RESUMO
The prevalence of presbyopia and nuclear cataracts (NUC) is reported to be higher in tropical areas than that in other regions, suggesting a potential influence of high temperatures on lens health. Transient receptor potential vanilloid (TRPV) channels play a crucial role in detecting ambient temperatures across various species, with TRPV1 and TRPV4 expressed in lens epithelial cells. In this study, we investigated whether ambient temperatures affect TRPV1 and TRPV4 activity in the lens, potentially contributing to the development of presbyopia and NUC. We conducted experiments using cultured human lens epithelial cell lines under different temperature conditions. Our results revealed that the mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) and p38 pathways, downstream molecules of TRPV1, were activated, while Src family kinase, a downstream molecule of TRPV4, was inhibited at 37.5 °C culture compared to 35.0 °C. Confocal microscope images demonstrated higher expression of TRPV1 in 3D-structured cells under high-temperature culture conditions. Additionally, in organ culture lenses, higher elasticity was observed at elevated temperatures compared to that at lower temperatures. These results suggest that high ambient temperatures may induce lens sclerosis via TRPV1 activation, potentially contributing to the development of presbyopia and NUC.
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Regulation of ion and water microcirculation within the lens is tightly controlled through aquaporin channels and connexin junctions. However, cataracts can occur when the lens becomes cloudy. Various factors can induce cataracts, including diabetes which is a well-known cause. The most common phenotype of diabetic cataracts is a cortical and/or posterior subcapsular opacity. In addition to the three main types and two subtypes of cataracts, a vacuole formation is frequently observed; however, their origin remains unclear. In this study, we focused on the aquaporins and connexins involved in diabetes-induced cataracts and vacuoles in Nile grass type II diabetes. The results showed that the expression of aquaporin 0 and aquaporin 5 increased, and that of connexin 43 decreased in diabetic rat lenses. Additionally, aquaporin 0 and 5 were strongly localized in peripheral of vacuoles, suggesting that aquaporins are involved in vacuoles formation. Transillumination photography revealed large vacuoles at the tip of the Y-suture in the anterior capsule of the diabetic lens, and several small vacuoles were observed in the posterior capsule. Within the vacuoles, cytoplasmic degradation and aggregation of fibrous material were observed. Our findings suggest that aquaporins are potential candidate proteins for preventing vacuole formation.
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Aquaporinas , Catarata , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Ratos , Animais , Vacúolos/metabolismo , Conexinas/genética , Conexinas/metabolismo , Aquaporinas/metabolismoRESUMO
When regenerated tissue is generated from induced pluripotent stem cells (iPSCs), it is necessary to track and identify the transplanted cells. Fluorescently-labeled iPSCs synthesize a fluorescent substance that is easily tracked. However, the expressed protein should not affect the original genome sequence or pluripotency. To solve this problem, we created a cell tool for basic research on iPSCs. Iris tissue-derived cells from GFP fluorescence-expressing mice (GFP-DBA/2 mice) were reprogrammed to generate GFP mouse iris-derived iPSCs (M-iris GFP iPSCs). M-iris GFP iPSCs expressed cell markers characteristic of iPSCs and showed pluripotency in differentiating into the three germ layers. In addition, when expressing GFP, the cells differentiated into functional recoverin- and calbindin-positive cells. Thus, this cell line will facilitate future studies on iPSCs.
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Células-Tronco Pluripotentes Induzidas , Iris , Neurônios Retinianos , Animais , Camundongos , Calbindinas/metabolismo , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/metabolismo , Iris/citologia , Camundongos Endogâmicos DBA , Recoverina/metabolismo , Neurônios Retinianos/metabolismoRESUMO
The incidence rate of post-cataract surgery posterior capsule opacification (PCO) and lens turbidity is about 20% in 5 years. Soemmering's ring, which is a type of PCO also called a regenerated lens with similar tissue structure to that of a human lens, is an important proxy for elucidating the mechanism of lens regeneration and maintenance of transparency. The authors created new human immortalized crystalline lens epithelial cells (iHLEC-NY1s) with excellent differentiation potential, and as a result of culturing the cells by static and rotation-floating methods, succeeded in producing a three-dimensional cell structure model (3D-iHLEC-NY1s) which is similar to Soemmering's ring in tissue structure and expression characteristics of αA-crystalline, ßB2-crystalline, vimentin proteins. 3D-iHLEC-NY1s is expected to be a proxy in vitro experimental model of Soemmering's ring to enable evaluation of drug effects on suppression of cell aggregate formation and transparency. By further improving the culture conditions, we aim to control the cell sequence and elucidate the mechanism underlying the maintenance of lens transparency.
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Opacificação da Cápsula/patologia , Linhagem Celular Transformada , Células Epiteliais/citologia , Cristalino/citologia , Idoso , Diferenciação Celular , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Feminino , Humanos , Cristalino/metabolismo , Modelos Biológicos , Vimentina , Cadeia A de beta-Cristalina , Cadeia B de beta-CristalinaRESUMO
We designed an intravitreal injection formulation containing lanosterol nanoparticles (LAN-NPs) via the bead mill method and evaluated the therapeutic effect of LAN-NPs on lens structure collapse and opacification using two rat cataract models (SCR-N, rats with slight lens structure collapse; SCR-C, rats with the combination of a remarkable lens structure collapse and opacification). The particle size of lanosterol in the LAN-NPs was around 50-400 nm. A single injection of LAN-NPs (0.5%) supplied lanosterol into the lens for 48 h, and no irritation or muddiness was observed following repeated injections of LAN-NPs for 6 weeks (once every 2 days). Moreover, LAN-NPs repaired the slight collapse of the lens structure in SCR-N. Although the remarkable changes in the lens structure of SCR-C were not repaired by LAN-NP, the onset of opacification was delayed. In addition, the increase of cataract-related factors (Ca2+ contents, nitric oxide levels, lipid peroxidation and calpain activity levels) in the lenses of SCR-C was attenuated by the repeated injection of LAN-NPs. It is possible that a deficiency of lanosterol promotes the production of oxidative stress. In conclusion, it is difficult to improve serious structural collapse with posterior movement of the lens nucleus with a supplement of lanosterol via LAN-NPs. However, the intravitreal injection of LAN-NPs was found to repair the space and structural collapse in the early stages in the lenses.
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Catarata/prevenção & controle , Lanosterol/uso terapêutico , Cristalino/patologia , Nanopartículas/uso terapêutico , Animais , Catarata/tratamento farmacológico , Linhagem Celular , Humanos , Injeções Intravítreas , Lanosterol/administração & dosagem , Masculino , Nanopartículas/administração & dosagem , Óxido Nítrico/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Transgênicos , Transtornos da Visão/prevenção & controleRESUMO
Atopic cataracts develop under the ages of 40 years, after which visual acuity rapidly declines. However, the mechanism underlying the development of atopic cataracts is not yet clear. We focused on the eosinophil granule major basic protein (MBP), which was detected in the aqueous humor of atopic cataracts previously, and which was cytotoxic. Specifically, we investigated its origin in this fluid and its effects on lens epithelial cells (LECs). MBP immunostaining was positive in atopic cataract-derived LECs, but negative in age-related cataract-derived LECs. MBP mRNA was not detected in either type of cataract, but protein was detected in the aqueous humor. Furthermore, the flare values associated with atopic cataracts were higher than those with age-related cataracts. When MBP was purified from eosinophils or recombinant MBP was added to LEC culture medium, cell viability decreased in a concentration-dependent manner, but an MBP antibody neutralized the cytotoxic effect of this protein towards these cells. These results were consistent with the flow of MBP into the aqueous humor from the blood due to a compromised blood-aqueous barrier. Thus, MBP could further penetrate the lens capsule and adhere to LECs, resulting in decreased cell viability and the development of atopic cataracts.
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Catarata/imunologia , Proteína Básica Maior de Eosinófilos/metabolismo , Eosinófilos/metabolismo , Proteoglicanas/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Humor Aquoso/imunologia , Humor Aquoso/metabolismo , Estudos de Casos e Controles , Catarata/sangue , Catarata/patologia , Extração de Catarata , Sobrevivência Celular/imunologia , Células Cultivadas , Proteína Básica Maior de Eosinófilos/análise , Proteína Básica Maior de Eosinófilos/imunologia , Proteína Básica Maior de Eosinófilos/isolamento & purificação , Eosinófilos/imunologia , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Feminino , Humanos , Cristalino/citologia , Cristalino/imunologia , Cristalino/patologia , Cristalino/cirurgia , Masculino , Cultura Primária de Células , Proteoglicanas/análise , Proteoglicanas/imunologia , Proteoglicanas/isolamento & purificação , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Adulto JovemRESUMO
Dendritic cell-based immunotherapy, which uses a patient's own immune cells, can be used for cancer treatment and allergy control, such as autoimmune disease and rejection associated with transplantation. However, these treatments create a burden on patients due to repeated blood collection. We used cell biological analysis of monocytes with few mutations obtained from minimal blood collection for genome recombination. Next, we established human peripheral blood monocyte-derived induced pluripotent stem cells (iPSCs) using a commercial vector and standard culture method. We found that when established iPSCs were induced to differentiate, monocytes showed phagocytic properties and expressed CD14 and CX3CR1. Further, the generated dendritic cells (DCs) expressed CCL17 and highly expressed HLA-DR following the addition of the mite antigen. Taken together, these data show that monocyte-derived iPS cells can be used to differentiate into monocytes and DCs. In addition, the use of these cells can be applied to the pathological analysis of dendritic cell therapy and monocyte diseases.
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Diferenciação Celular , Células Dendríticas/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Monócitos/fisiologia , Receptor 1 de Quimiocina CX3C/análise , Receptor 1 de Quimiocina CX3C/metabolismo , Células Cultivadas , Quimiocina CCL17/análise , Quimiocina CCL17/metabolismo , Células Dendríticas/transplante , Feminino , Citometria de Fluxo , Antígenos HLA-DR/análise , Antígenos HLA-DR/metabolismo , Voluntários Saudáveis , Humanos , Imunoterapia/métodos , Receptores de Lipopolissacarídeos/análise , Receptores de Lipopolissacarídeos/metabolismo , Masculino , Pessoa de Meia-Idade , Cultura Primária de Células/métodosRESUMO
Diabetes mellitus is a widespread metabolic disorder, and long-term hyperglycemia in diabetics leads to diabetic keratopathy. In the present study, we used a shotgun liquid chromatography/mass spectrometry-based global proteomic approach using the cornea of streptozotocin-induced diabetic (STZ) rats to examine the mechanisms of delayed corneal wound healing in diabetic keratopathy. Applying a label-free quantitation method based on spectral counting, we identified 188 proteins that showed expression changes of >2.0-fold in the cornea of STZ rats. In particular, the level of lumican expression in the cornea of STZ rats was higher than that of the normal rats. In the cornea of the normal rat, the expression level of lumican was elevated during the wound healing process, and it returned to the same expression level as before cornea injury after the wound was healed completely. On the other hand, a high expression level of lumican in the cornea of STZ rats was still maintained even after the wound was healed completely. In addition, adhesion deficiency in corneal basal cells and Bowman's membrane was observed in the STZ rat. Thus, abnormally overexpressed lumican may lead to adhesion deficiency in the cornea of STZ rats.
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Córnea/metabolismo , Córnea/patologia , Lesões da Córnea/metabolismo , Lesões da Córnea/patologia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Proteômica/métodos , Cicatrização , Animais , Modelos Animais de Doenças , Ontologia Genética , Hiperglicemia/metabolismo , Masculino , Anotação de Sequência Molecular , Ratos WistarRESUMO
We previously prepared ophthalmic formulations containing cilostazol (CLZ) nanoparticles by bead mill methods (CLZnano), and found that instillation of CLZnano into rat eyes supplies CLZ into the retina. In this study, we investigated changes in the electroretinograms (ERG) of streptozotocin-induced diabetic rats (STZ rats), a model of diabetes mellitus. In addition, we demonstrated that dispersions containing CLZ nanoparticles attenuate changes in the ERG of STZ rats. The instillation of CLZnano had no effect on body weight or plasma glucose and insulin levels. Furthermore, no corneal toxicity was observed in the in vivo study using STZ rats. The a-wave and b-wave levels in addition to oscillatory potentials (OP) amplitude decreased in STZ rats two weeks after the injection of streptozotocin, with the instillation of CLZnano attenuating these decreases. In addition, the level of vascular endothelial growth factor (VEGF) in the retinas of STZ rats was 9.26-fold higher than in in normal rats, with this increase also prevented by the instillation of CLZnano Thus, we have found that a-wave and b-wave levels in addition to OP amplitude are decreased in rats following the injection of excessive streptozotocin. Furthermore, the retinal disorders associated with diabetes mellitus are attenuated by the instillation of CLZnano. These findings provide significant information that can be used to design further studies aimed at developing anti-diabetic retinopathy drugs.
Assuntos
Retinopatia Diabética/etiologia , Retinopatia Diabética/fisiopatologia , Nanopartículas , Tetrazóis/administração & dosagem , Animais , Linhagem Celular , Sobrevivência Celular , Cilostazol , Diabetes Mellitus Experimental , Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/patologia , Modelos Animais de Doenças , Eletrorretinografia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Epitélio Corneano/citologia , Epitélio Corneano/metabolismo , Masculino , Ratos , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/patologia , Estreptozocina/efeitos adversosRESUMO
Retinopathy leads to irreparable vision loss via capillary closure and areas of nonperfusion. However, the current instillation systems do not allow a sufficient amount of drug required to treat retinopathy to reach the posterior segment (retina); therefore, a new formulation targeting the posterior segment is expected as therapy for retinopathy. We prepared ophthalmic formulations containing nilvadipine nanoparticles (NILnano), and demonstrated whether the instillation of NILnano can prevent retinal dysfunction in rats injected with excessive streptozotocin (STZ rats) in this study. NILnano (mean particle size, 77 nm) was prepared by wet bead mill treatment, with the inclusion of various additives (2-hydroxypropyl-ß-cyclodextrin, benzalkonium chloride, d-mannitol, and methylcellulose). Retinal dysfunction was observable two weeks after rats received intraperitoneal injections of streptozotocin (100 mg/kg × 2, consecutive days, STZ rat). Changes in retinal function were evaluated by electroretinogram (ERG) and immunological methods. The retinal thickness, measured as the distance between the ganglion cell layer and the distal border of the outer nuclear layer, increased two weeks after the injection of streptozotocin, resulting in decreases in the levels of a-waves, b-waves, and oscillatory potential amplitudes in ERG of rats. The instillation of NILnano allowed the topical supplement of nilvadipine into the retina, and repeated instillation of NILnano (2 times/day) attenuated the retinal disorders led by the excessive streptozotocin. In conclusion, we found that retinal dysfunction in rats injected with streptozotocin can be prevented by the NILnano instillation. These results are useful in further studies aimed at the therapeutic treatment of retinopathy.
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Diabetes Mellitus Experimental/tratamento farmacológico , Retinopatia Diabética/tratamento farmacológico , Nifedipino/análogos & derivados , Estreptozocina/efeitos adversos , Animais , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/patologia , Retinopatia Diabética/induzido quimicamente , Retinopatia Diabética/fisiopatologia , Eletrorretinografia , Humanos , Nanopartículas/administração & dosagem , Nanopartículas/química , Nifedipino/administração & dosagem , Nifedipino/química , Ratos , Retina/efeitos dos fármacos , Retina/fisiopatologiaRESUMO
In a previous study, a novel human corneal-like epithelium model utilizing an immortalized human corneal epithelial cell line (iHCE-NY1) was developed as an alternative to animal models to identify chemicals not classified under the United Nations Globally Harmonized System of Classification and Labeling of Chemicals (GHS) and was evaluated following the criteria of Test Guideline 492 of the Organization for Economic Co-operation and Development (OECD). In the present study, our aim was to establish an eye irritation test protocol using the iHCE-NY1 model to classify liquid chemicals under the GHS ocular hazard categories: no effect, no classification (No Cat.), Category 2 (Cat. 2) reversible effects, and Category 1 (Cat. 1) irreversible eye damage. The protocol involved exposing the iHCE-NY1 model to 31 liquid test chemicals for 5 min, followed by observation at post-incubation periods (PIPs) to assess recovery. Classification was based on cell viability, and histopathological findings on PIP days 7, 14, and 21. The outcomes were compared with an established database of classifications. All Cat. 1 liquid chemicals, 62.5% of No Cat., and 63.2% of Cat. 2 were correctly categorized. This study demonstrates that the iHCE-NY1 model can not only distinguish No Cat. test liquid chemicals but also differentiate between Cat. 2 and Cat. 1 liquid chemicals.
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BACKGROUND: Bronchial thermoplasty (BT) is a recently developed non-pharmacological therapy for refractory bronchial asthma. Although increasing evidence has suggested that BT is effective for various phenotypes of severe asthma, its safety and efficacy in patients with severe irreversible impaired lung function are unclear. OBJECTIVES: To assess the efficacy and safety of BT in patients with refractory asthma, including patients with a severely impaired forced expiratory volume in 1 second (FEV1). DESIGN: This was a single-center, retrospective, observational cohort study. METHODS: We retrospectively reviewed the medical records of 15 patients with refractory asthma (Global Initiative for Asthma step 4 or 5), including patients with severely impaired airflow limitation (% predicted pre-bronchodilator FEV1 <60%), who had undergone BT between June 2016 and January 2022. We analyzed the efficacy (change in asthma symptoms, exacerbation rate, pulmonary function, asthma medication, and serum inflammatory chemokine/cytokines before and after BT) and complications in all patients. We compared these data between patients with severe obstructive lung dysfunction [group 1(G1)] and patients with FEV1 ⩾ 60% [group 2 (G2)]. RESULTS: Six patients were in G1 and nine were in G2. Clinical characteristics, T2 inflammation, and concurrent treatment were equivalent in both groups. BT significantly improved asthma-related symptoms (measured using the Asthma Control Test and Asthma Quality of Life Questionnaire scores) in both groups. FEV1 was significantly improved in G1 but not in G2. Four patients in G2, but none in G1, experienced asthma exacerbation requiring additional systemic corticosteroids (including two requiring prolonged hospitalization) after BT. Long-term responders (patients who reduced systemic or inhaled corticosteroid without newly adding biologics in a follow-up > 2 years) of BT were identified in G1 and G2 (n = 2, 33.3% and n = 4, 44.4%, respectively). CONCLUSION: BT in patients with refractory asthma and severe airflow limitation is equally safe and efficacious as that in patients with moderate airflow limitation.
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Asma , Termoplastia Brônquica , Pulmão , Índice de Gravidade de Doença , Humanos , Masculino , Estudos Retrospectivos , Feminino , Termoplastia Brônquica/efeitos adversos , Asma/fisiopatologia , Asma/terapia , Pessoa de Meia-Idade , Volume Expiratório Forçado , Resultado do Tratamento , Idoso , Pulmão/fisiopatologia , Adulto , Recuperação de Função Fisiológica , Fatores de Tempo , Qualidade de VidaRESUMO
Enhancement of density via human lens epithelium (HLE) cell proliferation is the underlying cause of nuclear cataracts. Moreover, our previous epidemiological study demonstrated that the risk of nuclear cataract development is significantly higher under elevated environmental temperatures compared with under lower temperatures. The present study investigated the relationship between temperature and cell proliferation in terms of mitochondrial function, which is a nuclear cataractinducing risk factor, using two different HLE cell lines, SRA01/04 and immortalized human lens epithelial cells NY2 (iHLECNY2). Cell proliferation was significantly enhanced under the hightemperature condition (37.5ËC) in both cell lines. The cell growth levels of SRA01/04 and iHLECNY2 cells cultured at 37.5ËC were 1.20 and 1.16fold those in the lowtemperature cultures (35.0ËC), respectively. Moreover, the levels of cytochrome c oxidase mRNA (mitochondrial genome, cytochrome c oxidase13) and its activity in SRA01/04 and iHLECNY2 cells cultured at 37.5ËC were higher compared with those in cells cultured at 35.0ËC. In addition, adenosine5'triphosphate (ATP) levels in SRA01/04 and iHLECNY2 cells were also significantly higher at 37.5ËC compared with those at 35.0ËC. By contrast, no significant differences in Na+/K+ATPase or Ca2+ATPase activities were observed between HLE cells cultured at 35.0 and 37.5ËC. These results suggested that expression of the mitochondrial genome was enhanced in hightemperature culture, resulting in a sufficient ATP content and cell proliferation for lens opacity. Therefore, elevated environmental temperatures may increase the risk of nuclear cataracts caused by HLE cell proliferation via mitochondrial activation.
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Catarata , Complexo IV da Cadeia de Transporte de Elétrons , Humanos , Complexo IV da Cadeia de Transporte de Elétrons/genética , Mitocôndrias , Células Epiteliais , Catarata/etiologia , Trifosfato de Adenosina , Adenosina TrifosfatasesRESUMO
Induced pluripotent stem (iPS) cells are widely used as a research tool in regenerative medicine and embryology. In studies related to lens regeneration in the eye, iPS cells have been reported to differentiate into lens epithelial cells (LECs); however, to the best of our knowledge, no study to date has described their formation of three-dimensional cell aggregates. Notably, in vivo studies in newts have revealed that iris cells in the eye can dedifferentiate into LECs and regenerate a new lens. Thus, as basic research on lens regeneration, the present study investigated the differentiation of human iris tissue-derived cells and human iris tissue-derived iPS cells into LECs and their formation of three-dimensional cell aggregates using a combination of two-dimensional culture, static suspension culture and rotational suspension culture. The results revealed that three-dimensional cell aggregates were formed and differentiated into LECs expressing αA-crystallin, a specific marker protein for LECs, suggesting that the cell-cell interaction facilitated by cell aggregation may have a critical role in enabling highly efficient differentiation of LECs. However, the present study was unable to achieve transparency in the cell aggregates; therefore, we aim to continue to investigate the degradation of organelles and other materials necessary to make the interior of the formed cell aggregates transparent. Furthermore, we aim to expand on our current work to study the regeneration of the lens and ciliary body as a whole in vitro, with the aim of being able to restore focusing function after cataract surgery.
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Osteosarcoma is a malignant tumor that produces neoplastic bone or osteoid osteoma. In human multicentric osteosarcoma (HMOS), a unique variant of human osteosarcoma (HOS), multiple bone lesions occur simultaneously or asynchronously before lung metastasis. HMOS is associated with an extremely poor prognosis, and effective treatment options are lacking. Using the proteins in our previously generated HMOS cell lines as antigens, we generated antibodies using a human antibody phage library. We obtained antibody clones recognizing 95 independent antigens and developed a fluorescence probe-based enzyme-linked immunosorbent assay (ELISA) technique capable of evaluating the reactivity of these antibodies by fluorescence intensity, allowing simple, rapid, and high-throughput selection of antibody clones. These results were highly correlated with those using flow cytometry. Subsequently, the HMOS cell lysate was incubated with the antibody, the antigen-antibody complex was recovered with magnetic beads, and the protein bands from electrophoresis were analyzed using liquid chromatography-mass spectrometry (LC/MS). CAVIN1/polymerase I transcript release factor was specifically detected in the HMOS cells. In conclusion, we found via a novel high-throughput screening method that CAVIN1/PTRF is an HMOS-specific cell membrane biomarker and an antigen capable of producing human antibodies. In the future, antibody-drug conjugate targeting of these specific proteins may be promising for clinical applications.
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Regenerative medicine in ophthalmology that uses induced pluripotent stem cells (iPS) cells has been described, but those studies used iPS cells derived from fibroblasts. Here, we generated iPS cells derived from iris cells that develop from the same inner layer of the optic cup as the retina, to regenerate retinal nerves. We first identified cells positive for p75NTR, a marker of retinal tissue stem and progenitor cells, in human iris tissue. We then reprogrammed the cultured p75NTR-positive iris tissue stem/progenitor (H-iris stem/progenitor) cells to create iris-derived iPS (H-iris iPS) cells for the first time. These cells were positive for iPS cell markers and showed pluripotency to differentiate into three germ layers. When H-iris iPS cells were pre-differentiated into neural stem/progenitor cells, not all cells became positive for neural stem/progenitor and nerve cell markers. When these cells were pre-differentiated into neural stem/progenitor cells, sorted with p75NTR, and used as a medium for differentiating into retinal nerve cells, the cells differentiated into Recoverin-positive cells with electrophysiological functions. In a different medium, H-iris iPS cells differentiated into retinal ganglion cell marker-positive cells with electrophysiological functions. This is the first demonstration of H-iris iPS cells differentiating into retinal neurons that function physiologically as neurons.
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Fenômenos Eletrofisiológicos , Células-Tronco Pluripotentes Induzidas/fisiologia , Iris/citologia , Regeneração Nervosa/fisiologia , Neurônios Retinianos/fisiologia , Diferenciação Celular , Células Cultivadas , Reprogramação Celular , Corpos Embrioides/metabolismo , Células-Tronco Embrionárias/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Proteínas do Tecido Nervoso/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Recoverina/metabolismo , Reprodutibilidade dos Testes , Células Ganglionares da Retina/metabolismo , Neurônios Retinianos/citologia , Teratoma/patologiaRESUMO
General anesthesia was maintained by continuous administration of propofol, rocuronium and remifentanil. The dose of the medicine was determined by the effect site concentration calculated on a pharmacokinetics simulator respectively. Furthermore, a pharmacokinetics simulator enabled us to predict duration before the appearance of awareness.
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Androstanóis/administração & dosagem , Androstanóis/farmacocinética , Anestesia Geral , Simulação por Computador , Piperidinas/administração & dosagem , Piperidinas/farmacocinética , Propofol/administração & dosagem , Propofol/farmacocinética , Adulto , Humanos , Remifentanil , Rocurônio , SoftwareRESUMO
We attempted to prepare ophthalmic in situ gel formulations containing lanosterol (Lan) nanoparticles (LA-NPs/ISG) and investigated the characteristics, delivery pathway into the lens, and anti-cataract effects of LA-NPs/ISG using SCR-N (rats with slight lens structure collapse) and SCR-C (rats with a combination of remarkable lens structure collapse and opacification). LA-NPs/ISG was prepared by bead milling of the dispersions containing 0.5% Lan powder, 5% 2-hydroxypropyl-ß-cyclodextrin, 0.5% methylcellulose, 0.005% benzalkonium chloride, and 0.5% mannitol. The particle size distribution of Lan was 60-250 nm. The LA-NPs/ISG was gelled at 37 °C, and the LA-NPs/ISG was taken into the cornea by energy-dependent endocytosis and then released to the intraocular side. In addition, the Lan contents in the lenses of both SCR-N and SCR-C were increased by the repetitive instillation of LA-NPs/ISG (twice per day). The space and structure collapse in the lens of SCR-N with aging was attenuated by the instillation of LA-NPs/ISG. Moreover, the repetitive instillation of LA-NPs/ISG attenuated the changes in cataract-related factors (the enhancement of nitric oxide levels, calpain activity, lipid peroxidation levels, Ca2+ contents, and the decrease of Ca2+-ATPase activity) in the lenses of SCR-C, and the repetitive instillation of LA-NPs/ISG delayed the onset of opacification in the SCR-C. It is possible that the LA-NPs/ISG is useful in maintaining lens homeostasis.
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A mouthwash formulation of rebamipide (REB) is commonly used to treat oral mucositis; however, this formulation does not provide sufficient treatment or prevention in cases of serious oral mucositis. To improve treatment, we attempted to design a hydrogel incorporating REB nanocrystals (R-NPs gel). The R-NPs gel was prepared by a bead mill method using carbopol hydrogel, methylcellulose and 2-hydroxypropyl-ß-cyclodextrin, and another hydrogel incorporating REB microcrystals (R-MPs gel) was prepared following the same protocol but without the bead mill treatment. The REB particle size in the R-MPs gel was 0.15-25 µm, and while the REB particle size was 50-180 nm in the R-NPs gel. Next, we investigated the therapeutic effect of REB nanocrystals on oral mucositis using a hamster model. Almost all of the REB was released as drug nanocrystals from the R-NPs gel, and the REB content in the cheek pouch of hamsters treated with R-NPs gel was significantly higher than that of hamsters treated with R-MPs gel. Further, treatment with REB hydrogels enhanced the healing of oral wounds in the hamsters. REB accumulation in the cheek pouch of hamsters treated with the R-NPs gel was prevented by an inhibitor of clathrin-dependent endocytosis (CME) (40 µM dynasore). In conclusion, we designed an R-NPs gel and found that REB nanocrystals are taken up by tissues through CME, where they provide a persistent effect resulting in an enhancement of oral wound healing.
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The prevalence of nuclear cataracts was observed to be significantly higher among residents of tropical and subtropical regions compared to those of temperate and subarctic regions. We hypothesized that elevated environmental temperatures may pose a risk of nuclear cataract development. The results of our in silico simulation revealed that in temperate and tropical regions, the human lens temperature ranges from 35.0 °C to 37.5 °C depending on the environmental temperature. The medium temperature changes during the replacement regularly in the cell culture experiment were carefully monitored using a sensor connected to a thermometer and showed a decrease of 1.9 °C, 3.0 °C, 1.7 °C, and 0.1 °C, after 5 min when setting the temperature of the heat plate device at 35.0 °C, 37.5 °C, 40.0 °C, and 42.5 °C, respectively. In the newly created immortalized human lens epithelial cell line clone NY2 (iHLEC-NY2), the amounts of RNA synthesis of αA crystallin, protein expression, and amyloid ß (Aß)1-40 secreted into the medium were increased at the culture temperature of 37.5 °C compared to 35.0 °C. In short-term culture experiments, the secretion of Aß1-40 observed in cataracts was increased at 37.5 °C compared to 35.0 °C, suggesting that the long-term exposure to a high-temperature environment may increase the risk of cataracts.