The blood–brain barrier (BBB), which is comprised of brain capillary endothelial cells, plays a pivotal role in the transport of drugs from the blood to the brain. Therefore, an analysis of proteins ...in the endothelial cells, such as transporters and tight junction proteins, which contribute to BBB function, is important for the development of therapeutics for the treatment of brain diseases. However, gene transfection into the vascular endothelial cells of the BBB is fraught with difficulties, even in vitro. We report herein on the development of lipid nanoparticles (LNPs), in which mRNA is encapsulated in a nano-sized capsule composed of a pH-activated and reductive environment-responsive lipid-like material (ssPalm). We evaluated the efficiency of mRNA delivery into non-polarized human brain capillary endothelial cells, hCMEC/D3 cells. The ssPalm LNPs permitted marker genes (GFP) to be transferred into nearly 100% of the cells, with low toxicity in higher concentration. A proteomic analysis indicated that the ssPalm-LNP had less effect on global cell signaling pathways than a Lipofectamine MessengerMAX/GFP-encoding mRNA complex (LFN), a commercially available transfection reagent, even at higher mRNA concentrations.
We have previously shown that prophylactic oral administration of transgenic rice seeds expressing hypoallergenic modified antigens suppressed the development of allergic conjunctivitis induced by ...Japanese cedar pollen. We have now investigated the efficacy of oral immunotherapy with such transgenic rice for established allergic conjunctivitis in mice.
BALB/c mice were sensitized with two intraperitoneal injections of Japanese cedar pollen in alum, challenged with pollen in eyedrops, and then fed for 16 days with transgenic rice seeds expressing modified Japanese cedar pollen allergens Cry j 1 and Cry j 2 or with nontransgenic rice seeds as a control. They were then challenged twice with pollen in eyedrops, with clinical signs being evaluated at 15 min after the first challenge and the eyes, blood, spleen, and lymph nodes being isolated at 24 h after the second challenge.
The number of eosinophils in the conjunctiva and the clinical score for conjunctivitis were both significantly lower in mice fed the transgenic rice than in those fed nontransgenic rice. Oral vaccination with transgenic rice seeds also resulted in a significant increase in the production of IFN-γ by splenocytes, whereas it had no effect on the number of CD4+CD25+Foxp3+ regulatory T cells in the spleen or submandibular or mesenteric lymph nodes.
Oral administration of transgenic rice seeds expressing hypoallergenic allergens ameliorated allergic conjunctivitis in the established setting. Such a rice-based edible vaccine is potentially both safe and effective for oral immunotherapy in individuals with allergic conjunctivitis.
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The synthesis of cytokines and adhesion molecules by corneal fibroblasts contributes to the innate immune response to corneal infection. The effects of the antimicrobial peptide LL37 on cytokine and ...adhesion molecule expression induced by bacterial lipopolysaccharide (LPS) in human corneal fibroblasts were examined.
The release of the cytokines IL-6 and IL-8 into culture supernatants and the expression of intercellular adhesion molecule (ICAM)-1 at the cell surface were measured with ELISAs and by flow cytometry. The abundance of mRNAs was quantitated by RT and real-time PCR analysis, and the phosphorylation of signaling proteins was examined by immunoblot analysis. The subcellular localization of ICAM-1 and the transcription factor nuclear factor (NF)-κB was determined by immunofluorescence analysis. Neutrophil infiltration in a mouse model of LPS-induced keratitis was evaluated by immunohistofluorescence analysis.
The antimicrobial peptide LL37 inhibited the up-regulation of IL-6, IL-8, and ICAM-1 both at protein and mRNA levels in corneal fibroblasts induced by LPS without affecting those elicited by TNF-α. Furthermore, LL37 attenuated the LPS-induced phosphorylation of the NF-κB inhibitor IκBα and the mitogen-activated protein kinases extracellular signal-regulated kinase, p38, and c-Jun NH2-terminal kinase, as well as the translocation of NF-κB to the nucleus in corneal fibroblasts. Lipopolysaccharide-induced keratitis in mice was also suppressed by topical application of LL37.
The inhibition of LPS-induced cytokine and adhesion molecule expression in human corneal fibroblasts by LL37 suggests that this peptide might promote the resolution of corneal inflammation associated with bacterial infection.
Background/aims To determine whether oral immunotherapy with transgenic rice seeds expressing hypoallergenic modified antigens suppresses cedar pollen-induced allergic conjunctivitis by eliciting ...immune tolerance in mice. Methods BALB/c mice were fed once a day for 20 days with 220 mg of transgenic rice expressing modified Japanese cedar pollen allergens Cry j 1 and Cry j 2 or with non-transgenic rice seeds as a control. They were then sensitised with two intraperitoneal injections of Japanese cedar pollen in alum before challenge twice with pollen in eye drops. Twenty-four hours after the second challenge, the conjunctiva, spleen, and blood were isolated for histological analysis, cytokine production assays, and measurement of serum immunoglobulin E concentrations, respectively. Results The numbers of eosinophils and total inflammatory cells in the conjunctiva were significantly lower in mice fed the transgenic rice than in those fed non-transgenic rice. The clinical score evaluated at 15 min after antigen challenge was also significantly lower in mice fed the transgenic rice than in those fed non-transgenic rice. The serum concentrations of both total and allergen-specific immunoglobulin E were also significantly lower in mice fed the transgenic rice. Oral vaccination with transgenic rice resulted in significant down-regulation of the allergen-induced production of interleukin (IL)-2, IL-4, IL-5, IL-12p70, interferon-γ, and IL-17A by splenocytes. Conclusions Oral immunotherapy with transgenic rice expressing modified Japanese cedar pollen allergens suppressed pollen-induced experimental allergic conjunctivitis in mice by eliciting immune tolerance. This novel prophylactic approach is potentially safe and effective for allergen-specific oral immunotherapy in allergic conjunctivitis.
The Tsuruoka Metabolomics Cohort Study (TMCS) is an ongoing population-based cohort study being conducted in the rural area of Yamagata Prefecture, Japan. This study aimed to enhance the precision ...prevention of multi-factorial, complex diseases, including non-communicable and aging-associated diseases, by improving risk stratification and prediction measures. At baseline, 11,002 participants aged 35–74 years were recruited in Tsuruoka City, Yamagata Prefecture, Japan, between 2012 and 2015, with an ongoing follow-up survey. Participants underwent various measurements, examinations, tests, and questionnaires on their health, lifestyle, and social factors. This study uses an integrative approach with deep molecular profiling to identify potential biomarkers linked to phenotypes that underpin disease pathophysiology and provide better mechanistic insights into social health determinants. The TMCS incorporates multi-omics data, including genetic and metabolomic analyses of 10,933 participants, and comprehensive data collection ranging from physical, psychological, behavioral, and social to biological data. The metabolome is used as a phenotypic probe because it is sensitive to changes in physiological and external conditions. The TMCS focuses on collecting outcomes for cardiovascular disease, cancer incidence and mortality, disability and functional decline due to aging and disease sequelae, and the variation in health status within the body represented by omics analysis that lies between exposure and disease. It contains several sub-studies on aging, heated tobacco products, and women’s health. This study is notable for its robust design, high participation rate (89%), and long-term repeated surveys. Moreover, it contributes to precision prevention in Japan and East Asia as a well-established multi-omics platform.
Background Disruption of corneal epithelial barrier function by inflammation may contribute to the development of dry eye. The effects of rebamipide, a drug used for the treatment of dry eye, on ...barrier function and cytokine expression in a human corneal epithelial (HCE) cell line were examined. Methods Barrier function of HCE cells was evaluated by measurement of transepithelial electrical resistance (TER). The subcellular localisation of the tight junction protein zonula occludens (ZO)-1 was examined by immunofluorescence analysis. The release of cytokines was determined with ELISAs, and the intracellular abundance of cytokine mRNAs was quantitated by reverse transcription and real-time PCR analysis. Degradation of the nuclear factor-κB inhibitor IκBα was detected by immunoblot analysis. Results Rebamipide increased TER of HCE cells in a concentration-dependent manner as well as attenuating the loss of TER and the disappearance of ZO-1 from the cell surface induced by tumour necrosis factor α (TNFα). Rebamipide also suppressed TNFα-induced expression of interleukin-6 and interleukin-8 at the mRNA and protein levels and inhibited the TNFα-induced degradation of IκBα. Conclusions The upregulation of barrier function and the anti-inflammatory effects of rebamipide, together with its mucin secretagogue activity, may contribute to the effectiveness of this drug for the treatment of dry eye.
Background/aims The dry-eye drug rebamipide has mucin secretagogue activity in and anti-inflammatory effects on corneal epithelial cells. Corneal stromal fibroblasts (transdifferentiated keratocytes) ...function as immune modulators in the pathogenesis of chronic ocular allergic inflammation and in innate immune responses at the ocular surface. The possible anti-inflammatory effects of rebamipide on human corneal stromal fibroblasts were examined. Methods Serum-deprived cells were incubated for 1 h with rebamipide and then for various times in the additional absence or presence of cytokines or bacterial lipopolysaccharide (LPS). The release of chemokines into culture supernatants was determined with ELISAs. The intracellular abundance of chemokine mRNAs was quantitated by reverse transcription and real-time PCR analysis. Degradation of the nuclear factor κB (NFκB) inhibitor IκBα was detected by immunoblot analysis. Results Rebamipide suppressed the release of interleukin (IL)-8 and the upregulation of IL-8 mRNA induced by tumour necrosis factor α (TNF-α) or LPS in corneal fibroblasts. It also inhibited eotaxin-1 (CCL-11) expression at the protein and mRNA levels induced by the combination of TNF-α and IL-4. In addition, rebamipide attenuated the degradation of IκBα induced by TNF-α or LPS. Conclusions Rebamipide inhibited the synthesis of chemokines by corneal fibroblasts in association with suppression of NFκB signalling. Rebamipide may therefore prove effective for the treatment of corneal stromal inflammation associated with allergy or bacterial infection.
Severe ocular allergic diseases are characterized by pronounced conjunctival inflammation triggered by T helper 2 (Th2) cells and corneal epithelial damage induced by eosinophils. To examine the role ...of alarmins released by damaged corneal epithelial cells in tissue eosinophilia, we investigated the effects of a supernatant derived from necrotic human corneal epithelial (HCE) cells on expression of the chemokine CCL11 (eotaxin) and the adhesion molecule VCAM-1 in human corneal fibroblasts.
An alarmin preparation was obtained as the material released from HCE cells after three cycles of freezing and thawing. CCL11 released into culture medium and cell surface expression of VCAM-1 were measured with enzyme-linked immunosorbent assays, and the amounts of CCL11 and VCAM-1 mRNAs were quantitated by reverse transcription and real-time polymerase chain reaction analysis. Signaling by the transcription factor NF-κB was evaluated by immunoblot and immunofluorescence analyses.
The combination of the necrotic HCE cell supernatant and either interleukin (IL)-4 or IL-13 induced synergistic increases in CCL11 release, VCAM-1 expression, and the abundance of CCL11 and VCAM-1 mRNAs in corneal fibroblasts. The necrotic HCE cell supernatant also induced NF-κB activation in corneal fibroblasts, whereas an inhibitor of NF-κB and IL-1 receptor antagonist each attenuated CCL11 release induced by the alarmin preparation and either IL-4 or IL-13.
Alarmins including IL-1 released from necrotic corneal epithelial cells cooperate with Th2 cytokines to induce CCL11 production and VCAM-1 expression in corneal fibroblasts, and may thereby play an important role in tissue eosinophilia associated with ocular allergic diseases.
Antigen-specific immunotherapy is expected to be a desirable treatment for allergic diseases. Currently, antigen-specific immunotherapy is performed by administering disease-causing antigens ...subcutaneously or sublingually. These approaches induce long-term remission in patients with allergic rhinitis or asthma. The oral route is an alternative to subcutaneous and sublingual routes, and can also induce long-term remission, a phenomenon known as “oral tolerance.” The effectiveness of oral tolerance has been reported in the context of autoimmune diseases, food allergies, asthma, atopic dermatitis, and allergic rhinitis in both human patients and animal models. However, few studies have examined its efficacy in animal models of allergic conjunctivitis. Previously, we showed that ovalbumin feeding suppressed ovalbumin-induced experimental allergic conjunctivitis, indicating the induction of oral tolerance is effective in treating experimental allergic conjunctivitis. In recent years, transgenic rice has been developed that can induce oral tolerance and reduce the severity of anaphylaxis. The major Japanese cedar pollen antigens in transgenic rice, Cryptomeria japonica 1 and C. japonica 2, were deconstructed by molecular shuffling, fragmentation, and changes in the oligomeric structure. Thus, transgenic rice may be an effective treatment for allergic conjunctivitis.
► Analysis of CoQ10 by online supercritical fluid extraction (SFE)–SFC–MS. ► Only 0.05mg of dried photosynthesis bacterium was needed for the analysis. ► A higher ratio of the reduced form of CoQ10 ...was detected by online SFE. ► Highly accurate analysis of easily oxidized compounds can be performed.
The accurate profiling of oxidative stress markers is often disturbed by oxidation occurred during sample preparation. In this study, an online supercritical fluid extraction–supercritical fluid chromatography/mass spectrometry system was constructed utilizing supercritical fluid as an efficient solvent for extraction. A higher ratio of the reduced form of coenzyme Q10 (CoQ10) was detected by online extraction, rather than by offline extraction using a conventional organic solvent. The results suggest that the profile of easily oxidized compounds can be acquired with high accuracy using this system because the oxidation of the target compounds during sample preparation can be avoided when using the online extraction.
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