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We explore the interaction between corneal immunity and mesenchymal stem/stromal cells (MSCs) and their potential in treating corneal and ocular surface disorders. We outline the cornea's immune privilege mechanisms and the immunomodulatory substances involved. In this realm, MSCs are characterized by their immunomodulatory properties and regenerative potential, making them promising for therapeutic application. Therefore, we focus on the role of MSCs in immune-mediated corneal diseases such as dry eye disease, corneal transplantation rejection, limbal stem cell deficiency, and ocular graft-versus-host disease. Preclinical and clinical studies demonstrate MSCs' efficacy in promoting corneal healing and reducing inflammation in these conditions. Overall, we emphasize the potential of MSCs as innovative therapies in ophthalmology, offering promising solutions for managing various ocular surface pathologies.
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PURPOSE: To assess corneal sensitivity changes in patients with ocular graft-versus-host disease using a non-contact and Cochet-Bonnet esthesiometer. In addition, we evaluate the association between corneal sensitivity and subbasal nerve changes and epitheliopathy in these patients. METHODS: In this retrospective study, the clinical data and images were evaluated for 36 patients (19 female, 17 male) who fulfilled the inclusion criteria. The analyzed data included demographic and ocular surface parameters, including best-corrected visual acuity, corneal sensitivity with non-contact (mbar) and Cochet-Bonnet (cm) esthesiometer, corneal fluorescein staining (CFS) and symptoms scores, tear volume (Schirmer-I test, mm/5'), and subbasal nerve density (µm/mm2; assessed with in vivo confocal microscopy). RESULTS: The mean age of the study cohort was 59.9 ± 10.5 years. The mean corneal sensitivity assessed by Cochet-Bonnet and non-contact esthesiometer was 5.9 ± 0.3 cm and 7.3 ± 2.0 mbar, respectively. The ocular surface parameters included a corneal fluorescein staining (CFS) score, as per the National Eye Institute grading scheme, of 6.9 ± 3.5, and a Schirmer-I test result of 7.5 ± 6.2 mm/5 minutes.. Total corneal subbasal nerve density was inversely associated with CFS scores (r = -0.74; P < 0.001). Moreover, similar correlations between CFS scores and main trunk (r = -0.62; P < 0.001) and branch (r = -0.59; P < 0.001) nerve densities were observed. A significant correlation was found between reduced corneal sensitivity and higher CFS scores (r = 0.66; P < 0.001). Higher pressures were correlated with lower total (r = -0.83; P < 0.001), main trunk (r = -0.62; P < 0.001), and branch (r = -0.72; P < 0.001) nerve densities. The univariate analysis showed that corneal sensitivity loss (assessed with non-contact esthesiometer) was correlated with advanced age of the patients (P = 0.049) and inversely associated with total (P < 0.001), main trunk (P < 0.001), and branch (P < 0.001) nerve densities. In addition, sensitivity loss was inversely associated with punctal occlusion (cauterization (P = 0.001) or plug placement (P < 0.001). The multivariate analysis adjusted for age and punctal occlusion confirmed the associations in the univariate analysis. CONCLUSIONS: In this study, we observed that corneal sensitivity loss was associated with reduced main trunk, branch, and total nerve density in patients with ocular graft-versus-host disease. In addition, a significant correlation was observed between reduced corneal nerve density, corneal sensitivity, and severity of epitheliopathy.
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PURPOSE: To describe the frequency of postoperative complications in children undergoing penetrating keratoplasty (PK). METHODS: This retrospective cohort study included pediatric patients (aged 0-18 years) in the Intelligent Research in Sight (IRIS) Registry who underwent primary PK between January 2013 and December 2020. Patients were identified using Current Procedure Terminology codes. Postoperative complications were identified using International Classification of Diseases Revision 9 and 10 codes and categorized as complications affecting the anterior segment (epithelial defect, infectious keratitis, neurotrophic keratitis, and glaucoma) or posterior segment (vitreous hemorrhage, retinal detachment, choroid detachment, choroidal hemorrhage, endophthalmitis, epiretinal membrane, and proliferative vitreoretinopathy). RESULTS: Of the 544 pediatric patients undergoing PK, 259 (47.6%) experienced postoperative complications. Anterior segment complications (n = 222, 40.8%) were more common than posterior segment complications (n = 96, 17.6%). The most common anterior complication was infectious keratitis (n = 163, 30.0%) with a median onset time of 275 days (IQR 50-560 days) after surgery. Vitreous hemorrhage (n = 52, 9.6%) and retinal detachment (n = 48, 8.8%) were the most common posterior segment complications. CONCLUSIONS: Infectious keratitis was the most common postoperative complication in children undergoing PK. More complications occurred in the anterior segment (70%) of the eye as compared with the posterior segment (30%). These findings demonstrate the challenges associated with pediatric PK and may help guide postoperative monitoring and management.
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Fuchs endothelial corneal dystrophy is a heterogenous disease with multifactorial etiology, and genetic, epigenetic, and exogenous factors contributing to its pathogenesis. DNA damage plays a significant role, with ultraviolet-A (UV-A) emerging as a key contributing factor. We investigate the potential application of neuropeptide α-melanocyte stimulating hormone (α-MSH) in mitigating oxidative stress induced endothelial damage. First, we examined the effects of α-MSH on a cultured human corneal endothelial cell line (HCEnC-21T) exposed to hydrogen peroxide (H2O2) induced oxidative DNA damage. We performed immunofluorescence and flow cytometry to assess DNA damage and cell death in the cultured cells. Additionally, we used an established mouse model that utilizes ultraviolet light to induce corneal endothelial cell damage resulting in decreased CEnC number, increased cell size variability, and decreased percentage of hexagonal cells. This endothelial decompensation leads to an increase in corneal thickness. Following UV-A exposure, the mice were systemically treated with α-MSH, either immediately after exposure (early treatment) or beginning two weeks post-exposure (delayed treatment). To evaluate treatment efficacy, we analyzed CEnC density and morphology using in vivo confocal microscopy, and central corneal thickness using anterior segment optical coherence tomography. Our findings demonstrated that α-MSH treatment effectively protects HCEnC-21T from free-radical induced oxidative DNA damage and subsequent cell death. In vivo, α-MSH treatment, mitigated the loss of CEnC density, deterioration of cell morphology and suppression of the resultant corneal swelling. These results underline the potential application of α-MSH as a therapeutic agent for mitigating corneal endothelial damage.
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Dano ao DNA , Modelos Animais de Doenças , Endotélio Corneano , Distrofia Endotelial de Fuchs , Estresse Oxidativo , alfa-MSH , Animais , alfa-MSH/farmacologia , Camundongos , Endotélio Corneano/efeitos dos fármacos , Endotélio Corneano/patologia , Humanos , Distrofia Endotelial de Fuchs/patologia , Distrofia Endotelial de Fuchs/tratamento farmacológico , Dano ao DNA/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Raios Ultravioleta/efeitos adversos , Linhagem Celular , Peróxido de Hidrogênio/farmacologiaRESUMO
A four-electrode system and high-voltage amplitude stimulation (with a voltage of more than 1 V) are always used for nonlinear spectroscopy of biological solutions like yeast suspension. Multiple studies indicate that yeast membrane activities are the primary source of the harmonics detected from yeast suspensions. However, other investigations have demonstrated that at least part of the harmonics are mainly due to the nonlinear impedance of the electrode-electrolyte interface. Here, we want to find a mathematical model to analyze the odd harmonics generated by the interface. The KCl solution is the basis of the essential medium used in yeast culture. No study models the nonlinear behavior of the KCl solution using four-electrode. We used a sinusoidal voltage with a low frequency and a high amplitude to test an ionic solution with 1, 30, and 130 mM of potassium chloride. The voltage had no direct current component. Here, we show that the relationship between the amplitude of the excitation voltage and the third harmonic of the current can be accurately represented using the Butler-Volmer equation. Additionally, we analyze the impact of potassium chloride (KCl) concentration on the behavior of the third harmonic. This paper presents a method and model that can be used for nonlinear spectroscopy studies of biological solutions using a four-electrode system with the aim of deriving information from cells without considering the interface effect.
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PURPOSE: Vision loss associated with opacification of the cornea is one of the leading causes of blindness globally. However, the epidemiological data pertaining to the demographics, associated etiological causes and reduced vision in corneal opacity patients continue to be sparse. This study assesses the case frequencies, underlying etiologies, and vision outcomes in patients diagnosed with corneal opacity, in the United States. DESIGN: Retrospective cohort study PARTICIPANTS: Patients in the IRIS® Registry (Intelligent Research in Sight) who were diagnosed with corneal opacity between January 1st, 2013, and November 30th, 2020. METHODS: The IRIS Registry contains demographic and clinical data of 79,887,324 patients who presented to eye clinics during the study period. We identified patients with corneal opacity using International Classification of Disease (ICD) codes (ICD-9, and -10) of "371" (corneal scar) and "H17" (corneal opacity), respectively. The analyzed data included demographic parameters included age, sex, race, ethnicity, and geographical location. We evaluated clinical data including laterality, etiology, disease descriptors, and best-corrected visual acuity (VA) up to 1 year before the onset (± 30 days), at the time of diagnosis, and at one year following diagnosis (± 30 days). MAIN OUTCOME MEASURES: Case frequencies, etiology, and vision outcomes in patients diagnosed with corneal opacity. RESULTS: We identified 5,220,382 patients who were diagnosed with corneal opacity and scars using H17 (ICD-10) and 371.0 (ICD-9) codes over seven years. The case frequency of corneal opacity during the study period was 6,535 cases per 100,000 patients (6.5%). The mean age of the patients was 63.36±18.14 years and the majority were female (57.6%). In the cohort, 38.39% and 30.00% of patients had bilateral and unilateral corneal opacity, respectively. Most of the patients were White (69.13%), followed by Black or African American (6.84%), Asian (2.45%), American Indian or Alaska Native(0.34%), Native Hawaii or other Pacific Islander(0.19%). Among the patients with corneal opacity, 7.34% had Hispanic or Latino ethnicity. The primary etiologies associated with corneal opacity included corneal dystrophies (64.66%) followed by edema (18.25%), ulcer (7.78%), keratoconjunctivitis (7.18%), degeneration (5.62%), neovascularization (6.27%), and trauma (5.28%). Visual acuity of the patients significantly worsened due to corneal opacity (0.46±0.74 logMAR; â¼20/58 in Snellen) and did not improve to the baseline (0.37±0.68 logMAR, â¼20/46 in Snellen) post-management (0.43±0.77 logMAR, â¼20/54 in Snellen). The multiple linear regression analysis showed worse vision outcomes in females (compared to males), and Asian, Black or African American, and American Indian or Alaska Native (compared to White) patients. Additionally, worse vision outcomes were observed in patients with opacity associated with corneal malformation, degenerative disorders, edema, injury, and ulcer compared to those with hereditary corneal dystrophy. CONCLUSIONS: Our study shows that the corneal opacity was diagnosed in 6.5% of the patients in the IRIS Registry and it was primarily associated with corneal dystrophies. The final vision outcomes in corneal opacity patients were significantly worse compared to baseline. The worse vision outcomes were associated with sociodemographic differences that might be associated with disparities in access, utilization, and care patterns.
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Regulatory T cells (Tregs) play a critical role in maintaining immune homeostasis, and their dysfunction is implicated in the pathogenesis of various autoimmune disorders, including dry eye disease (DED). Treg dysfunction in DED allows T-helper cell 17 (Th17) mediated chronic inflammation at the ocular surface. In this study, the factors causing Treg dysfunction in DED were investigated. We observed reduced expression of Treg functional markers - FoxP3, CD25, and CTLA-4 in the cells isolated from DED mice (DED Tregs). Additionally, DED Tregs showed increased expression levels of receptors for pro-inflammatory cytokine receptors, namely IL-6R, IL-17RA, and IL-23R. An increased expression level of pro-inflammatory cytokine receptors was observed on exposing Tregs isolated from naïve mice (NTregs) to IL-6 or IL-17, but not IL-23, with a concomitant downregulation of FoxP3, CD25, and CTLA-4 in these cells. Furthermore, among these cytokines, IL-6 induced the most pronounced loss of Treg mediated suppression of Th17 proliferation and IL-10 secretion. In vitro and in vivo blockade of IL-6 effectively restored function in DED Tregs, leading to enhanced suppressive function against proliferating Th17 cells and ameliorating disease severity. In conclusion, this study provides insights into mechanisms of Treg dysregulation in DED, specifically delineating the effect of Th17-associated cytokines, with IL-6 emerging as the critical factor inducing Treg dysfunctionality. These findings highlight the potential for developing novel therapeutic interventions for DED through restoration of immunosuppressive function of Tregs.
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Modelos Animais de Doenças , Síndromes do Olho Seco , Fatores de Transcrição Forkhead , Interleucina-6 , Camundongos Endogâmicos C57BL , Linfócitos T Reguladores , Animais , Linfócitos T Reguladores/imunologia , Síndromes do Olho Seco/metabolismo , Síndromes do Olho Seco/imunologia , Camundongos , Interleucina-6/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Células Th17/imunologia , Antígeno CTLA-4/metabolismo , Feminino , Citometria de Fluxo , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Dessecação , Células CultivadasRESUMO
Substance P (SP) is a neuropeptide expressed by nerves and an array of cells that serves as a critical mediator of neuroinflammation. Our recent work has demonstrated that blocking the preferred receptor for SP, neurokinin-1 receptor (NK1R), effectively suppresses the induction of acute dry eye disease (DED) by preserving regulatory T cell (Treg) function, while inhibiting antigen-presenting cell (APC) maturation and subsequent generation of effector Th17 cells (eTh17). Clinically, DED is a chronic disorder characterized by sustained ocular surface inflammation which is mediated by long-lived memory Th17 cells (mTh17) demonstrated in our well-established chronic DED model. The present study aimed to further understand the function of SP in the chronic phase of DED and its role in regulating the underlying pathogenic mTh17. In vitro culture of effector T cells isolated from acute DED with SP led to an enhanced conversion of eTh17 to mTh17, while culturing memory T cells isolated from chronic DED with SP effectively preserved the mTh17 cells. In contrast, the addition of an NK1R antagonist in the cultures abolished the SP-mediated effects. Furthermore, in vivo treatment with the NK1R antagonist during the resolution phase of acute DED significantly suppressed mTh17 generation, and treatment in the chronic phase of DED disrupted the maintenance of mTh17. Taken together, our results demonstrate that increased expression of SP promotes mTh17 generation and maintenance in chronic DED, and thus blockade of SP represents a novel promising mTh17-targeting strategy in treating chronic ocular surface inflammation.
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Purpose: To evaluate the therapeutic efficacy of topical application of a neurokinin-1 receptor (NK1R) antagonist in a rabbit model of nonallergic ocular redness. Methods: Nonallergic ocular redness was induced in rabbits by a single, topical application of dapiparzole hydrochloride eye drops (0.5%, 1%, 2%, or 5%). The NK1R antagonist L-703,606 was topically applied to the eye at the same time of induction or 20 min after induction, and phosphate buffered saline (PBS) treatment served as the control. Superior bulbar conjunctival images were taken every 30 s for the first 2 min, followed by every 4 min for 8 min, and then every 10 min until 1 h. The severity of ocular redness was evaluated on the images using ImageJ-based ocular redness index (ORI) calculations. Results: The ORI scores were significantly increased after the application of 0.5%, 1%, 2%, or 5% dapiparzole at each time point evaluated, with the most severe redness induced by the 5% dapiprazole that led to a maximal mean increase in ORI score of 14 at 20 min post-induction and thus used for subsequent evaluation of therapeutic efficacy of NK1R antagonism. Topical L-703,606, when applied at the same time as dapiprazole induction, significantly suppressed the increase of ORI scores at all time points (â¼40% decrease). Furthermore, when applied at 20 min after dapiprazole induction, L-703,606 rapidly and effectively suppressed the increase of ORI scores at 30, 40, 50, and 60 min (â¼30% decrease). Conclusions: Topical blockade of NK1R effectively prevents and alleviates nonallergic ocular redness in a novel animal model.
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Graft-versus-host disease is among the most common clinical complications following allogeneic hematopoietic stem cell transplantation. It causes inflammation-mediated destruction and dysfunction of various organ systems including ocular tissues in 60-90% of the patients and is termed ocular GVHD (oGVHD). In oGVHD, donor-derived T-cells recognize host antigens as foreign, resulting in immune dysregulation, inflammation and fibrosis of lacrimal glands, meibomian glands, cornea, and conjunctiva. The clinical presentation in oGVHD patients range from mild dry eye symptoms to catastrophic inflammation mediated pathological changes which can cause corneal perforation and blindness. In this review article, we provide detailed insights into the impact of mucosal barrier disruption, the afferent and efferent phases of immunological response involving activation of antigen presenting cells and T cells, respectively. We evaluate the evidence outlining the effector phase of the disease leading to cellular destruction and eventually fibrosis in patients with oGVHD. Finally, we discuss the well-established criteria for the diagnosis of oGVHD.
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Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Humanos , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/imunologia , Oftalmopatias/etiologia , Oftalmopatias/imunologiaRESUMO
The tear fluid is a readily accessible, potential source for biomarkers of disease and could be used to monitor the ocular response to contact lens (CL) wear or ophthalmic pathologies treated by therapeutic CLs. However, the tear fluid remains largely unexplored as a biomarker source for RNA-based molecular analyses. Using a rabbit model, this study sought to determine whether RNA could be collected from commercial CLs and whether the duration of CL wear would impact RNA recovery. The results were referenced to standardized strips of filtered paper (e.g., Shirmer Strips) placed in the inferior fornix. By performing total RNA isolation, precipitation, and amplification with commercial kits and RT-PCR methods, CLs were found to have no significant differences in RNA concentration and purity compared to Schirmer Strips. The study also identified genes that could be used to normalize RNA levels between tear samples. Of the potential control genes or housekeeping genes, GAPDH was the most stable. This study, which to our knowledge has never been done before, provides a methodology for the detection of RNA and gene expression changes from tear fluid that could be used to monitor or study eye diseases.
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Lentes de Contato , RNA , Lágrimas , Lágrimas/metabolismo , Animais , Coelhos , RNA/isolamento & purificação , RNA/genética , RNA/análiseRESUMO
Purpose: To assess the effectiveness of topical and subconjunctival bevacizumab in suppressing vascularization in graft and host bed after high-risk corneal transplantation. Design: Secondary analysis of prospective, randomized, double-blind, placebo-controlled multicentric clinical trial. Participants: The study includes patients aged > 18 years who underwent high-risk penetrating keratoplasty, which was defined as corneal vascularization in ≥ 1 quadrants of the corneal graft and host bed, excluding the limbus. Methods: Patients were randomized to treatment and control groups. The patients in the treatment group received subconjunctival injection of bevacizumab (2.5 mg/0.1 ml) on the day of the procedure, followed by topical bevacizumab (10 mg/ml) 4 times per day for 4 weeks. The patients in control group received injection of vehicle (0.9% sodium chloride) on the day of procedure, followed by topical vehicle (carboxymethylcellulose sodium 1%) 4 times a day for 4 weeks. Main Outcome Measures: Vessel and invasion area of vessels in the corneal graft and host beds. Results: This study included 56 eyes of 56 patients who underwent high-risk corneal transplantation, with equal numbers in the bevacizumab and vehicle (control) treatment groups. The mean age of patients who received bevacizumab was 61.2 ± 15.9 years, and the mean age of those treated with vehicle was 60.0 ± 16.1 years. The vessel area at baseline was comparable in the bevacizumab (16.72% ± 3.19%) and control groups (15.48% ± 3.12%; P = 0.72). Similarly, the invasion areas were also similar in the treatment (35.60% ± 2.47%) and control (34.23% ± 2.64%; P = 0.9) groups at baseline. The reduction in vessel area was significantly higher in the bevacizumab-treated group (83.7%) over a period of 52 weeks compared with the control group (61.5%; P < 0.0001). In the bevacizumab-treated group, invasion area was reduced by 75.8% as compared with 46.5% in the control group. The vessel area was similar at 52 weeks postprocedure in cases of first (3.54% ± 1.21%) and repeat (3.80% ± 0.40%) corneal transplantation in patients who received bevacizumab treatment. In the vehicle-treated patients, the vessel area was significantly higher in repeat (9.76% ± 0.32%) compared with first (8.06% ± 1.02%; P < 0.0001) penetrating keratoplasty. In the bevacizumab treatment group, invasion areas at week 52 were comparable in first (11.70% ± 3.38%) and repeat (11.64% ± 1.74%) procedures, whereas invasion area was significantly higher in repeat (27.87% ± 2.57%) as compared with first (24.11% ± 2.17%) penetrating keratoplasty in vehicle-treated patients. Conclusions: In patients undergoing vascularized high-risk corneal transplantation, bevacizumab is efficacious in reducing vascularization of corneal graft and host bed, thereby reducing the risk of corneal graft rejection in vascularized host beds. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.
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PURPOSE: To determine the risk and incidence of keratitis following treatment with epidermal growth factor receptor inhibitors (EGFRi) and subtypes of EGFRi-associated keratitis. METHODS: This multi-center cohort study included EGFRi-treated patients and non-users with lung cancer between 2010 and 2023. EGFRi included first-generation agent gefitinib and erlotinib, second-generation agent afatinib, and third-generation agent osimertinib. The primary outcome was new-onset keratitis. Cox proportional hazard models with multivariable adjustment were applied to determine the effect of EGFRi on keratitis over time. Subgroup analyses were conducted, stratified by agents of EGFRi. Sub-outcome analyses were performed to identify the subtypes of EGFRi-associated keratitis. RESULTS: A total of 1549 EGFRi-treated patients and 6146 non-users were included. 38 (2.5%) EGFRi-treated patients developed keratitis. The incidence of keratitis in EGFRi-treated patients was significantly higher than that in controls (incidence rate, IR, per 1000 person-years = 14.7 vs 4.49, p < 0.0001). EGFRi-treated patients presented with an increased risk for keratitis (adjusted hazard ratio, aHR = 3.14, 95% CI = 1.85-5.35, p < 0.001). Erlotinib (aHR = 2.64, 95% CI = 1.35-5.15, p = 0.004), afatinib (aHR = 4.42, 95% CI = 2.17-9.02, p < 0.001), and osimertinib (aHR = 4.67, 95% CI = 1.60-13.64, p = 0.005), but not gefitinib (aHR = 2.30, 95% CI = 0.96-5.55, p = 0.063), significantly contributed to the risk of keratitis. Subtypes of EGFRi-associated keratitis included corneal ulcer (IR = 2.31 vs 0.166, p < 0.0001) and keratoconjunctivitis (IR = 9.27 vs 2.91, p < 0.0001). None of the EGFRi-treated patients developed perforated corneal ulcer, interstitial and deep keratitis, or corneal neovascularization. CONCLUSION: Treatment with EGFRi was associated with an increased risk of keratitis. Ocular toxicity of EGFRi was highest for third-generation agents, followed by second-generation agents, and then first-generation agents.
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Receptores ErbB , Ceratite , Neoplasias Pulmonares , Humanos , Masculino , Feminino , Neoplasias Pulmonares/tratamento farmacológico , Receptores ErbB/antagonistas & inibidores , Pessoa de Meia-Idade , Incidência , Ceratite/epidemiologia , Ceratite/tratamento farmacológico , Idoso , Estudos Retrospectivos , China/epidemiologia , Inibidores de Proteínas Quinases/uso terapêutico , Inibidores de Proteínas Quinases/efeitos adversos , Gefitinibe/uso terapêutico , Gefitinibe/efeitos adversos , Acrilamidas/uso terapêutico , Acrilamidas/efeitos adversos , Seguimentos , Fatores de Risco , Cloridrato de Erlotinib/uso terapêutico , Cloridrato de Erlotinib/efeitos adversos , Povo Asiático , Antineoplásicos/efeitos adversos , Antineoplásicos/uso terapêutico , População do Leste AsiáticoRESUMO
Myeloid derived suppressor cells (MDSCs) are a heterogenous population of immature hematopoietic precursors with known immunoregulatory functions. The immunosuppressive role of MDSCs has been highlighted in several inflammatory ophthalmic disorders; however, their therapeutic application in suppressing the immune-mediated changes in dry eye disease (DED) has not been studied. We observed significant reduction in antigen presenting cell (APC) frequencies and their maturation in the presence of MDSCs. Moreover, co-culturing MDSCs with T helper 17 cells (Th17) resulted in reduced Th17 frequencies and their IL-17 expression. On the contrary, MDSCs maintained regulatory T cell frequencies and enhanced their function in-vitro. Furthermore, we delineated the role of interleukin-10 (IL-10) secreted by MDSCs in their immunoregulatory functions. We confirmed these results by flow cytometry analysis and observed that treatment with MDSCs in DED mice effectively suppressed the maturation of APCs, pathogenic Th17 response, and maintained Treg function and significantly ameliorated the disease. The results in this study highlight the potential therapeutic application of MDSCs in treating refractory DED.
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Modelos Animais de Doenças , Síndromes do Olho Seco , Citometria de Fluxo , Camundongos Endogâmicos C57BL , Células Supressoras Mieloides , Linfócitos T Reguladores , Células Th17 , Animais , Células Supressoras Mieloides/imunologia , Síndromes do Olho Seco/imunologia , Síndromes do Olho Seco/metabolismo , Camundongos , Células Th17/imunologia , Linfócitos T Reguladores/imunologia , Células Apresentadoras de Antígenos/imunologia , Feminino , Progressão da Doença , Interleucina-10/metabolismo , Células Cultivadas , Técnicas de CoculturaRESUMO
Highly inflamed and neovascularized corneal graft beds are known as high-risk (HR) environments for transplant survival. One of the primary factors leading to this rejection is reduction in the suppressive function of regulatory T cells (Treg). Our results show that myeloid-derived suppressor cells (MDSC) counteract interleukin-6-mediated Treg dysfunction by expressing interleukin-10. Additionally, MDSC maintain forkhead box P3 stability and their ability to suppress IFN-γ+ Th1 cells. Administering MDSC to HR corneal transplant recipients demonstrates prolonged graft survival via promotion of Treg while concurrently suppressing IFN-γ+ Th1 cells. Moreover, MDSC-mediated donor-specific immune tolerance leads to long-term corneal graft survival as evidenced by the higher survival rate or delayed survival of a second-party C57BL/7 (B6) graft compared to those of third-party C3H grafts observed in contralateral low-risk or HR corneal transplantation of BALB/c recipient mice, respectively. Our study provides compelling preliminary evidence demonstrating the effectiveness of MDSC in preventing Treg dysfunction, significantly improving graft survival in HR corneal transplantation, and showing promising potential for immune tolerance induction.
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Diabetes mellitus (DM) is one of the most prevalent diseases globally, and its prevalence is rapidly increasing. Most patients with a long-term history of DM present with some degree of keratopathy (DK). Despite its high incidence, the underlying inflammatory mechanism of DK has not been elucidated yet. For further insights into the underlying immunopathologic processes, we utilized streptozotocin-induced mice to model type 1 DM (T1D) and B6.Cg-Lepob/J mice to model type 2 DM (T2D). We evaluated the animals for the development of clinical manifestations of DK. Four weeks post-induction, the total frequencies of corneal CD45+CD11b+Ly-6G- myeloid cells, with enhanced gene and protein expression levels for the proinflammatory cytokines TNF-α and IL-1ß, were higher in both T1D and T2D animals. Additionally, the frequencies of myeloid cells/mm2 in the sub-basal neural plexus (SBNP) were significantly higher in T1D and T2D compared to non-diabetic mice. DK clinical manifestations were observed four weeks post-induction, including significantly lower tear production, corneal sensitivity, and epitheliopathy. Nerve density in the SBNP and intraepithelial terminal endings per 40x field were lower in both models compared to the normal controls. The findings of this study indicate that DM alters the immune quiescent state of the cornea during disease onset, which may be associated with the progressive development of the clinical manifestations of DK.
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Doenças da Córnea , Diabetes Mellitus Tipo 1 , Diabetes Mellitus Tipo 2 , Humanos , Camundongos , Animais , Diabetes Mellitus Tipo 1/patologia , Córnea/patologia , Doenças da Córnea/patologia , Diabetes Mellitus Tipo 2/patologia , EstreptozocinaRESUMO
Alpha-melanocyte-stimulating hormone (α-MSH) and its binding receptors (the melanocortin receptors) play important roles in maintaining ocular tissue integrity and immune homeostasis. Particularly extensive studies have demonstrated the biological functions of α-MSH in both immunoregulation and cyto-protection. This review summarizes the current knowledge of both the physiological and pathological roles of α-MSH and its receptors in the eye. We focus on recent developments in the biology of α-MSH and the relevant clinical implications in treating ocular diseases.
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Melanocortinas , alfa-MSH , Humanos , alfa-MSH/farmacologia , alfa-MSH/metabolismo , Receptores de Melanocortina/metabolismo , Inflamação/tratamento farmacológico , Morte CelularRESUMO
In recent years, microscopy has revolutionized the study of dynamic living cells. However, performing long-term live cell imaging requires stable environmental conditions such as temperature, pH, and humidity. While standard incubators have traditionally provided these conditions, other solutions, like stagetop incubators are available. To further enhance the accessibility of stable cell culture environments for live cell imaging, we developed a portable CO2 cell culture mini-incubator that can be easily adapted to any x-y inverted microscope stage, enabling long-term live cell imaging. This mini-incubator provides and maintains stable environmental conditions and supports cell viability comparable to standard incubators. Moreover, it allows for parallel experiments in the same environment, saving both time and resources. To demonstrate its functionality, different cell lines (VERO and MDA-MB-231) were cultured and evaluated using various assays, including crystal violet staining, MTT, and flow cytometry tests to assess cell adhesion, viability, and apoptosis, respectively. Time-lapse imaging was performed over an 85-h period with MDA-MB-231 cells cultured in the mini-incubator. The results indicate that this device is a viable solution for long-term imaging and can be applied in developmental biology, cell biology, and cancer biology research where long-term time-lapse recording is required.
Assuntos
Dióxido de Carbono , Técnicas de Cultura de Células , Imagem com Lapso de Tempo , Técnicas de Cultura de Células/métodos , Incubadoras , Linhagem CelularRESUMO
Purpose: To evaluate whether fibrosis contributes to corneal transplant failure and to determine whether effector CD4+ T cells, the key immune cells in corneal transplant rejection, play a direct role in fibrosis formation. Methods: Allogeneic corneal transplantation was performed in mice. Graft opacity was evaluated by slit-lamp biomicroscopy, and fibrosis was assessed by in vivo confocal microscopy. Expression of alpha-smooth muscle actin (α-SMA) in both accepted and failed grafts was assessed by real-time PCR and immunohistochemistry. Frequencies of graft-infiltrating CD4+ T cells, neutrophils, and macrophages were assessed using flow cytometry. In vitro, MK/T-1 corneal fibroblasts were co-cultured with activated CD4+CD25- effector T cells isolated from corneal transplant recipient mice, and α-SMA expression was quantified by real-time PCR and ELISA. Neutralizing antibody was used to evaluate the role of interferon gamma (IFN-γ) in promoting α-SMA expression. Results: The majority of failed grafts demonstrated clinical signs of fibrosis which became most evident at week 6 after corneal transplantation. Failed grafts showed higher expression of α-SMA as compared to accepted grafts. Flow cytometry analysis showed a significant increase in CD4+ T cells in failed grafts compared to accepted grafts. Co-culture of activated CD4+CD25- effector T cells with corneal fibroblasts led to an increase in α-SMA expression by fibroblasts. Inhibition of IFN-γ in culture significantly suppressed this increase in α-SMA expression as compared to immunoglobulin G control. Conclusions: Fibrosis contributes to graft opacity in corneal transplant failure and is mediated at least in part by effector CD4+ T cells via IFN-γ.