RESUMEN
PURPOSE: To assess the efficacy and safety of human leukocyte antigen-matched allogeneic cultivated limbal epithelial stem cell grafts in the treatment of aniridia-associated keratopathy (AAK). METHODS: Six eyes of 6 patients with severe AAK received an allogeneic stem cell graft between January 2010 and March 2017. Anatomical and functional results were assessed at 6 months, 1 year, 2 years, and the final follow-up visit available. Safety analysis was performed by considering all perioperative and postoperative adverse events and additional surgeries required during the follow-up period. RESULTS: The mean follow-up was 53.6 months (range 24-104 months). In most patients (80%), there was an early improvement of the keratopathy postoperatively, which slowly regressed during longer follow-up. At the final follow-up, 4 of the eyes were graded as failure and 1 eye was graded as partial success. Grading the sixth eye was not possible because of an adverse event. None of the patients maintained a total anatomical success in the long-term. Only 1 patient maintained a modest improvement in best-corrected visual acuity from hand motion to counting fingers. Four serious adverse events were recorded in 2 patients. CONCLUSIONS: Severe AAK remains a challenging condition to manage. Transplantation of allogenic ex vivo cultivated limbal stem cells may provide a temporary improvement in ocular surface stability, but anatomical and functional results are poor in the long-term. The eyes are prone to adverse events, and any surgical treatment should take this into consideration.
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Aniridia/complicaciones , Enfermedades de la Córnea/cirugía , Epitelio Corneal/citología , Antígenos HLA/inmunología , Limbo de la Córnea/citología , Trasplante de Células Madre/métodos , Células Madre/inmunología , Adulto , Anciano , Células Cultivadas , Enfermedades de la Córnea/diagnóstico , Enfermedades de la Córnea/etiología , Epitelio Corneal/inmunología , Femenino , Estudios de Seguimiento , Supervivencia de Injerto , Humanos , Limbo de la Córnea/inmunología , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Células Madre/citología , Factores de Tiempo , Trasplante Autólogo , Agudeza Visual , Adulto JovenRESUMEN
Limbal stem cell (LSC) transplantation is the only efficient treatment for patients affected by LSC deficiency (LSCD). Allogeneic LSC transplantation is one of the most successful alternative for patients with bilateral LSCD. Nevertheless, the high variability of the human leukocyte antigens (HLA) remains a relevant obstacle to long-term allogeneic graft survival. This study characterized the immunologic properties of LSCs and proposed a genetic engineering strategy to reduce the immunogenicity of LSCs and of their derivatives. Hence, LSC HLA expression was silenced using lentiviral vectors encoding for short hairpin (sh) RNAs targeting ß2-microglobulin (ß2M) or class II major histocompatibility complex transactivator (CIITA) to silence HLA class I and II respectively. Beside the constitutive expression of HLA class I, LSCs showed the capability to upregulate HLA class II expression under inflammatory conditions. Furthermore, LSCs demonstrated the capability to induce T-cell mediated immune responses. LSCs phenotypical and functional characteristics are not disturbed after genetic modification. However, HLA silenced LSC showed to prevent T cell activation, proliferation and cytotoxicity in comparison to fully HLA-expressing LSCs. Additionally; HLA-silenced LSCs were protected against antibody-mediated cellular-dependent cytotoxicity. Our data is a proof-of-concept of the feasibility to generate low immunogenic human LSCs without affecting their typical features. The use of low immunogenic LSCs may support for long-term survival of LSCs and their derivatives after allogeneic transplantation.
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Antígenos HLA/inmunología , Trasplante de Células Madre Hematopoyéticas , Limbo de la Córnea/inmunología , Células Madre/inmunología , Células Cultivadas , Antígenos HLA/genética , Humanos , Limbo de la Córnea/citología , Trasplante HomólogoRESUMEN
Dry eye disease (DED) is a multifactorial disease of the ocular surface, characterized by loss of tear film homeostasis and ocular symptoms, in which neurosensory abnormalities have recently been shown to play an etiological role. Although the role of inflammation has been widely studied in DED, the kinetics of immune cells of the ocular surface in this complex disease are hereto unclear. Herein, we utilized intravital multiphoton imaging on transgenic mice to investigate the 3D morphology and kinetics of conventional dendritic cells (cDCs) and the role of ocular surface sensory nerves in regulating them in both the naïve state and experimental DED. Mice with DED had significantly lower tear secretion (p < 0.01), greater corneal fluorescein staining (p < 0.001), and higher cDC density in the ocular surface (p < 0.05), compared to naïve mice. cDCs in DED mice showed morphological alterations in the limbus, exhibiting smaller surface area (p < 0.001) and volume (p < 0.001) compared to naïve mice. Furthermore, corneal cDCs showed greater sphericity in DED mice compared to naïve mice (p < 0.01). In addition, limbal cDCs displayed significantly increased migratory kinetics in DED, including mean track speed, 3D instantaneous velocity, track length, and displacement, compared to naïve mice (all p < 0.05). In mice with DED, cDCs showed a higher meandering index in the limbus compared to central cornea (p < 0.05). In DED, cDCs were less frequently found in contact with nerves in the limbus, peripheral, and central cornea (p < 0.05). cDCs in contact with nerves demonstrated a larger surface area (p < 0.001) and volume (p < 0.001), however, they exhibited less sphericity (p < 0.05) as compared to cDCs not in contact with nerves in naïve mice. Importantly, cDCs in contact with nerves during DED had a decreased track length, displacement, mean track speed, and 3D instantaneous velocity compared to those not in contact with nerves (all p < 0.05). Taken together, we present in vivo evidence of altered cDC kinetics and 3D morphology in DED. Furthermore, apparent neuronal contact significantly alters cDC kinetics and morphological characteristics, suggesting that ocular surface nerves may play a direct role in mediating immune responses in DED.
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Células Dendríticas/inmunología , Síndromes de Ojo Seco/diagnóstico por imagen , Síndromes de Ojo Seco/inmunología , Microscopía Intravital/métodos , Limbo de la Córnea/diagnóstico por imagen , Limbo de la Córnea/inmunología , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Animales , Movimiento Celular/inmunología , Modelos Animales de Enfermedad , Femenino , Cinética , Limbo de la Córnea/inervación , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Nervio Óptico/inmunologíaRESUMEN
Corneal injuries and infections are the leading cause of blindness worldwide. Thus, understanding the mechanisms that control healing of the damaged cornea is critical for the development of new therapies to promptly restore vision. Innate lymphoid cells (ILCs) are a recently identified heterogeneous cell population that has been reported to orchestrate immunity and promote tissue repair in the lungs and skin after injury. However, whether ILCs can modulate the repair process in the cornea remains poorly understood. We identified a population of cornea-resident group 2 ILCs (ILC2s) in mice that express CD127, T1/ST2, CD90, and cKit. This cell population was relatively rare in corneas at a steady state but increased after corneal epithelial abrasion. Moreover, ILC2s were maintained and expanded locally at a steady state and after wounding. Depletion of this cell population caused a delay in corneal wound healing, whereas supplementation of ILC2s through adoptive transfer partially restored the healing process. Further investigation revealed that IL-25, IL-33, and thymic stromal lymphopoietin had critical roles in corneal ILC2 responses and that CCR2- corneal macrophages were an important producer of IL-33 in the cornea. Together, these results reveal the critical role of cornea-resident ILC2s in the restoration of corneal epithelial integrity after acute injury and suggest that ILC2 responses depend on local induction of IL-25, IL-33, and thymic stromal lymphopoietin.
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Lesiones de la Cornea/inmunología , Epitelio Corneal/lesiones , Subgrupos Linfocitarios/fisiología , Regeneración/inmunología , Traslado Adoptivo/métodos , Animales , Trasplante de Médula Ósea/métodos , Proliferación Celular/fisiología , Lesiones de la Cornea/fisiopatología , Citocinas/biosíntesis , Modelos Animales de Enfermedad , Epitelio Corneal/fisiología , Femenino , Inmunidad Innata , Interleucina-33/biosíntesis , Interleucinas/biosíntesis , Limbo de la Córnea/inmunología , Ratones Endogámicos C57BL , Cicatrización de Heridas/inmunología , Linfopoyetina del Estroma TímicoRESUMEN
Limbal stem cell (LSC) deficiency is a visually debilitating condition caused by abnormal maintenance of LSCs. It is treated by transplantation of donor-derived limbal epithelial cells (LECs), the success of which depends on the presence and quality of LSCs within the transplant. Understanding the immunobiological responses of these cells within the transplants could improve cell engraftment and survival. However, human corneal rings used as a source of LSCs are not always readily available for research purposes. As an alternative, we hypothesized that a human telomerase-immortalized corneal epithelial cell (HTCEC) line could be used as a model for studying LSC immunobiology. HTCEC constitutively expressed human leukocyte antigen (HLA) class I but not class II molecules. However, when stimulated by interferon-γ, HTCECs then expressed HLA class II antigens. Some HTCECs were also migratory in response to CXCL12 and expressed stem cell markers, Nanog, Oct4, and Sox2. In addition because both HTCECs and LECs contain side population (SP) cells, which are an enriched LSC population, we used these SP cells to show that some HTCEC SP cells coexpressed ABCG2 and ABCB5. HTCEC SP and non-side population (NSP) cells also expressed CXCR4, but the SP cells expressed higher levels. Both were capable of colony formation, but the NSP colonies were smaller and contained fewer cells. In addition, HTCECs expressed ΔNp63α. These results suggest the HTCEC line is a useful model for further understanding LSC biology by using an in vitro approach without reliance on a supply of human tissue. Stem Cells Translational Medicine 2017;6:761-766.
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Epitelio Corneal/citología , Limbo de la Córnea/citología , Limbo de la Córnea/inmunología , Modelos Biológicos , Células Madre/citología , Células 3T3 , Animales , Biomarcadores/metabolismo , Línea Celular , Línea Celular Transformada , Quimiotaxis , Ensayo de Unidades Formadoras de Colonias , Células Epiteliales/citología , Células Epiteliales/metabolismo , Antígenos HLA/metabolismo , Prueba de Histocompatibilidad , Humanos , Ratones , Receptores CXCR4/metabolismo , Células de Población Lateral/citología , Células Madre/metabolismo , Telomerasa/metabolismoRESUMEN
PURPOSE OF REVIEW: Vernal keratoconjunctivitis (VKC) is a severe bilateral chronic allergic inflammatory disease of the ocular surface. In most of the cases, the disease is limited to the tarsal conjunctiva and to the limbus. However, in the more severe cases, the cornea may be involved, leading to potentially sight threatening complications. Prompt recognition of these complications is crucial in the management of VKC, which is one of the most severe ocular allergic diseases. RECENT FINDINGS: A vicious cycle of inflammation occurs as a result of a set of reciprocal interactions between the conjunctiva and the cornea, which results in damage to the corneal epithelium and corneal stoma, and to the formation of shield ulcers and plaques, infectious keratitis, keratoconus, scarring, and limbal stem cell deficiency. These corneal complications can cause permanent decrease or loss of vision in children suffering from VKC. SUMMARY: Corneal complications in VKC are the result of an on-going process of uncontrolled inflammation. Proper recognition of the corneal complications in VKC is crucial, as most of these can be managed or prevented by a combination of medical and surgical measures.
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Conjuntiva/inmunología , Conjuntivitis Alérgica/inmunología , Córnea/inmunología , Limbo de la Córnea/inmunología , Animales , Comunicación Celular , Niño , Conjuntivitis Alérgica/complicaciones , Úlcera de la Córnea/etiología , Humanos , Queratitis/etiología , Queratocono/etiología , Visión OcularRESUMEN
BACKGROUND: CD4+ T helper type 2 cells play a central role in the pathogenesis of vernal keratoconjunctivitis (VKC), and antigen-presenting cells are required for the cell activation. In this study, we aimed to survey the density, distribution, and morphology of dendritic cells (DCs) in patients with VKC by in vivo confocal microscopy. METHODS: Thirty-five patients (mean, 12.4 ± 5.3 years) affected by VKC were included. All patients were treated with 0.1% fluorometholone eye drops and 0.5% cyclosporine A eye drops. The density and morphological and distributional characteristics of DCs in each right eye were evaluated by in vivo confocal microscopy before treatment and at 1, 3, and 6 months after treatment. Thirty-five age-matched normal subjects (mean, 16.5 ± 1.8 years) were studied as controls. RESULTS: There was significant difference in age between the VKC group and the control group (F = 18.17, P < 0.05). Compared with normal eyes, increased numbers of DCs were found in patients with VKC, with mean cell densities of 244.09 ± 59.76 cells/mm(2) at the bulbar conjunctiva, 574.53 ± 87.34 cells/mm(2) at the limbus, and 403.32 ± 106.59 cells/mm(2) at the peripheral cornea before treatment. These DCs exhibited a typical dendritic shape. At 3 months after treatment, the DC density at the conjunctiva decreased significantly (P < 0.05), approximating that in the controls. At 3 and 6 months, the DC densities at the limbus and peripheral cornea also decreased significantly (P < 0.05), but were still statistically higher than those in the controls. These DCs, with small dendritic processes or irregular shapes, were observed to gradually locate at the epithelial basal membrane and subbasal nerve plexus. CONCLUSIONS: In vivo confocal microscopy appears to be a valuable tool in evaluating the dynamic change of DCs at the conjunctiva and cornea. DCs play an essential role in VKC and therefore may constitute a target for therapeutic intervention for VKC.
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Conjuntivitis Alérgica/diagnóstico , Conjuntivitis Alérgica/inmunología , Células Dendríticas/inmunología , Células Dendríticas/patología , Adolescente , Adulto , Recuento de Células , Niño , Conjuntiva/inmunología , Conjuntiva/patología , Conjuntivitis Alérgica/tratamiento farmacológico , Córnea/inmunología , Córnea/patología , Femenino , Humanos , Limbo de la Córnea/inmunología , Limbo de la Córnea/patología , Masculino , Microscopía Confocal , Adulto JovenRESUMEN
BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) cultivated from the corneal limbus (L-MSCs) provide a potential source of cells for corneal repair. In the present study, we investigated the immunosuppressive properties of human L-MSCs and putative rabbit L-MSCs to develop an allogeneic therapy and animal model of L-MSC transplantation. METHODS: MSC-like cultures were established from the limbal stroma of human and rabbit (New Zealand white) corneas using either serum-supplemented medium or a commercial serum-free MSC medium (MesenCult-XF Culture Kit; Stem Cell Technologies, Melbourne, Australia). L-MSC phenotype was examined by flow cytometry. The immunosuppressive properties of L-MSC cultures were assessed using mixed leukocyte reactions. L-MSC cultures were also tested for their ability to support colony formation by primary limbal epithelial (LE) cells. RESULTS: Human L-MSC cultures were typically CD34â», CD45â» and HLA-DRâ» and CD73âº, CD90âº, CD105⺠and HLA-ABCâº. High levels (>80%) of CD146 expression were observed for L-MSC cultures grown in serum-supplemented medium but not cultures grown in MesenCult-XF (approximately 1%). Rabbit L-MSCs were approximately 95% positive for major histocompatibility complex class I and expressed lower levels of major histocompatibility complex class II (approximately 10%), CD45 (approximately 20%), CD105 (approximately 60%) and CD90 (<10%). Human L-MSCs and rabbit L-MSCs suppressed human T-cell proliferation by up to 75%. Conversely, L-MSCs from either species stimulated a 2-fold to 3-fold increase in LE cell colony formation. CONCLUSIONS: L-MSCs display immunosuppressive qualities in addition to their established non-immunogenic profile and stimulate LE cell growth in vitro across species boundaries. These results support the potential use of allogeneic L-MSCs in the treatment of corneal disorders and suggest that the rabbit would provide a useful pre-clinical model.
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Córnea/patología , Trasplante de Córnea , Limbo de la Córnea/citología , Células Madre Mesenquimatosas/citología , Animales , Proliferación Celular , Tratamiento Basado en Trasplante de Células y Tejidos , Córnea/inmunología , Humanos , Terapia de Inmunosupresión , Limbo de la Córnea/inmunología , Células Madre Mesenquimatosas/inmunología , Células Madre Mesenquimatosas/metabolismo , Conejos , Linfocitos T/citología , Linfocitos T/inmunologíaRESUMEN
BACKGROUND: Chronic inflammation in pterygium occurrence has not been explained. Whether damaged limbal basal epithelial cells are associated with pterygium occurrence in black Africans is not clear. OBJECTIVE: To explain chronic inflammation in pterygium, and to clarify whether damaged limbal basal epithelial cells were associated with pterygium occurrence in black Africans. METHODS: Chronic inflammatory changes and damaged limbal basal epithelial cells were assessed in 59 samples. RESULTS: Chronic inflammatory cells were present in 59 pterygia. Inflammatory cell count in 5 (27.8%) of 18 small pterygia was >200 (high) while in 22 (53.7%) of 41 large growths was <200 (low); p = 0.25. The proportion of pterygia with high counts tended to increase with pterygium extent. Twenty (33.9%) of 59 pterygia recurred after surgery. Ten (50%) of 20 samples had high cell counts and 10 (50%), low counts; p = 0.40. P53 expression was detected in 11 (18.6%) of 59 pterygium samples and 5 (71.4%) of 7 controls; p = 0.007. MMP 1 staining was present in 14 (23.7%) of 59 sections and 5 (71.4%) of 7 controls; p = 0.02. MMP2 in 16 (27.1%) cases and 5 (71.4%) controls; p = 0.03. MMP3 was overexpressed in 16 (27.1%) of 59 cases and 5 (71.4%) controls; p = 0.03. CONCLUSIONS: Mild chronic inflammation has a tendency to be more frequent than severe inflammation in pterygia. It is clear that damaged limbal basal epithelial cells are unlikely to be related to pterygium occurrence.
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Conjuntivitis/patología , Células Epiteliales/patología , Limbo de la Córnea/inmunología , Pterigion/inmunología , Adulto , Formación de Anticuerpos , Población Negra , Enfermedad Crónica , Conjuntivitis/inmunología , Femenino , Humanos , Inmunohistoquímica/métodos , Masculino , Persona de Mediana Edad , Pterigion/etnología , Pterigion/etiología , Pterigion/patología , Recurrencia , Sudáfrica/etnología , Proteína p53 Supresora de Tumor/metabolismo , Adulto JovenRESUMEN
Cornea transplantation has been considered to be different from other solid organ transplantation because of the assumed immune-privileged state of the anterior chamber of the eye. Three major lines of thought regarding the molecular mechanisms of immune privilege in the eye are as follows: (a) anatomical, cellular, and molecular barriers in the eye; (b) anterior chamber-associated immune deviation; and (c) immunosuppressive microenvironment in the eye. However, cornea transplants suffer allograft rejection when breached by vascularization. In recent developments, cellular corneal transplantation from cultivated limbal epithelial cells has shown impressive advances as a future therapy. The limbal stem cell niche contains stem cells that promote proliferation and migration and have immunosuppressive mechanisms to protect them from immunological reactions. Limbal stem cells are also noted to display an enhanced expression of genes for the antiapoptotic proteins, a property that is imperative for the survival of transplanted tissues. Further investigation of the molecular mechanisms regulating the immune regulation of limbal stem cells is relevant in the clinical setting to promote the survival of whole corneal and limbal stem cell transplantation.
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Limbo de la Córnea/citología , Limbo de la Córnea/inmunología , Trasplante de Células Madre , Células Madre/citología , Animales , Trasplante de Córnea , Rechazo de Injerto/inmunología , Rechazo de Injerto/prevención & control , Humanos , Complejo Mayor de Histocompatibilidad/inmunologíaRESUMEN
PURPOSE: We determined the plausible functional role of angiopoietin-like protein 2 (Angptl2) in inflammatory corneal hemangiogenesis and lymphangiogenesis in vivo. METHODS: Corneal hemangiogenesis and lymphangiogenesis were induced by suturing 10-0 nylon 1 mm away from the limbal vessel in Angptl2 knockout and K14-Angptl2 transgenic mice. We analyzed Angptl2 and interleukin 1ß (IL-1ß) expressions in normal and vascularized corneas by real-time RT-PCR and immunohistochemistry. Corneal hemangiogenic and lymphangiogenic responses, and macrophage infiltration were assessed by immunofluorescent microscopic studies using specific antibodies against CD31, LYVE-1, and F4/80, and compared to their corresponding background. Subconjunctival injection of Angptl2 siRNA to the sutured corneas was also performed. RESULTS: Angptl2 mRNA expression increased markedly in the neovascularized corneas compared to the normal cornea. Angptl2 protein was expressed strongly in the corneal epithelium and stroma of the vascularized cornea. The regions showing hemangiogenesis and lymphangiogenesis were increased significantly in K14-Angptl2 mice and reduced in Angptl2(-/-) mice compared to their corresponding background strains. In contrast to control mice, the number of F4/80-positive cells, as well as the expressions of F4/80 and IL-1ß were found to be higher in K14-Angptl2 mice and lower in Angptl2(-/-) mice. Subconjunctival injection of Angptl2 siRNA significantly inhibited hemangiogenesis and lymphangiogenesis in the sutured corneas. CONCLUSIONS: Our findings demonstrated Angptl2 to be upregulated in corneal inflammation, and highlight that corneal hemangiogenesis and lymphangiogenesis may be driven by Angptk2 overexpression via macrophage infiltration and IL-1ß expression. Angptl2 may be a novel therapeutic target for preventing blindness.
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Angiopoyetinas/genética , Angiopoyetinas/inmunología , Queratitis/inmunología , Linfangiogénesis/inmunología , Neovascularización Patológica/inmunología , Proteína 2 Similar a la Angiopoyetina , Proteínas Similares a la Angiopoyetina , Animales , Modelos Animales de Enfermedad , Interleucina-1beta/inmunología , Queratitis/genética , Queratitis/patología , Limbo de la Córnea/inmunología , Limbo de la Córnea/patología , Linfangiogénesis/genética , Macrófagos/inmunología , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Neovascularización Patológica/genética , Neovascularización Patológica/patología , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Suturas , Regulación hacia Arriba/inmunologíaRESUMEN
PURPOSE: Mesenchymal stromal stem cells (MSC) are non-hemopoietic cells with the capacity to self-renewal and to differentiate into various cell lineages of mesenchymal origin. More recently, the immune regulatory potential of MSC has been focused on. Furthermore, mesenchymal stem cells obtained from diverse tissues possess immunomodulatory properties and inhibit proinflammatory immune reactions. The aim of this study was to determine the immunosuppressive characteristics of mesenchymal stem cells isolated from human limbal (L-MSC) tissue. METHODS: L-MSC were enzymatically obtained from cadaveric sclero-corneal rims and expanded in vitro. The cells were characterized by flow cytometry using specific antibodies to mesenchymal stem cells markers. Clonogenic and tissue transdifferentiation in vitro assays were performed. The effect of L-MSC soluble factors on T cell proliferation was determined by flow cytometry. Cytokines such as transforming growth factor-b1 (TGF-ß1) and Interleukin-10 (IL-10) on supernatants from L-MSC were identified by enzyme-linked immunosorbent assay (ELISA). RESULTS: Herein, we described that L-MSC cells in vitro-expanded were positive for the expression of vimentin, CD29, CD34, CD39, CD73 and CD105 mesenchymal stem cells markers; meanwhile, this cell population was negative to CD45 and HLA-DR hemopoietic markers as well as to cytokeratin expression. Clonogenic assays showed that these cells were able to form colonies. In addition, this L-MSC population had the ability to transdifferentiate into neurons and chondrocytes and to form tubular networks on matrigel in the presence of vascular endothelial growth factor (VEGF). These results indicated that these cells were stem cells. Additionally, soluble factors secreted by L-MSC were capable of mediating the suppression of T-cell receptor (TCR)-engagement lymphocyte proliferation. In an attempt to identify the possible immunosuppressive factors secreted by L-MSC, TGFß1 and IL-10 cytokines were determined in the L-MSC supernatants by ELISA; interestingly, TGFß1 was constitutively secreted by this cell population; in contrast, IL-10 was not detectable. Moreover, TGFßRII neutralizing antibodies were able to revert the TCR-engagement lymphocyte proliferation inhibition mediated by L-MSC. Thus, TGFß1 secreted by L-MSC was able to suppress T cell proliferation. CONCLUSIONS: Taken together these results, explain in part the immunosuppressive features of this cell population obtained from the human limbus. All these characteristics make this cell population an excellent source to be used in the regenerative medicine.
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Limbo de la Córnea/inmunología , Células Madre Mesenquimatosas/inmunología , Factor de Crecimiento Transformador beta1/inmunología , Antígenos CD/genética , Antígenos CD/inmunología , Autopsia , Biomarcadores/metabolismo , Proliferación Celular/efectos de los fármacos , Transdiferenciación Celular/efectos de los fármacos , Condrocitos/citología , Condrocitos/metabolismo , Medios de Cultivo Condicionados/farmacología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Inmunosupresores/farmacología , Limbo de la Córnea/citología , Limbo de la Córnea/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Neuronas/citología , Neuronas/metabolismo , Cultivo Primario de Células , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Medicina Regenerativa , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Vimentina/genética , Vimentina/inmunologíaRESUMEN
BACKGROUND: The purpose of this study is to conduct a histopathological research of the conjunctival findings and eosinophilic inflammation of novel atopic keratoconjunctivitis in a NC/Nga mouse model using crude Dermatophagoides farina. METHODS: NC/Nga mice were sensitized by repeated topical applications of an ointment containing Dermatophagoides farinae body (Dfb). They were then divided into 4 groups depending on the following topical ophthalmic treatment: DFb group undergoing topical ophthalmic ointment containing Dfb; DFco group undergoing topical instillation of allergen extracts of Dermatophagoides farinae; Ba group undergoing topical ointment with substrate alone and NT group without after-topical ophthalmic treatment. At 24 hours after the last ophthalmic treatment, histopathological examination was performed. The density of the subepithelial infiltration of the eosinophils was determined. Serum total IgE and house-dust-mite (HDM)-specific IgE antibody concentrations were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: In the DFb group, the conjunctiva showed similar findings to those of atopic keratoconjunctivitis, i.e. intraepithelial pseudotubular formation, Torus-form infiltration due to massive lymphocytes in the palpebral conjunctiva and gelatinous hyperplasia in the limbus with subepithelial granuloma composed of lymphocytes and eosinophils. Subepithelial infiltration of eosinophil density in the DFb group [878.4 ± 399.7cells/mm2 (mean ± SD)] was significantly higher than in the other 2 groups (DFco 85.6 ± 40.1 Ba 49.2 ± 32.3) (P < 0.001). Total serum IgE concentration and HDM-specific serum IgE antibody concentration in the DFb group and the DFco group were significantly higher compared with those in the NT group. CONCLUSIONS: Topical application of an ointment containing DFb to both the skin and eyes of NC/Nga mice can induce an atopic keratoconjunctivitis (AKC) model in these mice.
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Antígenos Dermatofagoides/inmunología , Conjuntiva/inmunología , Dermatophagoides farinae/inmunología , Eosinófilos/inmunología , Queratoconjuntivitis/inmunología , Animales , Antígenos Dermatofagoides/administración & dosificación , Extractos Celulares/inmunología , Movimiento Celular , Modelos Animales de Enfermedad , Humanos , Inmunización , Inmunoglobulina E/sangre , Queratoconjuntivitis/sangre , Queratoconjuntivitis/complicaciones , Limbo de la Córnea/inmunología , Linfocitos/inmunología , Ratones , Ratones EndogámicosRESUMEN
LYVE-1(+) corneal lymphatics contribute to drainage and immunity. LYVE-1 is widely accepted as the most reliable lymphatic marker because of its continuous expression in lymphatic endothelium. LYVE-1 expression in corneal lymphatics has not been examined. In this study, we report intact CD31(+) corneal lymphatic capillary endothelial cells that do not express LYVE-1. The number of LYVE-1(-) gaps initially increased until 8 wk of age but was significantly reduced in aged mice. C57BL/6 mice showed a notably higher number of the LYVE-1(-)/CD31(+) lymphatic regions than BALB/c mice, which suggests a genetic predisposition for this histological feature. The LYVE-1(-) lymphatic gaps expressed podoplanin and VE-cadherin but not αSMA or FOXC2. Interestingly, the number of LYVE-1(-) gaps in FGF-2, but not VEGF-A, implanted corneas was significantly lower than in untreated corneas. Over 70% of the CD45(+) leukocytes were found in the proximity of the LYVE-1(-) gaps. Using a novel in vivo imaging technique for visualization of leukocyte migration into and out of corneal stroma, we showed reentry of extravasated leukocytes from angiogenic vessels into newly grown corneal lymphatics. This process was inhibited by VE-cadherin blockade. To date, existence of lymphatic valves in cornea is unknown. Electron microscopy showed overlapping lymphatic endothelial ends, reminiscent of microvalves in corneal lymphatics. This work introduces a novel corneal endothelial lymphatic phenotype that lacks LYVE-1. LYVE-1(-) lymphatic endothelium could serve as microvalves, supporting unidirectional flow, as well as immunological hot spots that facilitate reentry of stromal macropahges.
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Glicoproteínas/metabolismo , Limbo de la Córnea/metabolismo , Envejecimiento/inmunología , Envejecimiento/metabolismo , Animales , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Cadherinas/metabolismo , Conjuntiva/crecimiento & desarrollo , Conjuntiva/inmunología , Conjuntiva/metabolismo , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Leucocitos/citología , Limbo de la Córnea/crecimiento & desarrollo , Limbo de la Córnea/inmunología , Linfangiogénesis , Vasos Linfáticos/metabolismo , Masculino , Proteínas de Transporte de Membrana , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
PURPOSE: To investigate the effect of ex vivo culture on the expression of human leukocyte antigen (HLA) in human corneal limbal epithelial cells (LECs), and to evaluate the expression of HLA and costimulatory molecules on human corneal limbal stromal cells (LSCs). METHODS: The expression of HLA-ABC and HLA-DR were evaluated using flow cytometry on cultured human LECs and LSCs after serial passage and compared with that on freshly isolated cells. Additionally, the expression of costimulatory molecules CD80, CD86, CD40, and immunoregulatory molecule HLA-G were investigated on LSCs with or without an addition of interferon (IFN) γ (250 U/mL, 4 days). RESULTS: HLA-ABC and HLA-DR were expressed in 99.12 ± 1.02% and 25.86 ± 4.34% of freshly isolated LECs, respectively. After culture, the HLA-DR-expressing cells significantly decreased with serial passage, whereas the percentage of HLA-ABC-expressing cells did not change. More than 90% of LSCs expressed HLA-ABC and HLA-G, whereas the expression of HLA-DR, CD80, CD86, and CD40 was negligible. This expression profile did not change after serial passage. However, the treatment with IFN-γ induced a significant increase in HLA-DR (0.17% vs. 17.68%) and CD40 (0.19% vs. 13.19%) expression on LSCs. CONCLUSIONS: The immunogenicity of LECs can be lowered after ex vivo cultivation, whereas LSCs can be immunogenic in the presence of IFN-γ.
Asunto(s)
Sustancia Propia/inmunología , Antígenos HLA/análisis , Limbo de la Córnea/inmunología , Antígenos CD/análisis , Células Cultivadas , Sustancia Propia/citología , Citometría de Flujo , Antígenos HLA/efectos de los fármacos , Antígenos HLA-DR/metabolismo , Humanos , Interferón gamma/farmacología , Limbo de la Córnea/citología , Factores de TiempoRESUMEN
PURPOSE: Squamous metaplasia of the ocular surface epithelium in severe Sjögren syndrome (SS) dry eye has been implicated to be associated with chronic engagement of immune-mediated inflammation. While the detailed immunopathological mechanism underlying keratinization of the ocular muco-epithelium in this setting remains unclear, mice deficient in the autoimmune regulator gene (Aire) demonstrate SS-like pathological changes in the exocrine organs and ocular surface including squamous metaplasia. Using this murine model, we sought to determine the specific immune events that predict squamous metaplasia of the cornea in Aire deficiency. METHODS: Lissamine green staining, goblet cell density, and corneal small proline-rich protein 1B (SPRR1B) were compared in Aire-sufficient and -deficient mice at 4, 8, and 16 weeks of age. Corneal, limbal and conjunctival infiltration of CD4(+) and CD8(+) T cells as well as CD11c(+) and MHC class II (I-A(d+)) dendritic cells (DCs) were examined at the same time points. Ordinary least squares regression was used to model SPRR1B's relationship with lissamine green staining, goblet cell density, and immune cell infiltration. RESULTS: Lissamine green staining was present in Aire-deficient mice by four weeks of age and increased over time. Compared to Aire-sufficient controls, conjunctival goblet cell density (GCD) decreased and corneal SPRR1B increased in Aire-deficient mice with significant differences noted at both 8 and 16 weeks. Immune-mediated CD4(+) T cell infiltration of the conjunctiva and limbus peaked at eight weeks and then decreased. In contrast, corneal T cell infiltration continued to increase over time, reaching a maximum cell number at 16 weeks. CD11c(+) myeloid-derived DCs were found in the conjunctiva and limbus at all time points. As the mice aged, there was a notable increase in corneal CD11c(+) cell counts. Interestingly, the dynamic of activated MHC class II(+) DCs was nearly identical to that of CD4(+) T cells, peaking first in the limbus at eight weeks with maximum infiltration of the cornea by 16 weeks. Regression analysis showed that squamous metaplasia biomarker, SPRR1B, is strongly related to the lissamine green staining of the ocular surface. Corneal infiltration of activated DCs was most prognostic of corneal SPRR1B expression while the presence of precursor DCs, activated DCs, and CD4(+) T cells in the limbus were also significant predictors of SPRR1B. CONCLUSIONS: Aire-deficient mice represent a useful model to study Sjögren-like autoimmune-mediated ocular surface disease. Results of the current study suggest that squamous cell precursor protein, SPRR1B, provides an important readout to evaluate ocular surface damage and specific events related to immune-mediated inflammation. Results also define an appropriate time frame for interventional studies to develop more effective therapies for keratinizing ocular surface disease.
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Síndromes de Ojo Seco/inmunología , Síndromes de Ojo Seco/patología , Factores de Transcripción/deficiencia , Animales , Biomarcadores/metabolismo , Recuento de Células , Movimiento Celular , Conjuntiva/inmunología , Conjuntiva/patología , Proteínas Ricas en Prolina del Estrato Córneo/metabolismo , Células Dendríticas/inmunología , Epitelio Corneal/inmunología , Epitelio Corneal/patología , Células Caliciformes/inmunología , Células Caliciformes/patología , Aparato Lagrimal/inmunología , Aparato Lagrimal/patología , Limbo de la Córnea/inmunología , Limbo de la Córnea/patología , Colorantes Verde de Lisamina , Metaplasia/inmunología , Metaplasia/patología , Ratones , Membrana Mucosa/inmunología , Membrana Mucosa/patología , Propiedades de Superficie , Linfocitos T/inmunología , Factores de Tiempo , Factores de Transcripción/metabolismo , Proteína AIRERESUMEN
Limbal stem cell deficiency (LSCD) is clinically characterized by growth of fibrovascular pannus onto the corneal surface, chronic inflammation and impaired visual acuity. The aim of this study was to characterize the pannus tissue. Total RNA was isolated from six pannus samples and protein from three pannus samples from patients with LSCD caused by chemical burns. Normal human corneal tissue (n=6) and conjunctiva (n=6) were used as control tissues. The expression of the epithelial lineage markers keratin 3 (K3), K19 and MUC5AC, the inflammatory markers IL-1beta, ICAM-1 and VEGF was analyzed by Western Blotting and/or real-time PCR. Normal corneal tissue had a higher expression of K3 and a lower expression of K19 and MUC5AC in comparison to normal conjunctiva. A higher expression of inflammatory markers was seen in conjunctiva compared to corneal tissue. The pannus tissue showed a similar expression of lineage markers to conjunctiva but a higher expression of most inflammatory markers as compared to both control tissues. The analyzed samples of pannus in LSCD resembled conjunctiva and not cornea and showed a high expression of inflammatory markers indicating chronic inflammation.
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Epitelio Corneal/inmunología , Epitelio Corneal/patología , Quemaduras Oculares/inmunología , Quemaduras Oculares/patología , Limbo de la Córnea/inmunología , Limbo de la Córnea/patología , Células Madre/inmunología , Células Madre/patología , Adulto , Células Cultivadas , Epitelio Corneal/lesiones , Femenino , Humanos , Limbo de la Córnea/lesiones , Masculino , Persona de Mediana EdadRESUMEN
All published literature to date has identified the human corneo-scleral limbus as the site within which stem cells of the ocular surface reside. Recently we described a unique anatomical structure at the limbus, termed the Limbal Epithelial Crypt (LEC) that has features of a putative stem cell niche. In this study we examined infant limbus tissue (donor age 4 months) for evidence of LEC and performed immunohistological comparison between infant limbus and adult LEC. No defined LEC were detected in the infant limbus. However, the entire infant limbus has characteristics resembling adult LEC. Both infant limbus and LEC demonstrated negative expression for desmoglein 3. p63 and integrin beta1 expressions were located to the distal region of the infant limbus and to the basal region of the LEC. ABCG2 expression was positive throughout most of the infant limbus as was connexin 43. Infant limbus and in particular the distal region, appeared to house cells that are more "stem-like" in nature. The LEC may be a result of normal physiological developmental in order to protect and maintain stem cells at the ocular surface.
Asunto(s)
Limbo de la Córnea/inmunología , Adulto , Envejecimiento/inmunología , Proteínas del Ojo/metabolismo , Humanos , Inmunofenotipificación , Lactante , Células Madre/inmunologíaRESUMEN
PURPOSE: To evaluate the long-term outcome of HLA-matched lr-CLAL for bilateral ocular surface disorders. METHODS: A retrospective, non-comparative interventional case series study of 39 eyes of 32 patients with bilateral surface disorders and clinical diagnosis of limbal stem cell deficiency who underwent HLA-matched lr-CLAL. Visual acuity (VA), ambulatory vision (> 20/200) and ocular surface stability were evaluated as main outcomes. Donor limbus was obtained from a sibling or a parent of the patient, after an appropriate Class I and II HLA match. RESULTS: One year after surgery, VA improved in 46.2 percent, ambulatory vision was achieved in 48.7 percent and a stable corneal surface was achieved in 84.6 percent of the eyes. At the final follow-up (mean, 48.7 ± 30.6 months), 66.6 percent of the eyes that had gained VA one year after surgery maintained an improved VA (p=0.28), 94.7 percent of eyes that had achieved ambulatory vision one year after surgery maintained 20/200 or better (p<0.001) and 93.9 percent still had a stable corneal surface (p=0.043) at the final follow-up. CONCLUSIONS: HLA-matched lr-CLAL can be an adequate method of treatment for bilateral ocular surface disorders, with a reasonable percentage of success of long-term results.
OBJETIVO: Avaliar os resultados em longo prazo do transplante alogênico inter vivo de limbo conjuntival para doenças bilaterais da superfície ocular com compatibilidade HLA doador-receptor. MÉTODOS: Estudo retrospectivo, não comparativo e intervencionista de 39 olhos de 32 pacientes submetidos a transplante alogênico de limbo com compatibilidade HLA e diagnóstico de deficiência límbica. Foram analisados como desfechos principais acuidade visual, visão ambulatorial (> 20/200) e estabilidade da superfície ocular. Limbo doador foi obtido de parentes do paciente após estudo de compatibilidade HLA classe I e II. RESULTADOS: Com um ano de pós-operatório, a acuidade visual melhorou em 46,2 por cento, visão ambulatorial foi atingida em 48,7 por cento e estabilidade da superfície corneana em 84,6 por cento dos pacientes. Ao final do seguimento (média, 48,7 ± 30,6 meses), 66,6 por cento dos olhos que haviam ganho acuidade visual um ano após a cirurgia mantiveram esta melhora (p=0,28), 94,7 por cento dos olhos que haviam alcançado visão ambulatorial um ano após a cirurgia mantiveram visão de 20/200 ou melhor (p<0,001) e 93,9 por cento ainda tinham superfície corneana estável (p=0,043) ao final do seguimento. CONCLUSÕES: O transplante alogênico inter vivo de limbo conjuntival com compatibilidade HLA revelou-se método adequado no tratamento de doenças bilaterais da superfície ocular, com uma porcentagem significativa de sucesso a longo-prazo.
Asunto(s)
Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Humanos , Persona de Mediana Edad , Adulto Joven , Enfermedades de la Córnea/cirugía , Epitelio Corneal/trasplante , Limbo de la Córnea/inmunología , Trasplante de Células Madre , Agudeza Visual/fisiología , Atención Ambulatoria , Enfermedades de la Córnea/etiología , Enfermedades de la Córnea/inmunología , Epitelio Corneal/inmunología , Epitelio Corneal/patología , Estudios de Seguimiento , Rechazo de Injerto/inmunología , Prueba de Histocompatibilidad , Estimación de Kaplan-Meier , Donadores Vivos , Estudios Retrospectivos , Trasplante de Células Madre/métodos , Trasplante Homólogo , Resultado del Tratamiento , Adulto JovenRESUMEN
PURPOSE: To evaluate the long-term outcome of HLA-matched lr-CLAL for bilateral ocular surface disorders. METHODS: A retrospective, non-comparative interventional case series study of 39 eyes of 32 patients with bilateral surface disorders and clinical diagnosis of limbal stem cell deficiency who underwent HLA-matched lr-CLAL. Visual acuity (VA), ambulatory vision (> or = 20/200) and ocular surface stability were evaluated as main outcomes. Donor limbus was obtained from a sibling or a parent of the patient, after an appropriate Class I and II HLA match. RESULTS: One year after surgery, VA improved in 46.2%, ambulatory vision was achieved in 48.7% and a stable corneal surface was achieved in 84.6% of the eyes. At the final follow-up (mean, 48.7 +/- 30.6 months), 66.6% of the eyes that had gained VA one year after surgery maintained an improved VA (p=0.28), 94.7% of eyes that had achieved ambulatory vision one year after surgery maintained 20/200 or better (p<0.001) and 93.9% still had a stable corneal surface (p=0.043) at the final follow-up. CONCLUSIONS: HLA-matched lr-CLAL can be an adequate method of treatment for bilateral ocular surface disorders, with a reasonable percentage of success of long-term results.