RESUMEN
Different types of refractive surgeries often exhibit differences in wound healing responses. The current study investigated post-operative tear protein profiles in subjects who underwent LASIK and SMILE to elucidate global changes to the proteomic profile during the period the patient cornea undergoes healing. In this study, 10 patients underwent LASIK and SMILE surgery with a contralateral paired eye design. Tear samples were collected using Schirmer's strips preoperatively, at 1 month, 3 months and 6 months postoperatively. Quantitative ITRAQ labeled proteomics was performed and the tear protein ratios were normalized to pre-operative protein levels for each subject. Whole proteomics identified 1345 proteins in tears from LASIK and 1584 proteins in SMILE across time points. About 67 proteins were common in LASIK and SMILE tears across all the time points. Wound healing responses were differentially regulated between two refractive surgeries (SMILE and LASIK). The proteins Ceruloplasmin, Clusterin, Serotransferrin were upregulated at 1 month and 3 months and downregulated at 6 months post operatively in LASIK surgery where as in SMILE these were downregulated. Galectin 3 binding protein showed upregulation at 1 month and the levels decreased at 3 months and 6 months postop in LASIK tears whereas the levels increased at 3 months and 6 months post-op in SMILE tears. The levels of proteins that protect from oxidative stress were higher in SMILE as compared to LASIK postoperatively. The extracellular matrix proteins showed an increase in expression at 6 months in SMILE tears and was stabilized at 6 months in LASIK tears post operatively. Different refractive surgeries induce distinct wound healing responses as identified in tears. This study has implications in targeting key proteins for improving the clinical outcome postrefractive surgery.
Asunto(s)
Proteínas del Ojo , Queratomileusis por Láser In Situ , Miopía , Proteómica , Lágrimas , Cicatrización de Heridas , Humanos , Lágrimas/metabolismo , Queratomileusis por Láser In Situ/métodos , Cicatrización de Heridas/fisiología , Proteómica/métodos , Femenino , Masculino , Adulto , Proteínas del Ojo/metabolismo , Miopía/cirugía , Miopía/metabolismo , Cirugía Laser de Córnea/métodos , Adulto Joven , Láseres de Excímeros/uso terapéutico , Periodo Posoperatorio , Sustancia Propia/metabolismo , Sustancia Propia/cirugíaRESUMEN
To demonstrate viral proteins/inflammatory cytokines in a patient with unilateral keratouveitis. Retrospective case report. A 70-year-old Asian-Indian male presented with acute onset of blurring of vision in the left eye (OS) of 2 days duration. He had was coronavirus disease 2019 (COVID-19)-positive 3 months earlier. He had undergone cataract surgery/retinal laser photocoagulation in both the eyes. The corrected distance visual acuity (CDVA) (Snellen) in the right eye (RE) (OD) and left eye (LE) (OS) was 20/20 and 20/80, respectively. OS showed decreased corneal sensation, Descemet's folds, mild stromal edema, and fine and pigmented keratic precipitates with anterior chamber 1+ flare and 1+ cells. Fundus evaluation showed scattered laser marks in the OD and temporal sectoral laser marks in OS. He was diagnosed with viral keratouveitis in OS. Tear samples were collected on Schirmer's strips and tear wash for mass spectrometry and cytokines, which had 368 and 451 viral proteins in the RE and LE, respectively, using nano liquid chromatography-mass spectrometry, which were more than controls. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and varicella zoster virus proteins were detected. Cytokine analysis using flow cytometer analysis showed higher inflammation in OS as compared to OD. The patient was treated with oral acyclovir and topical steroids and resulted in resolution of his keratouveitis. SARS-CoV-2 proteins were present in the tear sample 3 months after COVID-19. The presence of viral proteins does not indicate causality.
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COVID-19 , Queratitis , Uveítis , Humanos , Masculino , Anciano , Estudios Retrospectivos , SARS-CoV-2 , Queratitis/diagnóstico , Proteínas ViralesRESUMEN
Purpose: Pediatric cataract is a major cause of preventable childhood blindness worldwide. Although genetic mutations or infections have been described in patients, the mechanistic basis of human cataract development remains poorly understood. Therefore, gene expression of structural, developmental, profibrotic, and transcription factors in phenotypically and etiologically distinct forms of pediatric cataracts were evaluated. Methods: This cross-sectional study included 89 pediatric cataract subjects subtyped into 1) prenatal infectious (cytomegalovirus, rubella, and combined cytomegalovirus with rubella infection), 2) prenatal non-infectious, 3) posterior capsular anomalies, 4) postnatal, 5) traumatic, and 6) secondary, and compared to clear, non-cataractous material of eyes with the subluxated lenses. Expression of lens structure-related genes (Aqp-0, HspA4/Hsp70, CrygC), transcription factors (Tdrd7, FoxE3, Maf, Pitx 3) and profibrotic genes (Tgfß, Bmp7, αSmA, vimentin) in surgically extracted cataract lens material were studied and correlated clinically. Results: In cataract material, the lens-related gene expression profiles were uniquely associated with phenotype/etiology of different cataracts. Postnatal cataracts showed a significantly altered FoxE3 expression. Low levels of Tdrd7 expression correlated with posterior subcapsular opacity, whereas CrygC correlated significantly with anterior capsular ruptures. The expression of Aqp0 and Maf was elevated in infectious cataracts, particularly in CMV infections, compared to other cataract subtypes. Tgfß showed significantly low expression in various cataract subtypes, whereas vimentin had elevated gene expression in infectious and prenatal cataracts. Conclusion: A significant association between lens gene expression patterns in phenotypically and etiologically distinct subtypes of pediatric cataracts suggests regulatory mechanisms in cataractogenesis. The data reveal that cataract formation and presentation is a consequence of altered expression of a complex network of genes.
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Catarata , Cristalino , Humanos , Niño , Vimentina/genética , Vimentina/metabolismo , Estudios Transversales , Transcriptoma , Catarata/genética , Catarata/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismoRESUMEN
Dry eye disease (DED) is a multi-factorial ocular surface condition driven by compromised ocular lubrication and inflammation which leads to itching, dryness, and vision impairment. The available treatment modalities primarily target the acquired symptoms of DED including tear film supplements, anti-inflammatory drugs, mucin secretagogues, etc., However, the underlying etiology is still an area of active research, especially in regard to the diverse etiology and symptoms. Proteomics is a robust approach that has been playing major role in understanding the causative mechanism and biochemical changes in DED by identifying the changes in protein expression profile in tears. Tears are a complex fluid composed of several biomolecules such as proteins, peptides, lipids, mucins, and metabolites secreted from lacrimal gland, meibomian gland, cornea, and vascular sources. Over the past two decades, tears have emerged as a bona-fide source for biomarker identification in many ocular conditions because of the minimally invasive and simple sample collection procedure. However, the tear proteome can be altered by several factors, which increases the complexity of the approach. The recent advancements in untargeted mass spectrometry-based proteomics could overcome such shortcomings. Also, these technological advancements help to distinguish the DED profiles based on its association with other complications such as Sjogren's syndrome, rheumatoid arthritis, diabetes, and meibomian gland dysfunction. This review summarizes the important molecular profiles found in proteomics studies to be altered in DED which have added to the understanding of its pathogenesis.
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Síndromes de Ojo Seco , Aparato Lagrimal , Síndrome de Sjögren , Humanos , Proteómica , Síndromes de Ojo Seco/etiología , Lágrimas/metabolismo , Síndrome de Sjögren/complicaciones , Síndrome de Sjögren/metabolismo , Mucinas/metabolismoRESUMEN
The differentiation of keratocytes to fibroblasts and myofibroblasts is an essential requisite during corneal wound closure. The aim of this study is to uncover factors involved in differentiation-dependent alteration in the protein profile of human corneal stromal cells using quantitative proteomics. Human corneal fibroblasts were cultured and differentiated into keratocytes in serum-free media and myofibroblasts through treatment with TGF-ß. The protein cell lysates from the donors were tryptic and were digested and labeled using a 3-plex iTRAQ kit. The labeled peptides were subjected to LCMS analysis. Biological functional analysis revealed a set of crucial proteins involved in the differentiation of human corneal stromal cells which were found to be significantly enriched. The selected proteins were further validated by immunohistochemistry. Quantitative proteomics identified key differentially expressed proteins which are involved in cellular signaling pathways. Proteins involved in integrin signaling (Ras-RAP1b, TLN and FN) and SLIT-ROBO pathways (PFN1, CAPR1, PSMA5) as well as extracellular matrix proteins (SERPINH1, SPARC, ITGß1, CRTAP) showed enhanced expression in corneal fibroblasts and myofibroblasts compared to keratocytes, indicating their possible role in wound healing. Corneal stromal cell differentiation is associated with the activation of diverse molecular pathways critical for the repair of fibroblasts and myofibroblasts. Identified proteins such as profilin 1 and talin could play a tentative role in corneal healing and serve as a potential target to treat corneal fibrosis.
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Lesiones de la Cornea , Proteómica , Diferenciación Celular/fisiología , Células Cultivadas , Córnea/metabolismo , Lesiones de la Cornea/metabolismo , Fibroblastos/metabolismo , Humanos , Profilinas/metabolismo , Células del Estroma/metabolismo , Cicatrización de Heridas/fisiologíaRESUMEN
Infection mediated ocular surface stress responses are activated as early defense mechanisms in response to host cell damage. Integrated stress responses initiate the host response to different types of infections and modulate the transcription of key genes and translation of proteins. The crosstalk between host and pathogen results in profound alterations in cellular and molecular homeostasis triggering specific stress responses in the infected tissues. The amplitude and variations of such responses are partly responsible for the disease severity and clinical sequelae. Understanding the etiology and pathogenesis of ocular infections is important for early diagnosis and effective treatment. This review considers the molecular status of infection mediated ocular surface stress responses which may shed light on the importance of the host stress-signaling pathways. In this review, we collated literature on the molecular studies of all ocular surface infections and summarize the results from such studies systematically. Identification of important mediators involved in the crosstalk between the stress response and activation of diverse signaling molecules in host ocular surface infection may provide novel molecular targets for maintaining the cellular homeostasis during infection. These targets can be then explored and validated for diagnostic and therapeutic purposes.
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Infecciones del Ojo , HumanosRESUMEN
We studied the early protein profile in the ocular tissue extracted after LASIK and SMILE surgery. SMILE and LASIK was performed in contralateral eyes and stromal tissue samples were collected from 10 eyes of 5 donors. The stromal tissue samples were analyzed using label free quantification approach and ITRAQ labelling approach in LC-MS/MS. Combined functional analysis revealed many differentially expressed proteins which were involved in important biological processes. About 117 unique differentially expressed proteins were identified using two different proteomic approaches. Collagens, proteoglycans, corneal crystallins were enriched and showed differential expression in SMILE and LASIK as compared to the non-surgical control. Apart from these, 14-3-3 class of proteins, Lysozyme (LYZ), Macrophage Migratory Inhibitory Factor protein (MIF), Pigment Epithelial Derived Factor (PEDF) were differentially expressed when compared between LASIK and SMILE. Peroxiredoxin 1 (PRDX1) expression was found to be reduced in LASIK as compared to SMILE. The expression of Lysozyme C and Macrophage Migratory Inhibitory Factor inflammatory response was found to be less in SMILE as compared to LASIK. Western blot validation of specific markers such as Collagen IV (COL4), Keratocan (KERA), Lumican (LUM), Aldehyde dehydrogenase 3 A1 (ALDH3A1), Lysozyme C (LYZC) confirmed the differences in the protein levels observed in SMILE and LASIK operated tissues as compared to non-surgical controls. In conclusion, this study revealed the early molecular changes occurring in the cornea resulting from these two surgical procedures which may have implications on managing post-operative complications.
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Sustancia Propia/cirugía , Proteínas del Ojo/metabolismo , Queratomileusis por Láser In Situ/métodos , Proteoma/metabolismo , Anciano , Anciano de 80 o más Años , Western Blotting , Cromatografía Liquida , Colágeno/metabolismo , Sustancia Propia/metabolismo , Cirugía Laser de Córnea , Cristalinas/metabolismo , Humanos , Masculino , Metaboloma , Persona de Mediana Edad , Estudios Prospectivos , Proteoglicanos/metabolismo , Espectrometría de Masas en TándemRESUMEN
PURPOSE: Corneal dystrophies (CD) are classified as rare eye diseases that results in visual impairment and requires corneal transplant in advanced stages. Ocular surface inflammatory status in different types of CD remains underexplored. Hence, we studied the levels of tear soluble factors in the tears of patients with various types of corneal dystrophies. METHODS: 17 healthy subjects and 30 CD subjects (including epithelial, stromal and endothelial CD) were included in the study. Schirmer's strips were used to collect the tear fluid in all subjects. 27 soluble factors including cytokines, chemokines, soluble cell adhesion molecules and growth factors were measured in the eluted tears by multiplex ELISA or single analyte sandwich ELISA. RESULTS: Percentages of subjects with detectable levels of tear soluble factors were significantly higher in CD compared to controls. Significant higher level of IL-2 was observed in both epithelial and stromal CD. IL-4, TGFß1 and IgE were significantly higher in stromal CD. VCAM, IL-13 and Fractalkine were significantly elevated in epithelial and macular CD. IL-1α, IL-8, IL-12, ANG, Eotaxin, MCP1, RANTES, ICAM1, L-selectin and P-selectin were significantly higher in epithelial CD. TGFBIp was significantly elevated in lattice CD and endothelial CD. CONCLUSION: Distinct set of the tear soluble factors were dysregulated in various types of CD. Increase in tear inflammatory factors was observed in majority of the CD subjects depending on their sub-types. This suggests a plausible role of aberrant inflammation in CD pathobiology. Hence, modulating inflammation could be a potential strategy in improving the prognosis of CD.
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Distrofias Hereditarias de la Córnea , Citocinas , Ensayo de Inmunoadsorción Enzimática , Ojo , Humanos , LágrimasRESUMEN
Database URL: www.tri-incardiome.org.
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Enfermedad de la Arteria Coronaria , Bases de Datos Factuales , Investigación Biomédica Traslacional , Clero , Enfermedad de la Arteria Coronaria/diagnóstico , Enfermedad de la Arteria Coronaria/tratamiento farmacológico , Enfermedad de la Arteria Coronaria/metabolismo , Enfermedad de la Arteria Coronaria/fisiopatología , HumanosRESUMEN
INTRODUCTION: Immunotherapy by inducing oral tolerance to atherogenic self-antigens is gaining importance as an alternative treatment modality for atherosclerosis. The use of live bacterial vectors to express the recombinant antigen in vivo will obviate the need for large-scale purification of recombinant protein and may also augment the efficacy of oral tolerance induction. AIM: The objective of the study was to explore the use of recombinant Mycobacterium smegmatis as a live vector for oral delivery of antigens to induce immune tolerance. METHOD AND RESULTS: We developed a M. smegmatis vector to secrete a recombinant tripeptide construct (AHC; peptides from Apolipoprotein B, Heat-shock protein 60 and Chlamydia pneumoniae outer membrane protein) expressed in a dendroaspin protein scaffold in pJH154 background. Immune response and oral tolerance to the cloned peptides were studied in C57/BL6 mice. The efficacy of this live vaccine to control atherosclerosis was studied in ApoE(-/-) knockout mice in C57/BL6 background. Oral administration of M. smegmatis secreting the cloned AHC antigen was found to induce tolerance to cloned protein and reduce the development of atherosclerosis by 24.0% compared to control. Protection against atherosclerosis was associated with increase in expression of regulatory T cell-associated markers including CTLA4 (1.8-fold), Foxp3 (2.6-fold), TGF-ß (2.8-fold), IL10 (2.9-fold), and reduction in lipids, macrophage infiltration, and expression of inflammatory mediators in aorta. CONCLUSIONS: Our results suggest that M. smegmatis can be developed as an oral carrier of recombinant proteins to treat inflammatory autoimmune diseases.
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Antígenos/administración & dosificación , Enfermedades de la Aorta/prevención & control , Apolipoproteínas E/deficiencia , Aterosclerosis/prevención & control , Vectores Genéticos , Inmunoterapia/métodos , Mycobacterium smegmatis/genética , Oligopéptidos/administración & dosificación , Administración Oral , Animales , Antígenos/genética , Antígenos/inmunología , Antígenos/metabolismo , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/inmunología , Enfermedades de la Aorta/metabolismo , Apolipoproteínas E/genética , Aterosclerosis/genética , Aterosclerosis/inmunología , Aterosclerosis/metabolismo , Antígeno CTLA-4/inmunología , Antígeno CTLA-4/metabolismo , Modelos Animales de Enfermedad , Femenino , Factores de Transcripción Forkhead/inmunología , Factores de Transcripción Forkhead/metabolismo , Predisposición Genética a la Enfermedad , Tolerancia Inmunológica , Inmunización , Mediadores de Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Interleucina-10/inmunología , Interleucina-10/metabolismo , Metabolismo de los Lípidos , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Mycobacterium smegmatis/inmunología , Mycobacterium smegmatis/metabolismo , Oligopéptidos/genética , Oligopéptidos/inmunología , Oligopéptidos/metabolismo , Fenotipo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Factores de Tiempo , Factor de Crecimiento Transformador beta/inmunología , Factor de Crecimiento Transformador beta/metabolismo , Vacunas Sintéticas/administración & dosificaciónRESUMEN
The objective of the study was to gain molecular insights into the progression of atherosclerosis in Apob(tm2Sgy)Ldlr(tm1Her) mice, using transcriptome profiles. Weighted gene co network analysis (WGCNA) and time course analysis using limma were used to study disease progression from 0 to 20weeks. Five co-expression modules were identified by WGCNA using the expression values of 2153 genes. Genes associated with autophagy, endoplasmic reticulum stress, inflammation and lipid metabolism were differentially expressed at early stages of atherosclerosis. Time course analysis highlighted activation of inflammatory gene signaling at 4weeks, cell proliferation and calcification at 8weeks, amyloid like structures and oxidative stress at 14weeks and enhanced production of inflammatory cytokines at 20weeks. Our results suggest that maximum gene perturbations occur during early atherosclerosis which could be the danger signals associated with subclinical disease. Understanding these genes and associated pathways can help in improvement of diagnostic and therapeutic targets for atherosclerosis.
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Apolipoproteínas B/genética , Aterosclerosis/genética , Inflamación/genética , Receptores de LDL/efectos de los fármacos , Animales , Aterosclerosis/patología , Autofagia/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Estrés del Retículo Endoplásmico/genética , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Inflamación/patología , Metabolismo de los Lípidos/genética , Ratones , Ratones Noqueados , Estrés Oxidativo/genéticaRESUMEN
BACKGROUND: Inflammatory immune response to atherogenic self-antigens plays an important role in the development of atherosclerosis. We evaluated the role of oral tolerance to three peptides in controlling atherosclerosis in New Zealand white rabbits. METHODS: Peptides derived from apolipoprotein B (ApoB), heat shock protein 60, and outer membrane protein from Chlamydia pneumoniae were expressed as part of the dendroaspin protein scaffold (AHC). Groups of 3-month-old rabbits were dosed orally with purified AHC protein either before the onset of disease or 2 months after inducing atherosclerosis; they were euthanized at the age of 7 months to study disease development and progression. RESULTS: Oral treatment with AHC resulted in a marked increase in regulatory T cells in the lymphoid organs and reduced the development and progression of atherosclerosis by 48.6% and 28.4%, respectively (P < 0.05). Oral tolerance decreased plaque inflammation, enhanced expression of anti-inflammatory and regulatory markers in the aorta, and attenuated the adaptive immune response to self-antigens. AHC treatment in rabbits with established disease significantly decreased vascular cell adhesion molecule 1 (VCAM-1) (6.2 fold) and monocyte chemoattractant protein-1(MCP-1) (3 fold) expression and reduced the infiltration of macrophages into the aorta. Collagen content and the smooth muscle cell-to-macrophage ratio were higher in treated animals, whereas markers of plaque vulnerability, including matrix metalloproteinase expression, were reduced. CONCLUSIONS: Our results suggest that oral tolerance to multiantigenic AHC molecule restores the immune balance and induces markers of plaque stability in rabbits.
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Apolipoproteínas B/administración & dosificación , Aterosclerosis/inmunología , Proteínas de la Membrana Bacteriana Externa/administración & dosificación , Chaperonina 60/administración & dosificación , Fragmentos de Péptidos/administración & dosificación , Inmunidad Adaptativa , Animales , Aorta/citología , Aterosclerosis/patología , Quimiocina CCL2/metabolismo , Chlamydophila pneumoniae , Colágeno/metabolismo , Citocinas/sangre , Progresión de la Enfermedad , Venenos Elapídicos , Glutatión Sintasa/administración & dosificación , Tolerancia Inmunológica/inmunología , Ganglios Linfáticos/citología , Macrófagos/metabolismo , Miocitos del Músculo Liso/citología , Placa Aterosclerótica/inmunología , Placa Aterosclerótica/patología , Conejos , Bazo/citología , Linfocitos T/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Inflammatory immune response to self-antigens plays an important role in the development of atherosclerosis. Restoring immune tolerance to self-proteins reduces the pro-inflammatory response. We previously showed that oral tolerance to a combination of two peptides is atheroprotective. In the present study we expressed epitopes from apolipoprotein B 100 (ApoB), human heat shock protein (HSP60) and Chlamydia pneumonia outer membrane protein (Cpn) in a single protein scaffold and used this multi-antigenic construct to induce tolerance to individual peptides by oral route in ApoBtm2Sgy/Ldlrtm1Her/J mice. Antigen specific tolerance to individual peptides was observed in treated animals as seen by an increase in regulatory T cells. Tolerance to the peptides resulted in a 46.5% (p=0.002) reduction in the development of atherosclerosis compared with control. Atheroprotection was associated with a significant (p<0.05) decrease in plaque inflammation and an increase in the expression of immune regulatory markers in the aorta. CD11c+ cells coexpressing CD11b and CD103 increased in lymphoid organs and were found to activate regulatory T cells and reduce effector T-cell response. Adoptive transfer of CD11c+ cells was atheroprotective. Our results suggest that atheroprotection by oral tolerance to a multi-antigenic construct is mediated by antigen specific regulatory T cells and CD11c+ cells with immune regulatory properties.