Cells In Conjunctiva Research Articles

In Vivo Confocal Microscopy of Conjunctival Roundish Bright Objects: Young, Older, and Sjögren Subjects

To investigate by laser scanning confocal microscopy (LSCM) the density of presumed epithelial, presumed goblet, and presumed inflammatory cells in the tarsal conjunctiva of healthy young and older subjects and in patients with Sjögren's syndrome (SS). To evaluate the interobserver variability and to compare the measured densities with known age-related and SS-related changes. The authors studied 24 eyes of 12 healthy young subjects (8 women, 4 men; average age, 26 years; age range, 21-30 years), 24 eyes of 12 healthy older subjects (10 women, 2 men; average age, 68 years; age range, 67-74 years), and 24 eyes of 12 patients with SS (10 women, 2 men; average age, 62 years; age range, 49-72 years). The inferior tarsal conjunctiva of each patient was examined in vivo by LSCM. The density of the three cell types was independently analyzed by two masked investigators. The density of presumed epithelial, presumed goblet, and presumed inflammatory cells was significantly higher in SS patients than in both control groups (P < 0.001; Mann-Whitney U test). The densities for presumed goblet cells calculated by the two investigators were significantly different from one another (P < 0.01, Mann-Whitney U test) and were not correlated. LSCM is a promising tool that should profoundly change the study of the ocular surface, but it requires accurate standardization before it is used in clinical practice.

Ameliorative Effect of Thymoquinone on Ovalbumin-induced Allergic Conjunctivitis in Balb/c Mice

Objective: Thymoquinone (TQ) is an active and potent compound in the oil of Nigella sativa, which has anti-inflammatory properties. The aim of this study was to evaluate the effects of TQ on ovalbumin (OVA)-induced allergic conjunctivitis (AC) in Balb/c.Methods: The mice were divided into seven experimental groups; PBS Con, OVA Con, Conj, 0.05%TQ, 0.1%TQ, 0.5%TQ and Dex group. The mice were immunized and exposed to OVA, and eyes were treated with TQ and dexamethasone. Ocular symptoms were observed after last exposure to OVA. Eosinophils count in blood and Ophthalmic lavage fluid (OLF), recruitment of inflammatory cells in conjunctiva, serum IgE and OVA-specific IgE were evaluated by Giemsa and HE staining, and Enzyme-Linked Immunosorbent Assay (ELISA) respectively. High Performance Liquid Chromatography (HPLC) was used to determine the histamine level in OLF. The mRNA expression and protein level of cytokines were examined by real time RT-PCR and ELISA respectively.Results: Ocular symptoms of AC and other characteristics of allergic inflammation including IgE and OVA-specific immunoglobulin E (IgE) level, recruitment of eosinophils, histamine level, mRNA expressions and protein level of cytokines were remarkably increased in OVA-exposed mice compared with the control groups. Administration of TQ suppressed the ocular symptoms, inflammatory cell infiltration in conjunctiva, blood and OLF, increased level of serum IgE and OVA-specific IgE, and OLF histamine level in OVA-exposed mice. Furthermore, TQ abrogated the mRNA expression and serum level of interleukin including 1L-4, IL-5, IL-13 and transforming growth factor beta (TGF-β) in mice immunized and exposed to OVA.Conclusion: Administration of TQ significantly reduced the ocular symptoms in AC by attenuating the recruitment of eosinophils, level of IgE, histamine and cytokines. Our findings suggest that TQ might be a useful intimation for the treatment and future research for AC.

Contact lens wear and the goblet cells of the human conjunctiva—A review

To review the reported effects of contact lens wear on the goblet cells of the human conjunctiva. A literature search was undertaken to identify reports on the conjunctival health after contact lens wear, principally as assessed using the conjunctival impression cytology (CIC) technique in which cells are examined ex vivo, after fixation and staining. Details of technique, data on duration of contact lens wear and then CIC outcome in terms of goblet cell density (GCD) were extracted. Of 24 reports identified, 22 examined the bulbar conjunctiva and 2 examined the tarsal conjunctiva. A decrease in GCD was considered, directly or indirectly, to be a consequence of contact lens wear in 18 of the studies, but there was no obvious overall relationship between duration of lens wear and the GCD changes. Conversely, four reports indicated an increase in GCD or goblet cell-related mucins. Two reports concluded that there was no change in goblet cells or their mucin, a result however that is consistent with a recent conclusion that no statistically significant change in GCD was detectable in contact lens wearers assessed by in vivo imaging of the human conjunctiva by confocal microscopy. The majority of published studies have concluded that contact lens wear results in a decrease in goblet cells in the conjunctiva. While there are reports that draw a very different conclusion, it should be noted that there has been limited consistency in technique or the method of reporting the results across the various studies.

Downregulation of c-Myc in pterygium and cultured pterygial cells

The aim of this study was to determine c-Myc expression in pterygial tissue and in cultured fibroblasts and epithelial cells of pterygia, using normal human conjunctival samples as a control. Expression of c-Myc in pterygium and normal human conjunctiva tissue was examined by immunohistochemical assay and Western blot. Fibroblasts and epithelial cells from primary pterygium and normal human conjunctiva were cultured in medium with or without transforming growth factor beta1 for up to 3 days. c-Myc protein expression was analysed by indirect immunofluorescence. Levels of c-Myc mRNA in fibroblasts were measured by reverse transcription polymerase chain reaction. c-Myc protein expression was reduced in human pterygial tissue compared with normal human conjunctiva in immunohistochemical staining. The decrease was further verified by Western blot analysis. In primary cultured cells c-Myc expression as stained by immunofluorescence was reduced in both pterygial epithelial and fibroblast cells when compared with cultured normal human conjunctiva cells. In addition, compared with cells from normal conjunctiva, reduced levels of c-Myc mRNA were observed in both cultured pterygial epithelial and fibroblast cells. Increased c-Myc expression in pterygial cells, but not in normal conjunctival cells, was found when the cultured cells were treated with transforming growth factor beta1. This increase of c-Myc mRNA appeared to be time and transforming growth factor beta1 dose dependent. c-Myc expression is reduced in pterygium compared with normal conjunctiva. Transforming growth factor beta1 can elevate the downregulated c-Myc in cultured pterygial cells.

Challenge and treatment strategy for ocular surface damage in patients with long term use of antiglaucoma drugs

Long term use of topical anti-glaucoma drugs has been shown to induce chronic conjunctivitis, superficial punctate keratitis (SPK) and dry eye symptom. Under these conditions, a loss of goblet cells in conjunctiva, epithelial squamous metaplasia and apoptosis were morphologically revealed. Benzalkonium Chloride (BKC), a most frequently used preservative in eye drops, has been found to be an important factor causing ocular surface damage. Furthermore, a big challenge for ophthalmologists is that toxic damage of medication to ocular surface tissues is mild, poor specificity, and delayed manifestation in patients, especially when coexisting with other ocular surface diseases. Impairment of ocular surface tissues greatly impacts the life quality of patients and subsequently influences compliance with glaucoma therapy. This paper emphasizes to take measures to prevent ocular surface tissue damage resulted from chronic use of topical anti-glaucoma drugs and further discusses the treatment strategy. Effective and long-lasting action drugs should always be selected for glaucomatous patients in order to decrease the frequency of topical instillation or at a more expensive medication, a fixed combination formula can be considered for glaucoma therapy. An early surgery or laser treatment is also proposed for the patients who require an IOP reduction with an existing ocular surface impairment. Future investigation and development of new medications with long-term efficacy and appropriate BKC are suggested and preservative-free or drugs with new preservative materials recommended.

In vivo laser scanning confocal microscopy of vernal keratoconjunctivitis

To demonstrate the morphological characteristics of vernal keratoconjunctivitis (VKC) by using in vivo laser scanning confocal microscope (LSCM). Descriptive analysis of VKC by LSCM performed in EENT Hospital of Fudan University. 26 patients diagnosed as VKC and 26 age-matched healthy controls were enrolled in the study. The HRTII/RCM was used to examine bulbar conjunctiva, tarsal conjunctiva, superior limbus and inferior limbus. The density of Langerhans cells and inflammatory cells in tarsal and bulbar conjunctiva were calculated. The morphology of Vogt Palisades was analysed and normal rate of Vogt Palisades at superior and inferior limbus was calculated, respectively. Infiltration of a larger number of Langerhans cells and inflammatory cells could be identified in both bulbar conjunctiva and tarsal conjunctiva compared with normal subjects. Furthermore, the density of Langerhans cells in tarsal conjunctiva was significantly higher in tarsal form and mix form than that in bulbar form (P = 0.048). Normal Vogt Palisades were visible only in 15 and 23 eyes at superior and inferior limbus respectively. Abnormal Vogt Palisades were characterized by atrophy or absence of stromal papillae and disappearance of bright basal cells, accompanied with the infiltration of more Langerhans cells. At superior limbal area, bulbar form had the highest abnormal rate, followed by mix form, both were significantly higher than tarsal form (P = 0.005). The in vivo morphological characteristics of conjunctiva and limbus in VKC included the infiltration of Langerhans cells and inflammatory cells in epithelium and stroma, and the destruction of Vogt Palisades.

In Vivo Confocal Microscopy of Long-standing Mixed-form Vernal Keratoconjunctivitis

Purpose: To report the morphological characteristics of long-term mixed-form vernal keratoconjunctivitis (VKC) using in vivo laser scanning confocal microscopy (LSCM).Design: Descriptive case report.Methods: In vivo LSCM was performed on a Chinese patient diagnosed as bilateral mixed-form VKC for 14 years. Central cornea, inferior limbus, bulbar conjunctiva, and upper tarsal conjunctiva of both eyes were examined.Results: Infiltration of numerous dendritic cells and inflammatory cells in both bulbar conjunctiva and tarsal conjunctiva were identified under in vivo LSCM, along with proliferation of fibrous tissue in the conjunctival stroma and damage of Vogt palisades. Apart from abnormal appearance of corneal epithelial cells, the infiltration of dendritic cells was occasionally found in the cornea.Conclusions: In vivo LSCM is a useful tool to evaluate the morphology of VKC.

Characterisation of the phenotype and function of monocyte-derived dendritic cells in allergic conjunctiva

BackgroundDendritic cells (DCs) are the most potent antigen-presenting cells involved in initiating the immune response, presenting antigens to T cells and leading to T cell proliferation. In an immature state,...

A Novel HLA (HLA-A*0201) Transgenic Rabbit Model for Preclinical Evaluation of Human CD8+ T Cell Epitope-Based Vaccines against Ocular Herpes

We introduced a novel humanized HLA-A*0201 transgenic (HLA Tg) rabbit model to assess the protective efficacy of a human CD8(+) T cell epitope-based vaccine against primary ocular herpes infection and disease. Each of the three immunodominant human CD8(+) T cell peptide epitopes from HSV-1 glycoprotein D (gD(53-61), gD(70-78), and gD(278-286)) were joined with a promiscuous human CD4(+) T cell peptide epitope (gD(49-82)) to construct three separate pairs of CD4-CD8 peptides. Each CD4-CD8 peptide pair was then covalently linked to an N(epsilon)-palmitoyl-lysine residue via a functional base lysine amino group to construct CD4-CD8 lipopeptides. HLA Tg rabbits were immunized s.c. with a mixture of the three CD4-CD8 HSV-1 gD lipopeptides. The HSV-gD-specific T cell responses induced by the mixture of CD4-CD8 lipopeptide vaccine and the protective efficacy against acute virus replication and ocular disease were determined. Immunization induced HSV-gD(49-82)-specific CD4(+) T cells in draining lymph node (DLN); induced HLA-restricted HSV-gD(53-61), gD(70-78), and gD(278-286)-specific CD8(+) T cells in DLN, conjunctiva, and trigeminal ganglia and reduced HSV-1 replication in tears and corneal eye disease after ocular HSV-1 challenge. In addition, the HSV-1 epitope-specific CD8(+) T cells induced in DLNs, conjunctiva, and the trigeminal ganglia were inversely proportional with corneal disease. The humanized HLA Tg rabbits appeared to be a useful preclinical animal model for investigating the immunogenicity and protective efficacy of human CD8(+) T cell epitope-based prophylactic vaccines against ocular herpes. The relevance of HLA Tg rabbits for future investigation of human CD4-CD8 epitope-based therapeutic vaccines against recurrent HSV-1 is discussed.

Normal human bulbar conjunctiva on confocal microscopy in vivo

To analyze the morphology of human bulbar conjunctiva by in vivo laser scanning confocal microscopy. This research was a cross-sectional study. From February to July 2008, 50 eyes of 50 healthy subjects were enrolled in this study. They had no history of ocular trauma, infection or contact lens wear and had no found after routine slit-lamp examinations. In vivo laser scanning confocal microscopic examinations were performed on the superior, inferior, nasal and temporal bulbar conjunctiva and the images were recorded. The morphology of bulbar conjunctiva was analyzed and the density of epithelial cells, dendritic cells and goblet cells were calculated. One-way analysis of variance (ANOVA) was used to compare the means of epithelial cell densities in different layers and goblet cell densities in different positions. Subsequently the datum between two groups were analyzed by least significant difference (LSD). Superficial epithelial cells of bulbar conjunctiva were characterized as large loose-arranged cells with a hyporeflective nucleus. The mean density is (1643 +/- 206) cells/mm(2). Intermediate epithelial cells were captured with features of oval small tight-arranged cells with a punctiform hyperreflective nucleus. The mean density is (4693 +/- 228) cells/mm(2). Basal epithelial cells appeared to be polygonal and regular-arranged within hyperreflective cell borders. The mean density is (4420 +/- 230) cells/mm(2). There was a significant difference among three kinds of conjunctival epithelium (F = 1160.312, P = 0.000). The presumed goblet cell was defined as a large hyperreflective oval-shaped cell with relatively homogeneous brightness, crowded in groups or mainly dispersed. The mean density is (432 +/- 72) cells/mm(2). The dendritic cell appeared to be hyperreflective corpuscular particles with dendritic processes scattered among conjunctival epithelial cells. The mean density is (22 +/- 25) cells/mm(2). The basement membrane, a prominent hyperreflective band, separated epithelial cells from subepithelial structure. Bulbar conjunctival substantia propria, beneath the basement membrane, was mainly composed of highly vascularized, loose connective tissues which were irregularly arranged fibers or a network of fibers, punctiform hyperreflective immune cells and sharp flows of blood vessels. In vivo laser scanning confocal microscopy is a useful tool in the analysis of the bulbar conjunctival morphology, which provided a fast and noninvasive method for the diagnosis of ocular surface diseases.

TGFb in fibroproliferative diseases in the eye

Transforming growth factor b (TGF beta) is believed to be the most important ligand in the pathogenesis of fibrotic diseases in the eye. Such ocular fibrotic diseases include scarring in the cornea and conjunctiva, fibrosis in the corneal endothelium, post-cataract surgery fibrosis of the lens capsule, excess scarring the tissue around the extraocular muscles in the strabismus surgery and proliferative vitreoretinopathy. In the proliferative stage of diabetic retinopathy, fibrogenic reaction causes tractional retinal detachment in association with contraction of the tissue. A myofibroblast, the major cellular component in the fibrotic lesions, is derived from both mesenchymal cells (in cornea and conjunctiva) and epithelial cell types (lens or retinal pigment epithelium or corneal endothelium) through epithelial-mesenchymal transition (EMT). The myofibroblasts cause excess accumulation of fibrogenic extracellular matrix with resultant tissue contraction and impaired functions. Although various cytokine signaling pathways are involved in the fibrogenic reaction in tissues, TGF beta/Smad signal is the critical one. Blocking Smad signal by chemical or natural inhibitors or anti-Smad gene introduction effectively suppress fibrogenic reaction; inhibition of both fibroblast-myofibroblast conversion or EMT. Such strategies can be clinically tested.

TGFb in fibroproliferative diseases in the eye

Transforming growth factor b (TGF beta) is believed to be the most important ligand in the pathogenesis of fibrotic diseases in the eye. Such ocular fibrotic diseases include scarring in the cornea and conjunctiva, fibrosis in the corneal endothelium, post-cataract surgery fibrosis of the lens capsule, excess scarring the tissue around the extraocular muscles in the strabismus surgery and proliferative vitreoretinopathy. In the proliferative stage of diabetic retinopathy, fibrogenic reaction causes tractional retinal detachment in association with contraction of the tissue. A myofibroblast, the major cellular component in the fibrotic lesions, is derived from both mesenchymal cells (in cornea and conjunctiva) and epithelial cell types (lens or retinal pigment epithelium or corneal endothelium) through epithelial-mesenchymal transition (EMT). The myofibroblasts cause excess accumulation of fibrogenic extracellular matrix with resultant tissue contraction and impaired functions. Although various cytokine signaling pathways are involved in the fibrogenic reaction in tissues, TGF beta/Smad signal is the critical one. Blocking Smad signal by chemical or natural inhibitors or anti-Smad gene introduction effectively suppress fibrogenic reaction; inhibition of both fibroblast-myofibroblast conversion or EMT. Such strategies can be clinically tested.

Localization of the low‐affinity nerve growth factor receptor p75 in human limbal epithelial cells

Biological effects of nerve growth factor (NGF) are mediated through receptors known as nerve growth factor receptors (NGFR), which include p75 and TrkA. This study was initiated after identifying NGFR as an up-regulated gene in the limbus by cDNA microarray analysis and we postulate that its expression may be indicative of a stem/progenitor cell phenotype. Immunohistochemistry was performed on normal human adult (n= 5) and foetal (n= 3) corneal tissue using antibodies directed against p75, TrkA, NGF, p63, ABCG2 and CK3/12. Limbal, conjunctival and pterygium tissue was obtained from patients (n= 10) undergoing pterygium resection and used for immunohistochemical assessment. Paraffin-embedded archival human skin specimens (n= 4) were also evaluated. In vitro expression of NGFR was determined in limbal, conjunctival and pterygium-derived epithelial cells. p75 was selectively expressed by basal epithelial cells in pterygia, conjunctiva and limbus, but was absent in the central cornea. These results were confirmed with two additional p75 specific antibodies. In contrast, TrkA was found in full-thickness pterygium, conjunctival, limbal and corneal epithelium in both adult and foetal eyes. p75 expression was identified in a small percentage, while TrkA was found on the entire population of cultured conjunctival, limbal and pterygium-derived epithelial cells. This receptor was also observed in selective regions of the human epidermis and hair follicle bulge. Our results illustrate the selective expression of p75 in basal pterygium, conjunctival and limbal epithelium, while staining was absent in adult and foetal central cornea. p75 may represent an additional ocular surface epithelial stem/progenitor cell signature gene.

Detection and Regulation of Cationic Amino Acid Transporters in Healthy and Diseased Ocular Surface

To evaluate the presence and role of human cationic amino acid transporters (hCATs) at the ocular surface in healthy and pathologic states and under experimental inflammatory conditions. Expression of mRNA for hCATs 1, 2, and 3 (SLC7A1, SLC7A2, and SLC7A3) was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) in healthy lacrimal gland, conjunctiva, cornea, and nasolacrimal ducts and in an SV40 immortalized human corneal epithelial (HCE) cell line. Localization of hCAT1 and hCAT2 was determined by immunohistochemistry in healthy tissues and in sections of different corneal abnormalities, including keratoconus, Fuchs dystrophy, and herpetic keratitis. Cultured corneal epithelial cells were stimulated with proinflammatory cytokines and supernatants of Staphylococcus aureus and Pseudomonas aeruginosa and were analyzed by real-time PCR. Expression of hCAT1 and hCAT2 mRNA, but not of hCAT3 mRNA, was detected in healthy conjunctiva, cornea, and nasolacrimal ducts. Human lacrimal gland revealed only hCAT2 mRNA expression. Immunohistochemistry demonstrated the presence of hCAT1 and hCAT2 in epithelial cells of cornea, conjunctiva, and nasolacrimal ducts. Goblet cells revealed no reactivity. Moreover, hCAT2 was visible in acinar cells of lacrimal gland. No changes in staining reactivity were obtained for hCAT1 in different corneal abnormalities. In contrast, hCAT2 showed increased subjective staining intensity in all corneal abnormalities. Cell culture experiments revealed that TNF-alpha and supernatant of S. aureus increased hCAT1 and hCAT2 expression significantly. Supernatant of P. aeruginosa led to an increase in hCAT2-expression only. These results show for the first time the distribution of hCATs in tissues of the ocular surface and lacrimal apparatus. Both transporters seem to be differently regulated under pathologic conditions of the ocular surface. Their physiological functions in amino acid transport make them potential candidates for therapeutic intervention.

Kinetics of immune cell migration at the human ocular surface

Aim:The conjunctiva has a resident population of intraepithelial and stromal immune cells. These cells play an active part in ocular surface defence and disease. Our aim was to study the...

The Effect of Extremely Low Frequency Magnetic Field on the Conjunctiva and Goblet Cells

Purpose: The aim of this study was to examine the effects of extremely low frequency magnetic field (ELF MF) on the conjunctiva and goblet cells density. Methods: Thirty adult female Spraque Dawley rats were assigned to one of two groups, each containing 15 rats. The experimental group received magnetic field 4 hr/day for 7 days. The second group received standard laboratory care and served as a control. The specimens were evaluated by light microscopy for goblet cell density, conjunctival edema, inflammation, and fibroblast proliferation. Results: In ELF MF exposure group rats, the number of goblet cells was less than the control group. We also observed a significant difference between ELF MF exposure group and control group in terms of goblet cell density (p = 0.010). The ELF MF exposure group manifested conjunctival edema and inflammation compared to the control group (p < 0.001 and p = 0.003). There was no statistical difference between the groups for fibroblast proliferation (p = 0.422). Conclusion: This study suggests that ELF MF exposure leads to morphological alterations of the conjunctiva and reductions in the number of goblet cells. We believe that ELF MF may cause dry eye symptoms.

Functional Foxp3+CD4+CD25(Bright+)“Natural” Regulatory T Cells Are Abundant in Rabbit Conjunctiva and Suppress Virus-Specific CD4+and CD8+Effector T Cells during Ocular Herpes Infection

We studied the phenotype and distribution of "naturally" occurring CD4(+) CD25(+) T regulatory cells (CD4(+) CD25(+) nT(reg) cells) resident in rabbit conjunctiva, the main T-cell inductive site of the ocular mucosal immune system, and we investigated their suppressive capacities using herpes simplex virus type 1 (HSV-1)-specific effector T (T(eff)) cells induced during ocular infection. The expression of CD4, CD25, CTLA4, GITR, and Foxp3 was examined by reverse transcription-PCR, Western blotting, and fluorescence-activated cell sorter analysis in CD45(+) pan-leukocytes isolated from conjunctiva, spleen, and peripheral blood monocyte cells (PBMC) of HSV-1-infected and uninfected rabbits. Normal conjunctiva showed a higher frequency of CD4(+) CD25((Bright+)) T cells than did spleen and PBMC. These cells expressed high levels of Foxp3, GITR, and CTLA4 molecules. CD4(+) CD25((Bright+)) T cells were localized continuously along the upper and lower palpebral and bulbar conjunctiva, throughout the epithelium and substantia propria. Conjunctiva-derived CD4(+) CD25((Bright+)) T cells, but not CD4(+) CD25((low)) T cells, efficiently suppressed HSV-specific CD4(+) and CD8(+) T(eff) cells. The CD4(+) CD25((Bright+)) T-cell-mediated suppression was effective on both peripheral blood and conjunctiva infiltrating T(eff) cells and was cell-cell contact dependent but independent of interleukin-10 and transforming growth factor beta. Interestingly, during an ocular herpes infection, there was a selective increase in the frequency and suppressive capacity of Foxp3(+) CD4(+) CD25((Bright+)) T cells in conjunctiva but not in the spleen or in peripheral blood. Altogether, these results provide the first evidence that functional Foxp3(+) CD4(+) CD25((Bright+)) T(reg) cells accumulate in the conjunctiva. It remains to be determined whether conjunctiva CD4(+) CD25(+) nT(reg) cells affect the topical/mucosal delivery of subunit vaccines that stimulate the ocular mucosal immune system.

Clusters of Corneal Epithelial Cells Reside Ectopically in Human Conjunctival Epithelium

The ocular surface is covered by two biologically distinct epithelia: corneal and conjunctival. The expression of keratin12 (K12) is currently considered a hallmark of cornea-type differentiation. In the current study, the biological features of K12-positive cells in human bulbar conjunctival epithelium were examined. Human conjunctival tissues were subjected to investigate the K12-positive cells in conjunctiva by immunostaining, in situ hybridization, Western blot analysis, reverse transcriptase-polymerase chain reaction (RT-PCR), and fluorescence-activated cell sorting (FACS). Gene expression profiling of these cells was performed with introduced amplified-fragment length polymorphism (iAFLP). To determine the presence of stem- or progenitor cells, immunostaining and colony-forming assays were performed. Western blot analysis, RT-PCR revealed that K12 was expressed in conjunctival epithelium. Immunostaining analysis showed that K12-positive cells reside mainly in clusters in conjunctival epithelium. FACS analysis showed that 0.2% to 1.7% of conjunctival epithelial cells collected from the inferior bulbar conjunctiva were K12 positive. iAFLP analysis revealed that the gene expression patterns of these cells were highly similar to that of corneal epithelial cells. p63 and ABCG2 were expressed beneath the K12-positive cells. Some colony-forming cells expressed K12. The K12-positive cells appear to be ectopically residing, self-maintaining corneal epithelial cells in the conjunctival epithelium.

M Cells in the Follicle-Associated Epithelium of the Rabbit Conjunctiva Preferentially Bind and Translocate Latex Beads

This study investigates the presence of M cells in the rabbit conjunctiva. Resolving whether the conjunctiva contains M cells is important, because at other mucosal sites, these antigen sampling cells are known to initiate the mucosal immune response and to act as a site of entry for opportunistic pathogens. Fluorescent 0.2-microm polystyrene latex beads were either instilled into the conjunctival sac for 20 to 120 minutes in vivo or applied to flat mounts in vitro. Specimens were assessed by epi-fluorescence stereomicroscopy, widefield fluorescence microscopy, confocal scanning laser microscopy, and transmission and scanning electron microscopy. Latex beads preferentially bound to a subpopulation of cells in the epithelium overlying mucosal lymphoid follicles in the fornix region. At 4 degrees C, the beads were associated with the apical surface of cells that had longer, more irregular microvilli than the surrounding epithelial cells. Within 20 minutes of an in vivo exposure, latex beads were internalized by the follicle-associated epithelial cells and with time moved into the underlying follicle region. After 120 minutes of in vivo exposure, latex beads could be found in cervical lymph nodes. This study demonstrates that the follicle-associated epithelium of the rabbit conjunctiva contains a cell with morphologic characteristics and the ability to bind and translocate latex beads, which make it indistinguishable from antigen sampling M cells in the rabbit cecum and tonsils. Consistent with its hypothesized antigen sampling role, beads that have been translocated by this cell are rapidly transferred to cervical lymph nodes.

Effect of pollen-mediated oxidative stress on immediate hypersensitivity reactions and late-phase inflammation in allergic conjunctivitis

Allergic eye diseases are complex inflammatory conditions of the conjunctiva that are becoming increasingly prevalent and present an increasing economic burden because of direct and indirect health expenditures. We sought to identify factors that may synergize with antigen-induced allergic inflammation and lead to allergic conjunctivitis. We used a murine model of allergic conjunctivitis to test the effect of oxidative stress generated by pollen oxidases using nicotinamide adenine dinucleotide (reduced) or nicotinamide adenine dinucleotide phosphate (reduced) (NAD[P]H) as an electron donor present in pollen grains. Reactive oxygen species (ROS) generation by hydrated Ambrosia artemisiifolia pollen (short ragweed pollen; RWP) grains was determined by using 2'-7'-dihydro-dichlorofluorescein diacetate, nitroblue tetrazolium reduction, and Amplex Red assay. The RWP-induced changes in intracellular ROS levels were examined in A549 cells, human primary bronchial epithelial cells, and murine conjunctiva. Ragweed pollen grains contain NAD(P)H oxidase activity, which is diphenyleneiodonium-sensitive and quinacrine-sensitive and sodium azide-resistant. These NAD(P)H oxidases generate a superoxide anion that can be converted to H2O2 by pollen grain-associated superoxide dismutase. These diffusible oxygen radicals from pollen grains increase intracellular ROS levels in cultured epithelial cells and murine conjunctiva. Similar phenomena were observed in sensitized and naive mice, indicating that the RWP-induced oxidative stress in conjunctival epithelium is independent of adaptive immunity. Inactivation of NAD(P)H oxidase activity in RWP decreases the immediate-type hypersensitivity and inflammatory cell infiltration into the conjunctiva. Our data suggest that ROS generated by NAD(P)H oxidases in pollen grains intensify immediate allergic reactions and recruitment of inflammatory cells in murine conjunctiva.