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Biomedical subjects

Michael E Stern

Publications and source records attributed to Michael E Stern.

At least 19 recordsLinked to original sources
PubMed2006

Corticosteroid and doxycycline suppress MMP-9 and inflammatory cytokine expression, MAPK activation in the corneal epithelium in experimental dry eye.

We investigated the effects of corticosteroid and doxycycline on expression of matrix metalloproteinase (MMP)-9 and inflammatory cytokines and activation of mitogen-activated protein kinase (MAPK) signaling pathways, c-jun N-terminal kinases (JNK), extracellular-regulated kinases (ERK) and p38, in experimental murine dry eye. Experimental dry eye (EDE) was created in C57BL6 mice, with or without or topical treatment consisting of 1% methylprednisolone, 0.025% doxycycline or balanced salt solution four times per day. MMP-9 expression in the cornea epithelia was evaluated by laser scanning confocal microscopy. Gelatinase activity in the cornea was evaluated by in situ zymography and MMP-9 activity in tear washings was evaluated by gelatin zymography. Total and phosphorylated MAPKs (JNK1/2, ERK1/2, p38) were detected by Luminex immunobead assay. Levels of MMP-9, interleukin (IL)-1alpha, IL-1beta and tumor necrosis factor (TNF)-alpha RNA transcripts were evaluated by real-time PCR. MMP-9 immunoreactivity was localized to the apical corneal epithelial cell membranes in normal control eyes. Desiccating stress significantly increased production of MMP-9 by the corneal epithelium and increased its activity in the corneal epithelium and tear fluid. Dryness also increased expression of IL-1alpha, IL-1beta and TNF-alpha mRNA and stimulated phosphorylation of JNK1/2, ERK1/2 and p38 MAPKs in the corneal epithelium. Both methylprednisolone and doxycycline reduced expression and activity of MMP-9, decreased levels of inflammatory cytokines transcripts and reduced activation of MAPKs in the corneal epithelium in response to EDE. Desiccating stress stimulates expression of MMP-9, IL-1alpha, IL-1beta and TNF-alpha mRNA , as well as activates MAPK signaling pathways in the corneal epithelium. Both corticosteroid and doxycycline suppressed this molecular stress response.

Animals↗
PubMed2006

Desiccating stress induces T cell-mediated Sjögren's Syndrome-like lacrimal keratoconjunctivitis.

Chronic dry eye syndrome affects over 10 million people in the United States; it is associated with inflammation of the lacrimal gland (LG) and in some cases involves T cell infiltration of the conjunctiva. We demonstrate that environmental desiccating stress (DS) elicits T cell-mediated inflammation of the cornea, conjunctiva, and LG, but not other organs in mice. The lacrimal keratoconjunctivitis (LKC) was mediated by CD4(+) T cells, which, when adoptively transferred to T cell-deficient nude mice, produced inflammation in the LG, cornea, and conjunctiva, but not in any other organ. Adoptively transferred CD4(+) T cells produced LKC even though recipients were not exposed to DS. LKC was exacerbated in euthymic mice depleted of CD4(+)CD25(+)forkhead/winged helix transcription factor(+) regulatory T cells. The results suggest that DS exposes shared epitopes in the cornea, conjunctiva, and LG that induce pathogenic CD4(+) T cells that produce LKC, which under normal circumstances is restrained by CD4(+)CD25(+)forkhead/winged helix transcription factor(+) regulatory T cells.

Animals↗
PubMed2006

Altered expression of neurotransmitter receptors and neuromediators in vernal keratoconjunctivitis.

BACKGROUND: There is growing evidence that autonomic innervation is involved in the pathogenesis of mucus hypersecretion, goblet cell hyperplasia, and conjunctival hyperreactivity. OBJECTIVE: To determine the expression of neurotransmitters and neurotransmitter receptors in vernal keratoconjunctivitis (VKC) tissues to evaluate whether neurogenic inflammation plays a role in this ocular atopic-related disorder. METHODS: Biopsy specimens of upper tarsal conjunctiva from 8 VKC patients with active inflammation and from 4 healthy subjects were processed for immunohistochemistry using anti-M1, anti-M2, and anti-M3 muscarinic receptors; beta1-adrenergic receptor; vasoactive intestinal peptide; nerve growth factor; and protein gene product 9.5, a marker of nerve fibers. RESULTS: In the conjunctival epithelium of VKC patients, M1 muscarinic receptor, nerve growth factor, and protein gene product 9.5 expression were decreased, whereas M2 and M3 muscarinic receptors and beta1-adrenergic receptor were irregularly distributed, compared with control subjects. Neurotransmitter receptors and vasoactive intestinal peptide expression were increased in the substantia propria-localized infiltrate of VKC compared with healthy tissue. Nerve growth factor and protein gene product 9.5 staining was also enhanced in the conjunctival stroma of VKC vs healthy conjunctiva. CONCLUSIONS: The inflamed conjunctiva of VKC patients demonstrated an obvious alteration in muscarinic and beta1-adrenergic receptor, vasoactive intestinal peptide, protein gene product 9.5, and nerve growth factor expression. These results substantiate the involvement of an autonomic dysfunction in the pathogenesis of VKC.

Adolescent↗
PubMed2006

Apical corneal barrier disruption in experimental murine dry eye is abrogated by methylprednisolone and doxycycline.

PURPOSE: To evaluate the mechanism of apical corneal epithelial barrier disruption in response to experimental ocular surface desiccation and the effects of two anti-inflammatory agents (methylprednisolone and doxycycline) on this process. METHODS: Experimental dry eye (EDE) was created in C57BL/6 mice, without or with topical therapy, 1% methylprednisolone, 0.025% doxycycline, or physiologic saline solution (PSS) four times per day. Corneal smoothness and Oregon green dextran (OGD) permeability were assessed. Desquamation of and cornified envelope protein (involucrin and small proline-rich protein [SPRR]-2) expression by the corneal epithelium was evaluated by laser scanning confocal microscopy. Levels of cornified envelope proteins mRNA were measured by real-time PCR. RESULTS: Corneal OGD permeability, surface irregularity, and the number of desquamating apical corneal epithelia significantly increased in EDE. Desiccating stress significantly increased expression of involucrin and SPRR-2 in the corneal epithelia. Treatment of EDE with methylprednisolone or doxycycline reduced corneal permeability to OGD, improved corneal smoothness, and decreased involucrin and SPRR-2 immunoreactivity compared with EDE+PSS. CONCLUSIONS: Disruption of apical corneal epithelial barrier function in dry eye is accompanied by increased apical desquamation and increased expression of cornified envelope proteins. Topical treatment of EDE with the anti-inflammatory agents methylprednisolone or doxycycline preserves apical corneal barrier function.

Animals↗
PubMed2006

Desiccating stress stimulates expression of matrix metalloproteinases by the corneal epithelium.

PURPOSE: To determine the effects of experimental dryness on production of matrix metalloproteinases (MMPs) and their physiological inhibitors (TIMPs) by the corneal epithelium. METHODS: Experimental dry eye (EDE) was created in two strains of mice: BALB/c and C57BL/6. Real-time PCR was performed with MMP and TIMP probes, and the results were analyzed by the comparative C(T) method, selecting the relative mRNA levels in untreated control samples as calibrator. Immunofluorescent staining with specific antibodies immunolocalized MMP proteins in situ. MMP enzymatic activity was evaluated in tears and corneal lysates. Corneal permeability to Oregon green dextran (OGD) and sodium fluorescein was measured. Corneal smoothness was evaluated by graded regularity of a ring reflected off the corneal surface. RESULTS: Desiccating stress significantly increased levels of MMP-1, -3, -9, and -10 transcripts in the corneal epithelium in C57BL/6 mice, but had no effect on levels of MMP transcripts in the corneal epithelium of BALB/c mice. There was no change in levels of TIMP transcripts except for TIMP-4 which significantly increased on day 10 in C57BL/6 mice. The MMP-1, -3, and -9 concentration in tears significantly increased compared with control levels after EDE for 4 and 6 days, respectively, in C57BL/6 and BALB/c. Changes in MMP protein expression detected by immunofluorescent staining were similar to changes in gene transcripts for most MMPs. EDE increased corneal permeability to OGD and fluorescein and corneal surface irregularity. CONCLUSIONS: Corneal dryness stimulates production of certain MMPs in a strain-dependent fashion and causes the disruption of the corneal barrier, thus increasing permeability and corneal irregularity.

Animals↗
PubMed2005

Pathways of corneal and ocular surface inflammation: a perspective from the cullen symposium.

The goal of this symposium was to coalesce information presented by 22 investigators in the field of corneal and ocular surface inflammation into common pathways of inflammation. The perspective elucidated in this article defines the components of the normal ocular surface immune architecture and describes the consensus reached on the mechanisms/pathways involved in 1) acute inflammation; 2) late-stage (chronic) response; and 3) allergic disease. Seven diagrams didactically illustrate mechanisms. This paper is the introductory article in a supplement containing 18 articles by the symposium participants.

Journal Article↗
PubMed2005

Variation in the expression of inflammatory markers and neuroreceptors in human conjunctival epithelial cells.

The purpose of this research was to investigate the role of neurogenic involvement in the etiopatho-genesis of ocular surface inflammation, with the final goal of identifying new potential anti-inflammatory agents. We describe the presence of two "classic" markers of inflammation (HLA-DR and ICAM-1) and some neuroreceptors in cultured human conjunctival epithelial cells under basal and pro-inflammatory conditions. Two markers of inflammation (HLA-DR, ICAM-1) and several neuroreceptor subtypes (M1-, M2-, and M3-muscarinic; alpha1A-, alpha1B-, alpha1D-, alpha2A-, alpha2B-, alpha2C-, beta1-, beta2-, and beta3-adrenergic) were analyzed in a normal human conjunctival epithelial cell line (IOBA-NHC). These markers were studied in basal conditions and under the influence of two pro-inflammatory cytokines: tumor necrosis factor alpha (TNF-alpha) and/or interferon gamma (IFN-gamma). Immunofluorescence (confocal microscopy), western blotting, or flow cytometry techniques were used. In basal conditions, epithelial cells expressed all inflammatory markers except HLA-DR. The addition of IFN-gamma enhanced expression of HLA-DR, ICAM-1, and M2-muscarinic receptor. TNF-alpha up-regulated the expression of ICAM-1. When epithelial cells were incubated in the presence of both cytokines together, the cell surface expression of HLA-DR, ICAM-1, alpha1B-, and alpha2B-adrenergic receptors was increased. Cultured human conjunctival epithelial cells have been shown to be susceptible to up-regulation of the expression of inflammatory markers and cell membrane expression of some neuroreceptors under pro-inflammatory conditions. Consequently, pharmacologic neuro-modulation could have a role in the comprehensive management of ocular surface inflammation.

Journal Article↗
PubMed2005

Stress-activated protein kinase signaling pathways in dry eye and ocular surface disease.

Inflammation is recognized as a key component in the pathogenesis of dry eye and a variety of ocular surface diseases. Stress-activated protein kinases have been identified as pathways signaling ocular surface stresses, such as increased tear film osmolarity and ultraviolet light exposure. Activation of these stress pathways results in transcription of stress related genes, including inflammatory cytokines (e.g. interleukin-1 and tumor necrosis factor (TNF)-alpha and matrix metalloproteinases (MMPs), such as MMP-9. Treatment of osmo-stressed cultured corneal epithelia with inhibitors of the stress associated kinase c-jun N-terminal kinases was found to decrease production of MMPs by these cells. These findings suggest that SAPKs may be key therapeutic targets for dry eye and ocular surface diseases.

Journal Article↗
PubMed2005

Evaluation of ocular surface inflammation in the presence of dry eye and allergic conjunctival disease.

The ocular inflammatory diseases dry eye and allergic conjunctivitis are mediated by CD4+ T cells. Th1 cells secrete interferon (IFN)-gamma and are implicated in mediating the disease process in dry eye. Allergic conjunctivitis has been classically defined as a Th2 disease because of the predominance of Th2 cytokines interleukin (IL)-4 and IL-13. A multi-hit antigen challenge mouse model of allergic conjunctivitis provides evidence that IFN-gamma, a Th1 cytokine, acts as an endothelium gatekeeper by regulating endothelial expression of vascular adhesion molecule-1 required for inflammatory conjunctival cell infiltration. Current research encourages an in-depth evaluation of the exact role Th1 and Th2 cells play in ocular inflammation.

Journal Article↗
PubMed2005

Neurogenic inflammation: a first line of defense for the ocular surface.

Neurogenic inflammation and innate immunity may work together to protect mucosal surfaces, including the ocular surface. When the eye is exposed to pathogens, chemical irritants, or mechanical disruption, neurogenic inflammation is produced through release of neuromodulators, such as substance P and calcitonin gene-related peptide. This leads to the breakdown of the blood-tissue barrier, edema, and release of polymorphonuclear leukocytes into the tears, thus allowing intervention of systemic immunity. Neurogenic mechanisms may have a significant role in the onset and chronicity of ocular surface inflammation in conditions such as dry eye.

Journal Article↗
PubMed2005

Alpha2-adrenergic receptors are present in normal human conjunctiva.

PURPOSE: Despite the passage of medications, including antiglaucoma drugs, through the ocular surface, and despite the increasing relevance of neurogenic inflammation in the ocular surface, the presence of some neuroreceptors in the conjunctiva has not been ascertained. This study describes the presence of alpha2-adrenergic receptors in normal human conjunctiva. METHODS: Immunofluorescence microscopy, electrophoresis, and Western blot analyses were done in human conjunctival biopsies and rat control tissues. Antibodies against alpha2-adrenergic receptor subtypes alpha2A, alpha2B, and alpha2C were used. RESULTS: Immunoreactivity for alpha2A- and alpha2B-, but not alpha2C-adrenergic receptors was evenly distributed in epithelial cells of human conjunctiva cryosections. Immunoreactive bands were detected for the three alpha2-adrenergic receptor subtypes: a major band of 48-50 kDa and fellow bands of 65-67 kDa. CONCLUSIONS: Normal human conjunctival epithelial cells express alpha2A-, alpha2B-, and alpha2C-adrenergic receptors. Further studies to determine the functional implications of these receptors in ocular surface homeostasis are warranted.

Aged↗
PubMed2005

The Th1/Th2 paradigm in ocular allergy.

PURPOSE OF REVIEW: The paradigm that diseases are either Th1 mediated or Th2 mediated has recently been challenged in a number of classical ocular diseases. The objective of this article is to highlight the importance of understanding the exact mechanisms of Th1 and Th2 cells in the pathology of ocular allergy. RECENT FINDINGS: Current research of Th1 and Th2 cytokines in an animal model of ocular allergy demonstrates the intricate complex regulation by both subsets of cytokines of the disease process. Th2 prone BALB/c wild type mice sensitized and topically challenged with short ragweed for seven consecutive days (multi-hit) developed a sustained, chronic conjunctival inflammation. Significantly, IFN-gamma knockout mice in the multi-hit antigen challenge model had a reduced conjunctival cellular infiltrate. Evaluation of adhesion molecules that actively regulate cellular infiltration into the conjunctiva revealed a lack of vascular cell adhesion molecule-1 in multi-hit antigen challenged IFN-gamma knockout mice. SUMMARY: Recent ocular allergy studies question the Th1/Th2 paradigm. These studies encourage further understanding of the intricate interactions of Th1 and Th2 cytokines in ocular inflammatory disease. The following components of Th1 and Th2 cells in the development of chronic inflammation associated with allergic conjunctivitis will be discussed: T helper subsets Th1 and Th2 in ocular inflammation, activation of T cells in the lymph node, and the role of IFN-gamma as the endothelium gatekeeper in the pathology of Th2-mediated allergic conjunctivitis.

Animals↗
PubMed2005

Topical cyclosporine inhibits conjunctival epithelial apoptosis in experimental murine keratoconjunctivitis sicca.

PURPOSE: Increased apoptosis in the conjunctival epithelium has been observed in experimental murine keratoconjunctivitis sicca (KCS). Topical cyclosporine (CsA) has been noted to reduce conjunctival epithelial apoptosis in chronic canine and human KCS. The purpose of this study is to determine if topical CsA treatment inhibits conjunctival epithelial apoptosis in a murine model of KCS. METHODS: Dry eye was induced in 3 groups of C57BL6 mice by subcutaneous injection of scopolamine TID and exposure to an air draft and low-humidity environment for 16 hours per day for 12 days. The dry eye control group received no topical treatment; a second group received 1 microL of 0.05% CsA topically TID (dry eye + CsA); and the third group received 1 microL of the castor oil vehicle of CsA topically TID (dry eye + vehicle). Normal mice were used as untreated controls. After 12 days, the mice were killed, and the right eyes and eyelids were excised, frozen, and cryosectioned. Transmission electron microscopy (TEM) was performed on conjunctival and corneal samples taken from the left eyes. Apoptosis was detected in frozen sections with the ApopTag (ISOL) In Situ Oligo Ligation Kit, which specifically detects DNA fragmentation. Immunohistochemical staining was performed to detect activated caspase-3. Conjunctival goblet cell number was counted in tissue sections stained with period acid Schiff (PAS) reagent. These assays were performed on 2 separate sets of mice. RESULTS: Compared with untreated controls and dry eye mice receiving CsA, the number of ISOL-positive epithelial cells in the bulbar and tarsal conjunctiva was significantly greater in the dry eye control and dry eye mice + vehicle groups (P < 0.01 for both groups). There was no significant difference in the number of ISOL-positive conjunctival epithelial cells between the dry eye control and dry eye + vehicle mice. There was no significant difference in ISOL-positive cells in the corneal epithelium between the untreated controls and the 3 treatment groups. Dry eye + CsA mice showed less activated caspase-3 staining than the dry eye control and the dry eye + vehicle groups. TEM showed loss of superficial differentiated cells and extensive nuclear fragmentation characteristic of apoptosis in the dry eye control and dry eye + vehicle groups but not in the dry eye + CsA group. There was significant loss of goblet cells in the bulbar and tarsal conjunctivae of the dry eye control and the dry eye + vehicle groups compared with untreated controls and the dry eye + CsA group. CONCLUSIONS: Topical CsA significantly reduced conjunctival epithelial apoptosis and protected against goblet cell loss in experimental murine KCS. Inhibition of apoptosis appears to be a key mechanism for the therapeutic effect of CsA for KCS.

Administration, Topical↗
PubMed2005

Expression of muscarinic and adrenergic receptors in normal human conjunctival epithelium.

PURPOSE: To study the presence of muscarinic and alpha- and beta-adrenergic receptors in a normal human conjunctival epithelial (IOBA-NHC) cell line. METHODS: Neurotransmitter receptors were determined in IOBA-NHC cells by flow cytometry, immunofluorescence, and Western blot analysis. Antibodies to M(1)-, M(2)-, and M(3)-muscarinic and to alpha(1A)-, alpha(1B)-, alpha(1D)-, alpha(2A)-, alpha(2B)-, alpha(2C)-, beta(1)-, beta(2)-, and beta(3)-adrenergic receptor subtypes were used. Different culture media were tested, including the addition of tumor necrosis factor (TNF)-alpha and/or interferon (IFN)-gamma. Normal human conjunctiva biopsy specimens and rat tissues were used in control experiments. RESULTS: By immunofluorescence microscopy, all receptor subtypes, except the alpha(2C)-adrenergic receptor, were detected in control biopsy specimens. By flow cytometry, the M(2)- and M(3)-muscarinic receptors and alpha(1A)-, alpha(1B)-, alpha(1D)-, alpha(2A)-, alpha(2B)-, alpha(2C)-, beta(1)-, and beta(3)-adrenergic receptors were detected intracellularly and in cell membranes of the IOBA-NHC cells. M(1)-muscarinic and beta(2)-adrenergic receptors were detected only intracellularly, but were mobilized to the cell membrane when cholera toxin and hydrocortisone were omitted from the culture medium. Confocal microscopy detected the M(2) and M(3)-muscarinic and alpha(1A)-, alpha(2A)-, alpha(2B)-, beta(1)- and beta(2)-adrenergic receptor subtypes. Western blot analyses showed bands for all receptors. M(2)-muscarinic and alpha(1B)- and alpha(2B)-adrenergic receptors expression was upregulated when cells were treated with the proinflammatory cytokines IFN gamma and/or TNF alpha. CONCLUSIONS: The IOBA-NHC cell line maintained expression of the neurotransmitter receptors expressed in normal human conjunctival epithelium. A proinflammatory medium upregulated expression of some receptors. Although the functional state of these receptors is unknown, these findings justify further use of the IOBA-NHC cell line to study the neural component of conjunctival inflammation.

Animals↗
PubMed2005

Role of interferon-gamma in a mouse model of allergic conjunctivitis.

PURPOSE: To characterize the effect of repeated topical exposure to allergen in a mouse model of allergic conjunctivitis and to determine the role of interferon-gamma (IFN-gamma) in the pathogenesis of allergic conjunctivitis. METHODS: Wild-type BALB/c mice and IFN-gamma knockout (KO) BALB/c mice were sensitized in the footpad with short ragweed (SRW) allergen and challenged topically for seven consecutive days with SRW allergen. The number of splenic CD4(+) Th2 cells was determined by flow cytometry, and the cytokine profile of CD4(+) T cells from SRW-sensitized mice was evaluated by ELISA. The role of IFN-gamma in allergic conjunctivitis was also examined by timed in vivo neutralization with anti-IFN-gamma antibody. Allergic conjunctivitis was evaluated clinically and histopathologically. RESULTS: Repeated topical challenge with SRW allergen induced allergic conjunctivitis that was characterized by lid edema, chemosis, redness, and tearing. Histopathological analysis revealed a marked conjunctival infiltrate that was predominantly neutrophils and eosinophils. IFN-gamma KO mice and normal mice treated with anti-IFN-gamma antibody displayed milder clinical symptoms of allergic conjunctivitis and a 70% reduction in the number of eosinophils that infiltrated the conjunctiva. Spleen cells from SRW-sensitized mice contained a large population of cells that expressed the Th2 surface marker T1/ST2 and produced IL-4, -5, and -10 and IFN-gamma after stimulation with SRW allergen. CONCLUSIONS: Repeated topical application of SRW allergen induces a form of murine allergic conjunctivitis that mimics the human counterpart. IFN-gamma appears to contribute to the pathogenesis of murine allergic conjunctivitis at the effector phase, but not during the initial sensitization stage.

Allergens↗
PubMed2004

The role of the lacrimal functional unit in the pathophysiology of dry eye.

The majority of dry eye symptoms are due to a chronic inflammation of the lacrimal functional unit resulting in a loss of tear film integrity and normal function. This leads to a reduction in the ability of the ocular surface to respond to environmental challenges. The underlying cause of tear film dysfunction is the alteration of tear aqueous, mucin, and lipid components. This may result from a systemic autoimmune disease or a local autoimmune event. A lack of systemic androgen support to the lacrimal gland has been shown to be a facilitative factor in the initiation of this type of pathophysiology. Tear secretion is controlled by the lacrimal functional unit consisting of the ocular surface (cornea, conjunctiva, accessory lacrimal glands, and meibomian glands), the main lacrimal gland and the interconnecting innervation. If any portion of this functional unit is compromised, lacrimal gland support to the ocular surface is impeded. Factors such as neurogenic inflammation and T cell involvement in the disease pathogenesis as well as newly developed animal models of ocular surface inflammation are discussed.

Androgens↗