and decreased CD8+T cells in peripheral blood and tumor tissues. Second, the proportion of CD8+T and Th1 cells in the splenocytes of Tespa1-/- mice was lower than that in WT mice. Third, Tespa1-/- CD8+ tumor-infiltrating lymphocytes (TILs) showed weakened proliferation, invasion, cytotoxicity, and protein expression of IL-2 signalling pathway components compared to WT CD8+TILs. Furthermore, PD-1 expression in CD8+TILs was higher in Tespa1-/- than in WT mice. Lastly, CD8+TILs in WT mice improved the antitumour ability of Tespa1-/- mice. In conclusion, these findings suggest that Tespa1 plays a critical role in the tumor immune system by regulating CD8+T cells.

Decorating hexahistidine-metal assemblies with tyrosine enhances the ability of proteins to pass through corneal biobarriers.

In recent decades, the use of protein drugs has increased dramatically for almost every clinical indication, including autoimmunity and cancer infection, given their high specificity and limited side effects. However, their easy deactivation by the surrounding microenvironment and limited ability to pass through biological barriers pose large challenges to the use of these agents for therapeutic effects; these deficits could be greatly improved by nanodelivery using platforms with suitable physicochemical properties. Here, to assess the effect of the hydrophobicity of nanoparticles on their ability to penetrate biological barriers, the hydrophobic amino acid tyrosine (Y) was decorated onto hexahistidine peptide, and two nanosized YHmA and HmA particles were generated, in which Avastin (Ava, a protein drug) was encapsulated by a coassembly strategy. In vitro and in vivo tests demonstrated that these nanoparticles effectively retained the bioactivity of Ava and protected Ava from proteinase K hydrolysis. Importantly, YHmA displayed a considerably higher affinity to the ocular surface than HmA, and YHmA also exhibited the ability to transfer proteins across the barriers of the anterior segment, which greatly improved the bioavailability of the encapsulated Ava and produced surprisingly good therapeutic outcomes in a model of corneal neovascularization. STATEMENT OF SIGNIFICANCE: Improving the ability to penetrate tissue barriers and averting inactivation caused by surrounding environments, are the keys to broaden the application of protein drugs. By decorating hydrophobic amino acid, tyrosine (Y), on hexahistidine peptide, YHmA encapsulated protein drug Ava with high efficiency by co-assembly strategy. YHmA displayed promising ability to maintain bioactivity of Ava during encapsulation and delivery, and protected Ava from proteinase K hydrolysis. Importantly, YHmA transferred Ava across the corneal epithelial barrier and greatly improved its bioavailability, producing surprisingly good therapeutic outcomes in a model of corneal neovascularization. Our results contributed to not only the strategy to overcome shortcomings of protein drugs, but also suggestion on hydrophobicity as a nonnegligible factor in nanodrug penetration through biobarriers.

Lumiflavin Reduces Cisplatin Resistance in Cancer Stem-Like Cells of OVCAR-3 Cell Line by Inducing Differentiation.

Ovarian cancer stem-like cells (CSCs) play a vital role in drug resistance and recurrence of ovarian cancer. Inducing phenotypic differentiation is an important strategy to enhance the effects of chemotherapy and reduce the drug resistance of CSCs. This study found that lumiflavin, a riboflavin decomposition product, reduced the development of CSC resistance and enhanced the chemotherapy effect of cisplatin (DDP) on CSCs in DDP-resistant ovarian cancer OVCAR-3 cell line (CSCs/DDP) and was related to the induction of CSC phenotypic differentiation. Results showed that the development of DDP-resistant OVCAR-3 cells was related to the increase in the proportion of CSCs/DDP, and the treatment with lumiflavin reduced the DDP-resistance levels of OVCAR-3 cells and proportion of CSCs/DDP. Further investigation found that lumiflavin synergistic with DDP increased apoptosis, decreased mitochondrial membrane potential, and inhibited the clonal formation of CSCs/DDP. Meanwhile, in vivo experiments showed that lumiflavin dose-dependently enhanced the chemotherapy effect of DDP on tumor-bearing nude mice inoculated by CSCs/DDP. Lumiflavin treatment also reduced the ratio of CD133+/CD177+ to CD44+/CD24 cells, which is the identification of CSCs, in CSCs/DDP. In addition, transcriptome sequencing results suggested that the role of lumiflavin was related to the notch and stem cell pathway, and Western blot analysis showed that lumiflavin inhibited the protein expression of notch signaling pathway in CSCs/DDP. In conclusion, lumiflavin reduces the development of the drug resistance of OVCAR-3 cell and increases the sensitivity of CSCs/DDP to DDP by inducing phenotypic differentiation, which may have a potential role in the chemotherapy treatment of ovarian cancer.

Lumiflavin Enhances the Effects of Ionising Radiation on Ovarian Cancer Stem-Like Cells by Inhibiting Autophagy.

BACKGROUND: The development of Cancer Stem-like Cells (CSCs) is one of the main causes of ovarian cancer tolerance to radiotherapy. Autophagy is an adaptive process by which cells damage due to radiation. As a metabolite of riboflavin, lumiflavin can enhance the chemotherapeutic effects of cisplatin on ovarian cancer CSCs. OBJECTIVE: This study aimed to investigate the synergistic effects of lumiflavin and ionising radiation on ovarian cancer CSCs and explore the association of this metabolite with autophagy. METHODS: CSCs of human ovarian cancer cell lines HO8910 were treated with lumiflavin and rapamycin and then subjected to irradiation at a cumulative dose of 8 Gy. Cell proliferation ability, clonal formation ability, apoptosis rate, autophagy changes and autophagy-related protein changes were detected. RESULTS: Lumiflavin and ionising radiation synergistically reduced cell vitality and clone formation and increased the apoptosis of CSCs compared with irradiation alone. In addition, ionising radiation increased autophagy and the expression of associated proteins, whereas lumiflavin reduced those changes in autophagy progression. Moreover, rapamycin, an autophagy inhibitor, was observed to block the synergistic effects of lumiflavin and ionising radiation on CSC apoptosis. CONCLUSION: Lumiflavin can enhance the effects of ionising radiation on ovarian cancer CSCs. The mechanism by which these effects are exerted is related to blocking the autophagy pathway.

Healing characteristics of acellular porcine corneal stroma following therapeutic keratoplasty.

BACKGROUND: Acellular porcine corneal stroma (APCS) has proven to be a promising alternative to traditional corneal grafts. This prospective case series was conducted to further investigate the healing characteristics of APCS following keratoplasty. METHODS: Twenty-seven patients undergoing APCS implantation to treat infectious keratitis were included. The patients were followed up for 12 months after surgery. The main outcome measures included visual acuity, corneal transparency, graft thickness, and cellular and nerve regeneration. RESULTS: In the operated eyes, the best-corrected visual acuity (BCVA, in logarithm of the minimal angle of resolution [logMAR] units) increased from 1.23 ± 0.95 logMAR before surgery to 0.23 ± 0.18 logMAR at 12 months after surgery (P < .001). The contrast sensitivity was still evidently reduced, especially at higher spatial frequencies. Gradual transparency improvement was observed in APCS grafts post-operatively. After implantation, the APCS graft thickness initially increased (day 1 = 592.41 ± 52.69 µm) but then continuously decreased until 3 months after surgery (1 month = 449.26 ± 50.38 µm; 3 months = 359.63 ± 34.14 µm, P < .001). Graft reepithelialization was completed within 1 week. In the in vivo confocal microscopy scans, host keratocytes began to repopulate the APCS grafts between 3 and 6 months post-operatively; subbasal nerve regeneration was only noted in 18.52% (5/27) of the eyes by 12 months after surgery. CONCLUSIONS: Acellular porcine corneal stroma functions as an effective alternative to human corneal tissue in lamellar keratoplasty. However, APCS is somewhat different from fresh human cornea in term of the post-operative healing process, which warrants the attention of both clinicians and patients.

Lumiflavin increases the sensitivity of ovarian cancer stem-like cells to cisplatin by interfering with riboflavin.

Here, we used lumiflavin, an inhibitor of riboflavin, as a new potential therapeutic chemosensitizer to ovarian cancer stem-like cells (CSCs). This study demonstrates that the enrichment of riboflavin in CSCs is an important cause of its resistance to chemotherapy. Lumiflavin can effectively reduce the riboflavin enrichment in CSCs and sensitize the effect of cisplatin Diamminedichloroplatinum (DDP) on CSCs. In this study, CSCs of human ovarian cancer cell lines HO8910 were separated using a magnetic bead (CD133+). We also show the overexpression of the mRNA and protein of riboflavin transporter 2 and the high content of riboflavin in CSCs compared to non-CSCs (NON-CSCs). Moreover, CSCs were less sensitive to DDP than NON-CSCs, whereas, the synergistic effect of lumiflavin and DDP on CSCs was more sensitive than NON-CSCs. Further research showed that lumiflavin had synergistic effects with DDP on CSCs in increasing mitochondrial function damage and apoptosis rates and decreasing clonic function. In addition, we found that the combination of DDP and lumiflavin therapy in vivo has a synergistic cytotoxic effect on an ovarian cancer nude mice model by enhancing the DNA-damage response and increasing the apoptotic protein expression. Notably, the effect of lumiflavin is associated with reduced riboflavin concentration, and riboflavin could reverse the effect of DDP in vitro and in vivo. Accordingly, we conclude that lumiflavin interfered with the riboflavin metabolic pathways, resulting in a significant increase in tumour sensitivity to DDP therapy. Our study suggests that lumiflavin may be a novel treatment alternative for ovarian cancer and its recurrence.

Tacrolimus interacts with voriconazole to reduce the severity of fungal keratitis by suppressing IFN-related inflammatory responses and concomitant FK506 and voriconazole treatment suppresses fungal keratitis.

Purpose: To investigate the expression and roles of type I and II interferons (IFNs) in fungal keratitis, as well as the therapeutic effects of tacrolimus (FK506) and voriconazole on this condition. Methods: The mRNA and protein expression levels of type I (IFN-α/ß) and II (IFN-γ) IFNs, as well as of related downstream inflammatory cytokines (interleukin (IL)-1α, IL-6, IL-12, and IL-17), were detected in macrophages, neutrophils, lymphocytes, and corneal epithelial cells (A6(1) cells) stimulated with zymosan (10 mg/ml) for 8 or 24 h. A fungal keratitis mouse model was generated through intrastromal injection of Aspergillus fumigatus, and the mice were then divided into four groups: group I, the PBS group; group II, the voriconazole group; group III, the FK506 group; and group IV, the voriconazole plus 0.05% FK506 group. Corneal damage was evaluated with clinical scoring and histological examination. In addition, the mRNA and protein expression levels of type I (IFN-α/ß) and type II (IFN-γ) IFNs, as well as related inflammatory cytokines, were determined at different time points using quantitative real-time PCR (qRT-PCR) and western blotting. Results: After zymosan stimulation of mouse neutrophils, lymphocytes, macrophages, and A6(1) cells, the IFN mRNA and protein expression levels were markedly increased until 24 h, peaking at 8 h (p<0.001). The mRNA and protein expression levels of inflammatory cytokines (IL-1α, IL-6, IL-12, and IL-17) were also upregulated after zymosan stimulation. Moreover, type I (IFN-α/ß) and type II (IFN-γ) IFN expression levels were increased and positively correlated with the progression of fungal keratitis in vivo. FK506 administered with voriconazole reduced the pathological infiltration of inflammatory cells into the cornea and downregulated the expression levels of IFNs and related inflammatory cytokines. Conclusions: In conclusion, this study demonstrated that type I and II IFN levels were markedly increased in fungal keratitis and that FK506 combined with voriconazole decreased the severity of fungal keratitis by suppressing type I and II IFNs and their related inflammatory responses.

Aspirin modulates the inflammatory response in a thrombus­stimulated LMVEC model.

The purpose of the present study was to examine whether aspirin interferes with the inflammatory response in a thrombus­stimulated lung microvascular endothelial cell (LMVEC) model. The LMVECs were randomly divided into eight groups: Normal group (group N), model group (group M), model + ASP group (group M+A), model+CX3CL1­short hairpin (sh)RNA group (group M+SH), model + CX3CL1­overexpression vector group (group M+CX3), model + ASP + shRNA group (group M+A+SH), model + ASP + CX3CL1­overexpression vector group (group M+A+CX3), and normal + virus control group (group N+V). The endothelial cells were cultured, and a thrombus was added to the cells. Briefly, 12 h following the precipitation of the thrombus, data from ELISA, reverse transcription­quantitative polymerase chain reaction analysis and confocal microscopy revealed that the levels of tumor necrosis factor (TNF)­α, interleukin (IL)­6, CX3C chemokine ligand 1 (CX3CL1), CX3C chemokine receptor 1 (CX3CR1) and nuclear factor­κB (NF­κB) in group M were increased, compared with those in group N (P<0.01). These levels, with the exception of TNF­α, were significantly lower in group M+SH, compared with those in group M (P<0.01). Furthermore, the levels of IL­6 in groups M+A, M+CX3 and M+A+CX3 were decreased, compared with those in group M (P<0.01); the level of TNF­α in group M+A+SH was decreased, compared with that in group M (P<0.01); the level of CX3CR1 waslower in groups M+A and M+A+SH, compared with that in group M (P<0.01), and the level of NF­κB in group M+SH was decreased, compared with the level in group M and group M+A (P<0.05). In conclusion, the thrombus­stimulated LMVEC model exhibited induced production of TNF­α, IL­6, CX3CL, CX3CR1, NF­κB and intercellular adhesion molecule­1. Furthermore, it was confirmed that the signaling pathways involving CX3CL1­NF­κB, IL­6 and TNF­α were partly inhibited by aspirin.

New Grading System for Limbal Dermoid: A Retrospective Analysis of 261 Cases Over a 10-Year Period.

PURPOSE: To establish a new scoring system for limbal dermoid, in order to unify the diagnostic criteria and assess the prognosis. METHODS: A retrospective study was conducted on 261 patients with limbal dermoid. The basic information, clinical features, and pathology of dermoids were recorded, and the prognosis at 1 year after keratoplasty was assessed at follow-up. A new visual scoring system was created for the area of corneal involvement, the area of conjunctival involvement, and the surface shape. RESULTS: There were 154 females and 107 males with mean age of 4 ± 3 years at surgery. After scoring, 59% (136) of patients were classified as grade I, 26% (60) as grade II, and 14% (33) as grade III. The pathological results were 124 dermoid cases, 76 lipodermoid, 5 complex choristoma, and 10 epibulbar osseous choristoma. Moreover, patients with lower clinical scores presented a better prognosis; the mean logarithm of the minimum angle of resolution (logMAR) best-corrected visual acuity in grade I patients was 0.38 ± 0.05, which was better than the grade II value of 0.61 ± 0.09 (P < 0.05) and the grade III value of 0.94 ± 0.11 (P < 0.001). CONCLUSIONS: New grading systems for limbal dermoid were useful for clinical diagnosis and may have prognostic value in predicting visual acuity. A lower-grade dermoid exhibited better vision postoperatively.

Tacrolimus downregulates inflammation by regulating pro­/anti­inflammatory responses in LPS­induced keratitis.

Lipopolysaccharide (LPS)­induced keratitis is a progressive infectious ocular disease in which innate inflammatory responses often cause clinical tissue damage and vision loss. The present study aimed to investigate the effects of tacrolimus, an effective immunomodulator, on LPS­induced innate immune responses. The effects of tacrolimus on the apoptotic rate and viability of human corneal epithelial cells (HCECs), polymorphonuclear neutrophils (PMNs) and monocytes (THP­1 cells) were examined using flow cytome-try and MTT assays. Subsequently, the role of tacrolimus on LPS­induced inflammation in HCECs, PMNs and THP­1 cells was evaluated by detecting the expression levels of pro­inflammatory cytokines, including interleukin (IL)­1ß, IL­6 and matrix metallopeptidase 9; anti­inflammatory cytokines, including IL­10 and transforming growth factor­ß; and proangiogenic factors, including vascular endothelial growth factor and tumor necrosis factor­α using quantitative polymerase chain reaction. The results demonstrated that tacrolimus had good biocompatibility with HCECs, while promoting apoptosis and decreasing the viability of PMNs and THP­1 cells. Furthermore, tacrolimus effectively reduced the expression levels of pro­inflammatory cytokines and increased anti­inflammatory cytokines in LPS­induced keratitis in vitro. Notably, tacrolimus decreased the levels of proangiogenic factors, which are highly increased following LPS stimulation. Conclusively, tacrolimus appears to be a safe and effective treatment to suppress neutrophil and monocyte activity, modulate the balance of pro­/anti­inflammatory cytokines, and reduce the inflammatory response and angiogenic activity in LPS­induced bacterial keratitis.

Influence of aspirin on the CX3CL1/CX3CR1 signaling pathway in acute pulmonary embolism.

The present study aimed to explore the influence of aspirin on the CX3CL1/CX3CR1 signaling pathway in acute pulmonary embolism (APE) in rats. Our previous study found that CX3CL1/CX3CR1 was increased in APE. However, the effect of this signaling pathway on APE remains unclear. CX3CL1-shRNA adenovirus and CX3CL1-overexpression vector were constructed. Male Sprague-Dawley rats were randomly divided into 9 groups (n=10): normal group (group N), sham operation group (group Sham), sham operation + aspirin group (group ASP), model group (group M), model + ASP group (group M+A), model + shRNA group (group M+SH), sham operation + CX3CL1-overexpression vector group (group Sham+Cx3), model + ASP + shRNA group (group M+A+SH), and model + ASP + CX3CL1-overexpression vector group (group M+A+CX3). Arterial pressure detection, hematoxylin and eosin staining, reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, and laser confocal scanning microscopy were applied. Aspirin significantly decreased pulmonary artery pressure, improve pathological changes in the embolism, and decreased the expression of CX3CL1/CX3CR1 and CX3CL1/NF-κB. Moreover, the adenovirus-overexpression CX3CL1 vector aggravated the inflammatory changes in APE, which were improved by aspirin. However, the intervention of the adenovirus CX3CL1 vector reduced the change, while its combination with aspirin significantly improved the change. In conclusion, aspirin improved pathological changes in rats with APE via the CX3CL1/CX3CR1 signaling pathway.

Effects of aspirin on the ERK and PI3K/Akt signaling pathways in rats with acute pulmonary embolism.

Inflammation contributes to acute pulmonary embolism (APE). However, the contributions of the extracellular signal­regulated protein kinases (ERK) and phosphoinositide 3 kinase/protein kinase B (PI3K/Akt) signaling pathways have not yet been elucidated. The aim of this study was to examine the effects of aspirin on ERK and PI3K/Akt signaling in a rat model of APE and evaluate the prognostic values of brain natriuretic peptide (BNP), troponin (TnT) and D­Dimer. A total of 108 Sprague­Dawley rats were assigned into the control, sham, model and low­, medium­ and high­dose aspirin (150, 300 and 600 mg/kg, respectively) groups. In each group, six rats were sacrificed 6, 24 and 72 h subsequent to the induction of APE to collect the lungs and serum. Western blot analysis was used to assess ERK, PI3K and Akt expression; enzyme­linked immunosorbent assay (ELISA) was used to analyze BNP, TnT and D­Dimer levels; and changes in lung pathology were evaluated using hematoxylin and eosin (H&E) staining. The results showed that ERK and PI3K levels were decreased in the control, sham and the three aspirin groups at all time­points compared with the model group (P<0.01). The exception was in the medium­dose aspirin group at 24 h. The serum levels of BNP, TnT and D­Dimer were lower in the control and sham groups at all time­points compared with the model group (P<0.05). Furthermore, the levels of BNP, TnT and D­Dimer levels were decreased in the aspirin­treated groups (P<0.05) and markedly increased in the model group (P<0.05) at 24 h compared with the levels at 6 h. Pulmonary embolism, alveolar wall necrosis and hemorrhage were observed in the model group 6, 24 and 72 h subsequent to the induction of the model. However, congestion and inflammation were attenuated following aspirin treatment. In conclusion, aspirin reduces lung damage and improves prognosis. Decreased ERK, PI3K and Akt expression in the lungs and reduced levels of BNP, TnT and D­Dimer may be important factors in the effects observed.

Suppression of NF-κB pathway by crocetin contributes to attenuation of lipopolysaccharide-induced acute lung injury in mice.

Crocetin, a carotenoid compound, has been shown to reduce expression of inflammation and inhibit the production of reactive oxygen species. In the present study, the effect of crocetin on acute lung injury induced by lipopolysaccharide (LPS) was investigated in vivo. In the mouse model, pretreatment with crocetin at dosages of 50 and 100 mg/kg reduced the LPS-induced lung oedema and histological changes, increased LPS-impaired superoxide dismutase (SOD) activity, and decreased lung myeloperoxidase (MPO) activity. Furthermore, treatment with crocetin significantly attenuated LPS-induced mRNA and the protein expressions of interleukin-6 (IL-6), macrophage chemoattractant protein-1 (MCP-1), and tumour necrosis factor-α (TNF-α) in lung tissue. In addition, crocetin at different dosages reduced phospho-IκB expression and NF-κB activity in LPS-induced lung tissue alteration. These results indicate that crocetin can provide protection against LPS-induced acute lung injury in mice.