RESUMO
Patients with tear dysfunction often experience increased irritation symptoms when subjected to drafty and/or low humidity environmental conditions. The purpose of this study was to investigate the effects of low humidity stress (LHS) on corneal barrier function and expression of cornified envelope (CE) precursor proteins in the epithelium of C57BL/6 and c-jun N-terminal kinase 2 (JNK2) knockout (KO) mice. LHS was induced in both strains by exposure to an air draft for 15 (LHS15D) or 30 days (LHS30D) at a relative humidity <30%RH. Nonstressed (NS) mice were used as controls. Oregon-green-dextran uptake was used to measure corneal barrier function. Levels of small proline-rich protein (SPRR)-2, involucrin, occludin, and MMP-9 were evaluated by immunofluorescent staining in cornea sections. Wholemount corneas immunostained for occludin were used to measure mean apical cell area. Gelatinase activity was evaluated by in situ zymography. Expression of MMP, CE and inflammatory cytokine genes was evaluated by qPCR. C57BL/6 mice exposed to LHS15D showed corneal barrier dysfunction, decreased apical corneal epithelial cell area, higher MMP-9 expression and gelatinase activity and increased involucrin and SPRR-2 immunoreactivity in the corneal epithelium compared to NS mice. JNK2KO mice were resistant to LHS-induced corneal barrier disruption. MMP-3,-9,-13, IL-1α, IL-1ß, involucrin and SPRR-2a RNA transcripts were significantly increased in C57BL/6 mice at LHS15D, while no change was noted in JNK2KO mice. LHS is capable of altering corneal barrier function, promoting pathologic alteration of the TJ complex and stimulating production of CE proteins by the corneal epithelium. Activation of the JNK2 signaling pathway contributes to corneal epithelial barrier disruption in LHS.
Assuntos
Síndromes do Olho Seco/enzimologia , Epitélio Corneano/enzimologia , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Estresse Fisiológico , Animais , Transporte Biológico , Proteínas Ricas em Prolina do Estrato Córneo/genética , Proteínas Ricas em Prolina do Estrato Córneo/metabolismo , Dessecação , Síndromes do Olho Seco/patologia , Epitélio Corneano/patologia , Técnica Indireta de Fluorescência para Anticorpo , Umidade , Aparelho Lacrimal/fisiologia , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 9 Ativada por Mitógeno/genética , Ocludina , Compostos Orgânicos/metabolismo , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Novel approaches circumventing blood-ocular barriers in systemic drug delivery are lacking. We hypothesize receptor-mediated delivery of curcumin (CUR) across intestinal and ocular barriers leads to decreased inflammation in a model of lens-induced uveitis. CUR was encapsulated in double-headed polyester nanoparticles using gambogic acid (GA)-coupled polylactide-co-glycolide (PLGA). Orally administered PLGA-GA2-CUR led to notable aqueous humor CUR levels and was dosed (10 mg/kg twice daily) to adult male beagles (n = 8 eyes) with induced ocular inflammation. Eyes were evaluated using a semiquantitative preclinical ocular toxicology scoring (SPOTS) and compared to commercial anti-inflammatory treatment (oral carprofen 2.2 mg/kg twice daily) (n = 8) and untreated controls (n = 8). PLGA-GA2-CUR offered improved protection compared with untreated controls and similar protection compared with carprofen, with reduced aqueous flare, miosis, and chemosis in the acute phase (<4 hours). This study highlights the potential of PLGA-GA2 nanoparticles for systemic drug delivery across ocular barriers.
Assuntos
Curcumina , Nanopartículas , Uveíte , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Curcumina/farmacologia , Cães , Portadores de Fármacos , Inflamação/tratamento farmacológico , Masculino , Uveíte/tratamento farmacológico , Uveíte/etiologiaRESUMO
In both humans and animal models, the development of Sjögren syndrome (SS) and non-SS keratoconjunctivitis sicca (KCS) increases with age. Here, we investigated the ocular surface and lacrimal gland (LG) phenotype of NOD.B10.H2b mice at 7-14, 45-50, and 96-100 weeks. Aged mice develop increased corneal permeability, CD4+ T-cell infiltration, and conjunctival goblet cell loss. Aged mice have LG atrophy with increased lymphocyte infiltration and inflammatory cytokine levels. An increase in the frequency of CD4+Foxp3+ T regulatory cells (Tregs) was observed with age in the cervical lymph node (CLN), spleen, and LG. These CD4+CD25+ cells lose suppressive ability, while maintaining expression of Foxp3 (forkhead box P3) and producing interleukin-17 (IL-17) and interferon-γ (IFN-γ). An increase of Foxp3+IL-17+ or Foxp3+IFN-γ+ cells was observed in the LG and LG-draining CLN. In adoptive transfer experiments, recipients of either purified Tregs or purified T effector cells from aged donors developed lacrimal keratoconjunctivitis, whereas recipients of young Tregs or young T effector cells failed to develop disease. Overall, these results suggest inflammatory cytokine-producing CD4+Foxp3+ cells participate in the pathogenesis of age-related ocular surface disease.
Assuntos
Envelhecimento/imunologia , Olho/imunologia , Ceratoconjuntivite Seca/imunologia , Aparelho Lacrimal/imunologia , Síndrome de Sjogren/imunologia , Linfócitos T Reguladores/imunologia , Animais , Movimento Celular , Células Cultivadas , Modelos Animais de Doenças , Olho/patologia , Fatores de Transcrição Forkhead/metabolismo , Humanos , Tolerância Imunológica , Interferon gama/metabolismo , Interleucina-17/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Linfócitos T Reguladores/transplanteRESUMO
PurposeTo develop a hyaluronan hydrogel scaffold-based xeno-free culture system for ex vivo cultivation of human corneal epithelial stem cells (CESCs).Patients and MethodsCESCs were cultivated from donor limbal explants on the HyStem-C Hydrogel bio-scaffold in 12-well plates for 3 weeks. Group A used the traditional supplemented hormonal epidermal medium (SHEM) and group B used the defined SHEM (without fetal bovine serum and toxin A, adding 20% serum replacement). The growth and morphology of the cultured cells were assessed by phase contrast microscope. The expressions of specific cell markers were assessed by immunofluorescence staining and quantitative real-time PCR (qRT-PCR).ResultsSuccessful cultures of CESCs were obtained in both groups, resulting in multilayered stratified epithelia. Comparing to group A, the cells in group B was grown slightly slower and formed less cellular layers at the end of culture. The corneal specific cytokeratin (K) 12 and differentiation markers, involucrin, and connexin 43, were mainly expressed in the superficial cellular layers in both groups. Interestingly, certain basal cells were immune-positive to proposed stem cell markers such as K19, ABCG2, and integrin ß1 in both groups. There was no significant difference between the two groups with regard to the gene expression levels of all these selected corneal markers (all P>0.05).ConclusionsThe hyaluronan hydrogel scaffold-based xeno-free culture system may support the expansion of regenerative CESCs without the risk of xeno component contamination. The regenerated epithelium maintains similar characteristics of native corneal epithelium.
Assuntos
Epitélio Corneano/citologia , Ácido Hialurônico/farmacologia , Hidrogel de Polietilenoglicol-Dimetacrilato/farmacologia , Limbo da Córnea/citologia , Células-Tronco/citologia , Alicerces Teciduais , Idoso , Técnicas de Cultura de Células/métodos , Células Cultivadas , Conexina 43/biossíntese , Conexina 43/genética , Meios de Cultivo Condicionados , Epitélio Corneano/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Queratina-12/biossíntese , Queratina-12/genética , Limbo da Córnea/metabolismo , Masculino , Pessoa de Meia-Idade , Precursores de Proteínas/biossíntese , Precursores de Proteínas/genética , RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/metabolismoRESUMO
PURPOSE: In primary Sjögren's syndrome (SS), ocular surface changes within the conjunctival epithelium include lymphocytic infiltration, squamous cell metaplasia, and a reduction in goblet cell number. These changes may be the simple result of increased mechanical abrasion secondary to dryness. Alternatively, they may represent a local response to ocular and/or systemic inflammatory processes, perhaps in response to Epstein-Barr viral (EBV) infection, an agent recently implicated in the etiology of SS. To determine whether inflammatory processes or local infection by EBV contribute to the ocular surface pathology of SS, we examined the expression of inflammatory cell surface markers, cytokines, and EBV gene products within the ocular conjunctiva of patients with SS. METHODS: Ocular conjunctival tissue was isolated from patients with primary SS and nondry eye control patients by impression cytology or direct biopsy. These specimens were examined by immunofluorescence microscopy and reverse-transcriptase polymerase chain reaction (RT-PCR) for the expression of various markers. RESULTS: The authors found the frequency of expression of HLA-DR (P < 0.0001), ICAM-1 (P < 0.035), and IL-6 (P < 0.0001) to be significantly elevated in patients with primary SS versus nondry eye control patients. The IL-2 receptor and cytokines IL-1 beta and IL-8 were each found to be expressed with relatively high frequency in both patient populations, whereas mRNAs encoding cytokines IL-2, IFN-gamma, GM-CSF, and TGF-beta were not reproducibly detectable in either population. Messenger RNA encoding a marker for passive-latent EBV infection (EBNA-1) was detected with high frequency in both SS and normal populations. The EBV IL-10 analog BCRF-1 was expressed with low frequency in the SS population; however, these levels were not significantly different from the control population. The expression of two other markers of EBV infection, latent membrane protein (LMP, a lytic and latent marker), and BZLF-1 (putative latent-lytic switch gene) was undetectable in either study population. CONCLUSION: Based on the increased expression of the cell surface molecules HLA-DR and ICAM-1, and the inflammatory cytokine IL-6, the authors propose that local inflammatory processes contribute to the ocular surface changes and ocular surface dryness associated with primary SS.
Assuntos
Túnica Conjuntiva/microbiologia , Citocinas/análise , Herpesvirus Humano 4/isolamento & purificação , Síndrome de Sjogren/microbiologia , Sequência de Bases , Túnica Conjuntiva/imunologia , Citocinas/genética , Primers do DNA , Epitélio/imunologia , Epitélio/microbiologia , Infecções Oculares Virais/imunologia , Infecções Oculares Virais/microbiologia , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/microbiologia , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/imunologia , Humanos , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , RNA Mensageiro/análise , Síndrome de Sjogren/imunologiaRESUMO
PURPOSE: To compare the expression of the pro- and anti-inflammatory forms of interleukin (IL)-1 in the tear fluid and conjunctival epithelium of normal eyes and those with dry-eye disease. METHODS: The concentrations of IL-1 alpha, IL-1 beta (precursor and mature forms), and IL-1 receptor antagonist (IL-1Ra) were measured by ELISA in tear fluid samples obtained from normal individuals and patients with dry eye who had rosacea-associated meibomian gland disease (MGD) or Sjögren's syndrome (SS) aqueous tear deficiency (ATD). These cytokines were also measured in normal tear fluid before and after nasal stimulation to induce reflex tearing. The relative expression of these cytokines was evaluated in conjunctival impression cytology specimens and conjunctival biopsy tissue obtained from normal subjects and SS ATD-affected patients using immunofluorescent staining. Matrix metalloproteinase (MMP)-9 concentration and activity in the tear fluid were evaluated with gelatin zymography and with an MMP-9 activity assay kit, respectively. RESULTS: Compared with normal subjects, the concentration of IL-1 alpha and mature IL-1 beta in the tear fluid was increased, and the concentration of precursor IL-1 beta was decreased in patients with MGD (P < 0.05, P = 0.02, and P < 0.01, respectively) and SS ATD (P < 0.001, P = 0.02, and P < 0.001, respectively). There was no significant change in the concentration of IL-1 alpha, precursor IL-1 beta, and IL-1Ra in reflex tear fluid, indicating that the lacrimal glands may secrete these cytokines. The activity of MMP-9, a physiological activator of IL-1 beta, was significantly elevated in the tear fluid of both dry-eye groups compared with normal subjects. A strong positive correlation was observed between the intensity of corneal fluorescein staining and the tear fluid IL-1 alpha concentration (r(2) = 0.17, P < 0.02) and the mature-to-precursor IL-1 beta ratio (r(2) = 0.46, P < 0.001). Positive immunofluorescent staining for IL-1 alpha, mature IL-1 beta, and IL-1Ra was observed in a significantly greater percentage of conjunctival cytology specimens from eyes with SS ATD than in those from normal eyes (P < 0.01 for IL-1 alpha, P < 0.009 for mature IL-1 beta, and P < 0.05 for IL-1Ra). CONCLUSIONS: Dry-eye disease is accompanied by an increase in the proinflammatory forms of IL-1 (IL-1 alpha and mature IL-1 beta) and a decrease in the biologically inactive precursor IL-1 beta in tear fluid. Increased protease activity on the ocular surface may be one mechanism by which precursor IL-1 beta is cleaved to the mature, biologically active form. The conjunctival epithelium appears to be one source of the increased concentration of IL-1 in the tear fluid of patients with dry-eye disease. These results suggest that IL-1 may play a key role in the pathogenesis of keratoconjunctivitis sicca.
Assuntos
Túnica Conjuntiva/metabolismo , Síndromes do Olho Seco/metabolismo , Proteínas do Olho/metabolismo , Interleucina-1/metabolismo , Sialoglicoproteínas/metabolismo , Lágrimas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Síndromes do Olho Seco/patologia , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Fluorofotometria , Humanos , Proteína Antagonista do Receptor de Interleucina 1 , Lactoferrina/metabolismo , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Pessoa de Meia-IdadeRESUMO
The human lacrimal gland (LG) and ocular surface contain discrete regions of epithelial cells with specific functions and at different stages of cellular differentiation. Epithelial cells contain cytoskeletal antigens that show a differentiation-dependent pattern of expression. The objective of this study was to characterize the various epithelial cell populations in normal human ocular mucosa-associated lymphoid tissue (MALT; LG, conjunctiva, and cornea) based on their immunohistochemical staining patterns with anticytoskeletal monoclonal antibodies (MoAbs) reactive with cytokeratins (AE-1, AE-2, AE-3, AE-5, AE-14, PKK1, and 34 beta E12), muscle-specific actin (HHF35), and vimentin. AE-1 stained LG (acini, ducts, and myoepithelia) and the full thickness of corneal and conjunctival epithelia. It stained only the superficial and basal limbal epithelium. AE-2 weakly stained all epithelia, except LG acini and proximal intralobular ducts. AE-3 and 34 beta E12 MoAbs had strong immunoreactivity with all MALT epithelia. AE-5 strongly stained the inner cells (suprabasal) of LG central intra- and interlobular ducts and the suprabasal epithelial layers of the cornea. It weakly stained LG myoepithelia and the superficial conjunctival epithelium. AE-14 stained the outer (basal) cells of LG central intra- and interlobular ducts, LG myoepithelia, basal epithelial layers of the limbus and conjunctiva, and all corneal epithelia. PKK1 stained all epithelia, except the basal limbus. HHF35 and the antivimentin MoAbs stained only the LG myoepithelia. The results of these studies indicate that the different epithelia in human ocular MALT may be differentiated by specific patterns of immunoreactivity with anticytoskeletal MoAbs. These MoAbs may be useful molecular markers for identifying ocular MALT epithelia.
Assuntos
Túnica Conjuntiva/metabolismo , Córnea/metabolismo , Proteínas do Citoesqueleto/análise , Aparelho Lacrimal/metabolismo , Anticorpos Monoclonais , Biomarcadores , Túnica Conjuntiva/imunologia , Córnea/imunologia , Epitélio/química , Humanos , Técnicas Imunoenzimáticas , Aparelho Lacrimal/imunologia , Tecido Linfoide/imunologia , Tecido Linfoide/metabolismo , Mucosa/imunologia , Mucosa/metabolismoRESUMO
PURPOSE: To correlate tear fluorescein clearance with interleukin-1alpha (IL-1alpha) concentration and gelatinase B (matrix metalloproteinase [MMP]-9) activity in the tear fluid of patients with ocular rosacea and normal control subjects. METHODS: Gelatinase activity was evaluated by gelatin zymography in tear fluid obtained from 13 patients with ocular rosacea (including 1 patient with recurrent epithelial erosion, 2 with recurrent peripheral corneal infiltrates and vascularization, and 2 patients with epithelial basement membrane dystrophy) and 13 normal subjects with normal aqueous tear production and no irritation symptoms. Tear fluorescein clearance was evaluated by measuring fluorescence in tear fluid collected from the inferior meniscus 15 minutes after instillation of 5 microl of 2% Na-fluorescein with a CytoFluor II fluorometer. Pro-MMP-9 and IL-1alpha concentrations in the tear fluid were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Compared with normal control subjects, patients with ocular rosacea had a greater delay of tear fluorescein clearance (P < 0.001), a higher tear IL-1alpha concentration (P < 0.001), and a greater pro-gelatinase B (92 kDa) activity (P < 0.001) in their tear fluid. The 84-kDa active form of gelatinase B was observed in 46% of the rosacea tear samples and none of the controls. The zymographic results were confirmed by ELISA that showed a significantly greater concentration of pro-MMP-9 (92 kDa) in the tear fluid of rosacea patients than controls. Delayed tear clearance was correlated with elevated tear IL-1alpha concentration (p=0.67, P < 0.001) and increased tear gelatinase B activity (p=0.84, P < 0.001). Tear IL-1alpha concentration was correlated with tear gelatinase B activity (p=0.58, P < 0.002). CONCLUSIONS: Gelatinase B (MMP-9) activity is greater in patients with ocular rosacea than in normal eyes. The majority of this activity is due to 92-kDa proform of this enzyme. This activity is correlated with delayed tear clearance and tear fluid concentration of interleukin-1alpha, a proinflammatory cytokine that has been reported to stimulate gelatinase B production. Elevated gelatinase B activity in ocular rosacea may be involved in the pathogenesis of the irritation symptoms, recurrent epithelial erosions, vascularization, and epithelial basement membrane dystrophy that develops in the corneas of patients with this condition.
Assuntos
Colagenases/metabolismo , Doenças da Túnica Conjuntiva/metabolismo , Doenças da Córnea/metabolismo , Fluoresceína/farmacocinética , Interleucina-8/metabolismo , Rosácea/metabolismo , Lágrimas/enzimologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Ensaio de Imunoadsorção Enzimática , Feminino , Fluorofotometria , Humanos , Masculino , Metaloproteinase 9 da Matriz , Pessoa de Meia-IdadeRESUMO
PURPOSE: To evaluate the effect of doxycycline on the regulation of interleukin (IL)-1 expression and activity in human cultured corneal epithelium. METHODS: Human corneal limbal epithelium (HLE) was cultured from explants prepared from limbal rings of donor corneas. Primary cultured limbal epithelial cells were treated with either 10 microg/ml lipopolysaccharide (LPS), LPS with 10 microg/ml doxycycline, or LPS with 0.1 mg/ml methylprednisolone (MP) for 24 hours. The intracellular and supernatant protein amounts of IL-1alpha, the precursor and mature forms of IL-1beta, IL-1 receptor antagonist (IL-1 RA), and the intracellular level of IL-1beta-converting enzyme (ICE) were measured with enzyme-linked immunosorbent assays (ELISAs). Western blot analysis was performed to evaluate IL-1 RA protein. mRNA steady state amounts were determined by RNase protection assay (RPA) for IL-1alpha, IL-1beta, IL-1 RA, and ICE. RESULTS: LPS increased the mRNA and protein amounts of intracellular and released IL-1alpha, mature IL-1beta, and IL-1 RA. Doxycycline inhibited the LPS-induced IL-1beta increase in the mRNA and protein amounts in the corneal epithelium and upregulated the expression of the anti-inflammatory IL-1 RA protein. In addition, doxycycline reduced the steady state level of the cellular ICE protein but did not affect the level of ICE transcripts. IL-1beta secreted to the conditioned media of HLE was functionally active in inducing matrix metalloproteinase (MMP)-1 and MMP-3 in cultured corneal fibroblasts. Doxycycline significantly decreased IL-1beta bioactivity in the supernatants from LPS-treated corneal epithelial cultures. These effects were comparable to those induced by the corticosteroid, MP. CONCLUSIONS: Doxycycline can suppress the steady state amounts of mRNA and protein of IL-beta and decrease the bioactivity of this major inflammatory cytokine. These data may partially explain the clinically observed anti-inflammatory properties of doxycycline. The observation that doxycycline was equally potent as a corticosteroid, combined with the relative absence of adverse effects, makes it a potent drug for a wide spectrum of ocular surface inflammatory diseases.
Assuntos
Antibacterianos/farmacologia , Doxiciclina/farmacologia , Epitélio Corneano/efeitos dos fármacos , Interleucina-1/antagonistas & inibidores , Western Blotting , Caspase 1/genética , Caspase 1/metabolismo , Células Cultivadas , Primers do DNA/química , Combinação de Medicamentos , Ensaio de Imunoadsorção Enzimática , Epitélio Corneano/citologia , Epitélio Corneano/metabolismo , Glucocorticoides/farmacologia , Humanos , Proteína Antagonista do Receptor de Interleucina 1 , Interleucina-1/genética , Interleucina-1/metabolismo , Limbo da Córnea/citologia , Lipopolissacarídeos/farmacologia , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Metilprednisolona/farmacologia , RNA Mensageiro/metabolismo , Serratia marcescens , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismoRESUMO
PURPOSE: To evaluate human corneal epithelial culture supernatant and tear fluid for the presence of activators and inhibitors of matrix metalloproteinase (MMP)-9, MMP-3, and tissue inhibitor of metalloproteinase (TIMP)-1, respectively, and to evaluate the effect of MMP-3 on the activation of MMP-9 in these specimens. METHODS: Unstimulated tear fluid was collected from patients with ocular rosacea and normal control subjects. Levels of MMP-9, MMP-3, and TIMP-1 were determined by enzyme-linked immunosorbent assay (ELISA) and/or immunoblot analysis. Supernatants from primary human corneal epithelial cultures and human tear fluid were incubated with MMP-3. Cultured epithelial cells and their supernatants were also treated with doxycycline before MMP-3 was added. Gelatin zymography was used to identify activated 82-kDa MMP-9. MMP-9 activity was assessed with a commercial MMP-9 activity assay system. RESULTS: MMP-9 and TIMP-1 were detected at significantly higher concentrations in rosacea-affected than in normal tear fluids. MMP-3 was detected exclusively in the tear fluid of patients with ocular rosacea who had corneal epithelial disease. Treatment of the supernatant and tear fluid with MMP-3 resulted in two bands with molecular weights of 92 kDa and 82 kDa, representing pro-MMP-9 and activated MMP-9, respectively. Doxycycline added to the conditioned media did not affect activation of MMP-9 by MMP-3. However, 24-hour treatment of corneal epithelial cultures with doxycycline resulted in a lower concentration and activity of MMP-9 in their supernatants. CONCLUSIONS: MMP-9 and TIMP-1 are produced by the human corneal epithelium and are present in tear fluid. MMP-3 alone is sufficient to activate MMP-9 on the ocular surface. Doxycycline does not directly inhibit this activation by MMP-3, but it decreases MMP-9 activity when added to corneal epithelial cultures.
Assuntos
Epitélio Corneano/enzimologia , Metaloproteinase 9 da Matriz/metabolismo , Lágrimas/enzimologia , Western Blotting , Células Cultivadas , Doxiciclina/farmacologia , Ativação Enzimática/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Epitélio Corneano/efeitos dos fármacos , Epitélio Corneano/patologia , Doenças Palpebrais/enzimologia , Doenças Palpebrais/etiologia , Humanos , Metaloproteinase 3 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/farmacologia , Glândulas Tarsais/enzimologia , Glândulas Tarsais/patologia , Rosácea/complicações , Rosácea/enzimologia , Inibidor Tecidual de Metaloproteinase-1/metabolismoRESUMO
PURPOSE: To evaluate human ocular surface epithelium and tear fluid for the presence of sialomucin complex (MUC4), a high-molecular-weight heterodimeric glycoprotein composed of mucin (ASGP-1) and transmembrane (ASGP-2) subunits. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analysis assays were used to identify sialomucin complex RNA in ocular surface epithelia. Immunoprecipitation and immunoblot analysis were used to identify immunoreactive species in human tears and in the corneal and conjunctival epithelia using antibodies specific for carbohydrate and peptide epitopes on the sialomucin complex subunits. Immunofluorescence staining was used to detect sialomucin complex in frozen sections and impression cytology specimens of human cornea and conjunctival epithelia. RESULTS: ASGP-1- and ASGP-2-specific sequences were amplified from RNA extracted from both conjunctival and corneal epithelial biopsies by RT-PCR. Sialomucin complex transcripts were also detected in these tissues by Northern blot analysis, with a greater level of RNA detected in the peripheral than the central corneal epithelium. Sialomucin complex was immunoprecipitated from tear fluid samples and both corneal and conjunctival epithelia and detected by immunoblot analysis with specific anti-ASGP-1 and anti-ASGP-2 antibodies. The ASGP-1 peptide antibody HA-1 stained the full thickness of the corneal and conjunctival epithelia. In contrast, antibody 15H10, which reacts against a carbohydrate epitope on ASGP-1, stained only the superficial epithelial layers of these tissues. No staining was observed in the conjunctival goblet cells. CONCLUSIONS: Sialomucin complex was originally identified in rat mammary adenocarcinoma cells and has recently been shown to be produced by the ocular surface epithelia of rats. Furthermore, it has been identified as the rat homologue of human MUC4 mucin. The present studies show that it is expressed in the stratified epithelium covering the surface of the human eye and is present in human tear fluid. Expression of a carbohydrate-dependent epitope on the mucin subunit (ASGP-1) of sialomucin complex occurs in a differentiation-dependent fashion. Sialomucin complex joins MUC1 as another membrane mucin produced by the human ocular surface epithelia but is also found in the tear fluid, presumably in a soluble form, as found on the rat ocular surface.
Assuntos
Túnica Conjuntiva/química , Epitélio Corneano/química , Mucinas/análise , Lágrimas/química , Especificidade de Anticorpos , Northern Blotting , Epitélio/química , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Mucina-4 , Mucinas/genética , RNA/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sialoglicoproteínas/análiseRESUMO
Patients with human immunodeficiency virus infection are predisposed to fungal, parasitic, and viral infections. Bacterial infection can also be seen, although ocular bacterial infections have not been reported in patients with acquired immunodeficiency syndrome until recently. We present two cases of Pseudomonas corneoscleritis and one case of Pseudomonas keratitis in patients with human immunodeficiency virus infection that failed to respond to antibiotic treatment. Predisposing factors included extended-wear soft contact lens use in one patient and exposure secondary to Bell's palsy in another patient. All three patients had neutropenia that may have contributed to their poor response to treatment. Enucleation was required to treat two patients with overwhelming infection. Enucleation has been rarely required for treatment of corneoscleritis in immunocompetent patients treated at our institution. Pseudomonas keratitis in human immunodeficiency virus-infected patients represents a serious ocular infection requiring early diagnosis and aggressive treatment.
Assuntos
Infecções Oculares Bacterianas/complicações , Infecções por HIV/complicações , Ceratite/microbiologia , Infecções por Pseudomonas/complicações , Esclerite/microbiologia , Síndrome da Imunodeficiência Adquirida/complicações , Adulto , Lentes de Contato/efeitos adversos , Enucleação Ocular , Paralisia Facial/complicações , Humanos , Masculino , Pessoa de Meia-Idade , PrognósticoRESUMO
We describe a technique to accurately align the circumferences of the donor button and host cornea during penetrating keratoplasty. The donor cornea is trephined on a block into which four narrow, intersecting, equally spaced radial cuts are made. Suction is applied to the donor cornea through the four cuts, resulting in four radial marks in the donor corneal epithelium. Four radial marks are also made in the host cornea before trephination, using a radial keratotomy marker. When the donor button is placed within the recipient opening, the radial marks are aligned and used as guides for the cardinal sutures. This simple technique allows for matching of the donor and host circumferences, even if there is some collapse of the peripheral host cornea and sclera at surgery, and should minimize astigmatism resulting from donor/host misalignment.
Assuntos
Transplante de Córnea , Oftalmologia/instrumentação , Instrumentos Cirúrgicos , Astigmatismo/prevenção & controle , Córnea/ultraestrutura , Humanos , Métodos , Complicações Pós-Operatórias/prevenção & controle , Sucção , Doadores de TecidosRESUMO
The medical records of 48 patients with culture-positive streptococcal endophthalmitis diagnosed between January 1977 and May 1990 were reviewed. The viridans group streptococci were isolated in 24 (50%) of the 48 cases, enterococci in 13 cases (27.1%), Streptococcus pneumoniae in six cases (12.5%), and beta-hemolytic streptococci in six (12.5%) of 48 cases. The clinical statuses of endophthalmitis cases by etiology were postoperative in 40 patients (83.3%), posttraumatic in six patients (12.5%), and miscellaneous in two patients (4.2%). Overall, 15 (31.2%) patients achieved 20/400 or better visual acuity. The streptococcal isolates demonstrated a 32.6% in vitro resistance to gentamicin sulfate, whereas all isolates were sensitive to vancomycin hydrochloride. The enterococci were often resistant to the cephalosporins, whereas the other streptococcal species were not.
Assuntos
Endoftalmite/microbiologia , Infecções Oculares Bacterianas , Infecções Estreptocócicas , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Criança , Pré-Escolar , Resistência Microbiana a Medicamentos , Endoftalmite/tratamento farmacológico , Infecções Oculares Bacterianas/tratamento farmacológico , Feminino , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Fenótipo , Complicações Pós-Operatórias , Infecções Estreptocócicas/tratamento farmacológico , Streptococcus/isolamento & purificação , Resultado do Tratamento , VitrectomiaRESUMO
The medical records of 52 patients (53 eyes) with culture-proven gram-negative endophthalmitis between January 1982 and December 1990 were reviewed. Pseudomonas aeruginosa (23% [12/53]) and Haemophilus influenzae (19% [10/53]) were the most frequent isolates in this series. Overall, 26 (49%) of 53 treated patients achieved 20/400 or better visual acuity. Fifty-two (98%) of the original 53 gram-negative isolates were sensitive to the aminoglycoside antibiotics. To determine their sensitivity to recently developed antibiotics, 35 of the isolates were again grown on culture media and their sensitivities to ceftazidime, ciprofloxacin, and imipenem were obtained. Only ceftazidime demonstrated in vitro efficacy for all the organisms tested.
Assuntos
Endoftalmite/microbiologia , Infecções Oculares Bacterianas , Infecções por Bactérias Gram-Negativas , Aminoglicosídeos , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Endoftalmite/tratamento farmacológico , Seguimentos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/isolamento & purificação , Humanos , Testes de Sensibilidade Microbiana , Estudos Retrospectivos , Resultado do Tratamento , Acuidade Visual , VitrectomiaRESUMO
Some of the gram-positive isolates from exogenous bacterial endophthalmitis cases treated at our institution have been found to be resistant to either cefazolin sodium, gentamicin sulfate, or both. However, all of these isolates have been sensitive to vancomycin. These findings prompted us to reevaluate the retinal toxicity and clearance of intravitreal vancomycin in pigmented rabbits. Doses up to 2 mg were found to be nontoxic in phakic and aphakic-vitrectomized eyes. Clearance was determined in phakic and aphakic-vitrectomized rabbit eyes with or without intact lens capsules. The antibiotic was cleared most slowly in phakic eyes. Aphakic-vitrectomized eyes without an intact lens capsule cleared antibiotic most rapidly, while aphakic-vitrectomized eyes with intact capsules exhibited an intermediate clearance rate. In addition, the interaction between vancomycin and gentamicin on gram-positive endophthalmitis isolates was found to be additive or synergistic depending on the bacterial species. Based on these data, we recommend the combination of vancomycin and an aminoglycoside as the initial antibiotic therapy for exogenous bacterial endophthalmitis.
Assuntos
Retina/efeitos dos fármacos , Vancomicina/toxicidade , Animais , Afacia Pós-Catarata/metabolismo , Sinergismo Farmacológico , Quimioterapia Combinada , Endoftalmite/tratamento farmacológico , Gentamicinas/administração & dosagem , Lentes Intraoculares , Epitélio Pigmentado Ocular/efeitos dos fármacos , Epitélio Pigmentado Ocular/ultraestrutura , Coelhos , Retina/metabolismo , Vancomicina/administração & dosagem , Vancomicina/metabolismo , Vitrectomia , Corpo Vítreo/efeitos dos fármacosRESUMO
We used a high-magnification shadow photogrammetric system to evaluate corneal buttons trephined from human donor globes. Whole eyes were trephined with one of five instruments: nonguarded blades held by hand or placed on a handle, Barraquer-Mateus motorized trephine, Hessberg-Barron suction trephine, or Hanna microkeratotrephine. Corneoscleral buttons were punched with one of three punches: Cottingham, Katena Lieberman guillotine, or a modified Lieberman guillotine with an increased impact force (BPEI 2). The precision (how closely buttons approximated the trephine diameter), accuracy (repeatability), acircularity (deviation from roundness), and straightness (verticality of edges) of cut were calculated from the diameter and edge profile angle measurements of buttons cut by the different instruments. These results were statistically compared. The Hanna microkeratotrephine instrument had the greatest precision and accuracy, least acircularity, and straightest edges. Of the corneal punches evaluated, the Cottingham instrument had the greatest precision; however, the BPEI 2 punch cut with the greatest accuracy and the straightest edges.
Assuntos
Córnea/cirurgia , Transplante de Córnea , Fotografação , Manejo de Espécimes , Córnea/anatomia & histologia , Substância Própria/anatomia & histologia , Endotélio Corneano/anatomia & histologia , Epitélio/anatomia & histologia , Humanos , Instrumentos CirúrgicosRESUMO
BACKGROUND: Conjunctival intraepithelial neoplasia has traditionally been found at the limbus in elderly individuals. Recently, this ocular tumor has been observed in younger patients. OBJECTIVE: To investigate the potential association of human immunodeficiency virus infection with the emergence of this atypical presentation of conjunctival intraepithelial neoplasia. DESIGN, SETTING, AND PARTICIPANTS: Records of patients at the Bascom Palmer Eye Institute (Miami, Fla) in whom conjunctival intraepithelial neoplasia was diagnosed between January 1, 1991, and December 31, 1993, were reviewed. Attempts were made to contact those patients younger than 50 years for clinical evaluation and human immunodeficiency virus serologic testing. RESULTS: Conjunctival intraepithelial neoplasia was diagnosed in 73 patients during the study period. Of the nine patients younger than 50 years, six were available for serologic testing. Three (50%) of these individuals were found to be positive for human immunodeficiency virus. CONCLUSION: Human immunodeficiency virus testing and counseling should be considered in patients younger than 50 years in whom conjunctival intraepithelial neoplasia is diagnosed.
Assuntos
Carcinoma in Situ/patologia , Neoplasias da Túnica Conjuntiva/patologia , Infecções por HIV/diagnóstico , Adulto , Biomarcadores , Contagem de Linfócito CD4 , Carcinoma in Situ/complicações , Neoplasias da Túnica Conjuntiva/complicações , Feminino , Infecções por HIV/complicações , Soropositividade para HIV/complicações , Soropositividade para HIV/diagnóstico , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Acuidade VisualRESUMO
Members of the herpesvirus family, cytomegalovirus (CMV), Epstein-Barr virus (EBV), and herpes simplex virus (HSV), have been recognized as causal agents of chorioretinal inflammatory diseases. We investigated the use of the polymerase chain reaction for the detection of CMV, HSV, and EBV genomes in aqueous, subretinal fluid, and vitreous specimens in patients with clinically diagnosed CMV retinitis. Cytomegalovirus but not HSV or EBV genomic sequences were detected in all of these clinical specimens. We also investigated 18 normal aqueous and eight normal vitreous specimens obtained from patients undergoing cataract or vitrectomy surgery. Cytomegalovirus, HSV, and EBV DNA were not detected in any of the normal aqueous specimens. There was one weakly positive CMV normal vitreous, but none was HSV or EBV positive by the polymerase chain reaction. These results indicate that the polymerase chain reaction may be useful as a rapid and sensitive diagnostic technique to aid in the confirmation of clinical observations.
Assuntos
Humor Aquoso/microbiologia , DNA Viral/análise , Herpesviridae/isolamento & purificação , Reação em Cadeia da Polimerase , Corpo Vítreo/microbiologia , Síndrome da Imunodeficiência Adquirida/microbiologia , Anticorpos Antivirais/sangue , Autorradiografia , Citomegalovirus/genética , Citomegalovirus/isolamento & purificação , Infecções por Citomegalovirus/microbiologia , Herpesviridae/genética , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/isolamento & purificação , Humanos , Reação em Cadeia da Polimerase/métodos , Retinite/microbiologia , Simplexvirus/genética , Simplexvirus/isolamento & purificaçãoRESUMO
Although the rate of infectious endophthalmitis following penetrating ocular injury is generally less than 10%, certain settings may carry a greater risk of infection. One such setting is penetrating injury resulting from eating utensils contaminated with oral flora. We reviewed six of these injuries. Culture-positive bacterial endophthalmitis developed in four of the six eyes; only one of the eyes retained reading visual acuity (greater than 20/50) and two eyes lost light perception. The potential for infection and limited visual outcome in this series warrants aggressive prophylaxis and treatment. The unexpected isolation of Haemophilus influenzae in two of the four infections suggests that broad-spectrum antibiotic treatment should be considered in all such injuries since less common organisms may be encountered.